TY - JOUR AB - Manipulation of glucose uptake plays a critical role in muscle glucose disposal. We have shown that the secreted isoform of endoplasmic reticulum membrane protein complex subunit 10 (scEMC10) impairs glucose tolerance in mice and serum scEMC10 is positively associated with insulin resistance and hyperglycemia in humans. In this study, we attempt to investigate whether modulation of muscle glucose uptake implicates in the scEMC10-impacted glucose homeostasis. In mouse models, Emc10 gene knockout elevated, while recombinant scEMC10 treatment reduced, muscle glucose uptake and GLUT4 expression. In myoblasts, scEMC10 inhibited both GLUT4 expression and membrane translocation, and downregulated expression of genes associated with intracellular glucose metabolism. Mechanistically, scEMC10 suppressed the activation of muscle AMPK and insulin signaling cascades. Inhibition of scEMC10 via a neutralizing antibody enhanced muscle glucose uptake in mice, in parallel with heightened GLUT4 expression and membrane translocation, which accounts for an improved whole-body glucose homeostasis. In conclusion, this work identifies scEMC10 as a novel suppressor of muscle glucose uptake, and suggests inhibition of scEMC10 as a therapeutic strategy for type 2 diabetes. AU - Jin, S.* AU - Wu, W.* AU - Liu, S.* AU - Wang, Y.* AU - Huang, Q.* AU - He, K.* AU - Ni, Y.* AU - Chen, K.* AU - Huang, J.* AU - Liu, L.* AU - Dai, J.* AU - Zhan, C.* AU - Wang, X.* AU - Guan, Y.* AU - Blüher, M. C1 - 74846 C2 - 57634 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Secreted EMC10 inhibits muscle GLUT4 activity and glucose uptake in mice. JO - J. Biol. Chem. VL - 301 IS - 7 PB - Elsevier PY - 2025 SN - 0021-9258 ER - TY - JOUR AB - Deoxynivalenol (DON) is a toxicologically relevant trichothecene mycotoxin frequently found in cereal products. It is a virulence factor produced by the plant pathogen Fusarium graminearum during cereal crop infections. Investigating plant defense mechanisms is crucial for understanding plant resistance to F. graminearum and identifying new biocatalysts for DON detoxification. Previous studies identified DON-thiol adducts in cereal samples, indicating partial DON detoxification by glutathione transferases (GSTs). DON possesses two electrophilic centers for thiol conjugation, resulting in either epoxide opening at C13 or Michael addition at C10. At present, information on plant GSTs that catalyze these reactions is limited. In this study, Fusarium-inducible wheat GSTs were identified by analyzing the transcriptome of Fusarium-infected wheat heads. Twelve highly induced genes of the tau and phi GST classes were heterologously expressed and purified, biochemically characterized with model substrates, and assayed for activity with DON. Use of liquid chromatography coupled to mass spectrometry showed that four of the selected tau class GSTs conjugated DON to glutathione (GSH) by epoxide opening (DON-13-GSH) and/or the reversible Michael addition reaction (DON-10-GSH). The crystal structure of a wheat GST (herein designated "TaGST-10") in complex with DON-13-GSH was solved at a resolution of 2.3 Å and provided insights into the binding of DON at the active site of tau class GSTs. Our results corroborate the hypothesis that enzyme-catalyzed, glutathione-mediated DON detoxification may be involved in plant response to Fusarium infection. AU - Michlmayr, H.* AU - Siller, M.* AU - Kenjeric, L.* AU - Doppler, M.* AU - Malachová, A.* AU - Hofer, M.J.* AU - Hametner, C.* AU - Schweiger, W.* AU - Steiner, B.* AU - Kugler, K.G. AU - Mayer, K.F.X. AU - Buerstmayr, H.* AU - Schuhmacher, R.* AU - Krska, R.* AU - Labrou, N.E.* AU - Papageorgiou, A.C.* AU - Adam, G.* C1 - 75357 C2 - 58138 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Detoxification of deoxynivalenol by pathogen-inducible tau-class glutathione transferases from wheat. JO - J. Biol. Chem. VL - 301 IS - 10 PB - Elsevier PY - 2025 SN - 0021-9258 ER - TY - JOUR AB - Cellular plasticity is crucial for adapting to ever-changing stimuli. As a result, cells consistently reshape their translatome, and, consequently, their proteome. The control of translational activity has been thoroughly examined at the stage of translation initiation. However, the regulation of ribosome speed in cells is widely unknown. In this study, we utilized a timed ribosome runoff approach along with proteomics and transmission electron microscopy, to investigate global translation kinetics in cells. We found that ribosome speeds vary amongst various cell types, such as astrocytes, induced pluripotent human stem cells, human neural stem cells, and human and rat neurons. Of all cell types studied, mature cortical neurons exhibit the highest rate of translation. This finding is particularly remarkable because mature cortical neurons express eEF2 at lower levels than other cell types. Neurons solve this conundrum by inactivating a fraction of their ribosomes. As a result, the increase in eEF2 levels leads to a reduction of inactive ribosomes and an enhancement of active ones. Processes that alter the demand for active ribosomes, like neuronal excitation, cause increased inactivation of redundant ribosomes in an eEF2-dependent manner. Our data suggest a novel regulatory mechanism in which neurons dynamically inactivate ribosomes to facilitate translational remodelling. These findings have important implications for developmental brain disorders characterised by, among other things, aberrant translation. AU - Popper, B.* AU - Bürkle, M.* AU - Ciccopiedi, G.* AU - Marchioretto, M.* AU - Forné, I.* AU - Imhof, A.* AU - Straub, T.* AU - Viero, G.* AU - Götz, M. AU - Schieweck, R.* C1 - 69749 C2 - 55249 TI - Ribosome inactivation regulates translation elongation in neurons. JO - J. Biol. Chem. VL - 300 IS - 2 PY - 2024 SN - 0021-9258 ER - TY - JOUR AB - Soluble amyloid-β oligomers (AβOs) are proposed to instigate and mediate the pathology of Alzheimer's disease (AD), but the mechanisms involved are not clear. In this study, we reported that AβOs can undergo liquid-liquid phase separation (LLPS) to form liquid-like droplets in vitro. We determined that AβOs exhibited an α-helix conformation in a membrane-mimicking environment of sodium dodecyl sulfate (SDS). Importantly, SDS is capable of reconfiguring the assembly of different AβOs to induce their LLPS. Moreover, we found that droplet formation of AβOs was promoted by strong hydrated anions and weak hydrated cations, suggesting that hydrophobic interactions play a key role in mediating phase separation of AβOs. Finally, we observed that LLPS of AβOs can further promote Aβ to form amyloid fibrils, which can be modulated by (-)-epigallocatechin gallate (EGCG). Our study highlights amyloid oligomers as an important entity involved in protein liquid-to-solid phase transition and reveals the regulatory role of LLPS underlying amyloid protein aggregation, which may be relevant to the pathological process of AD. AU - Gui, X.* AU - Feng, S.* AU - Li, Z.* AU - Li, Y.* AU - Reif, B. AU - Shi, B.* AU - Niu, Z.* C1 - 67257 C2 - 54196 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Liquid-liquid phase separation of amyloid-β oligomers modulates amyloid fibrils formation. JO - J. Biol. Chem. VL - 299 IS - 3 PB - Elsevier PY - 2023 SN - 0021-9258 ER - TY - JOUR AB - Inosine 5′ monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report the identification of two additional missense variants in IMPDH2 from affected individuals and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation. AU - O'Neill, A.G.* AU - Burrell, A.L.* AU - Zech, M. AU - Elpeleg, O.* AU - Harel, T.* AU - Edvardson, S.* AU - Mor-Shaked, H.* AU - Rippert, A.L.* AU - Nomakuchi, T.* AU - Izumi, K.* AU - Kollman, J.M.* C1 - 68385 C2 - 54622 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Neurodevelopmental disorder mutations in the purine biosynthetic enzyme IMPDH2 disrupt its allosteric regulation. JO - J. Biol. Chem. VL - 299 IS - 8 PB - Elsevier PY - 2023 SN - 0021-9258 ER - TY - JOUR AB - The heat shock protein 90 (Hsp90) is a molecular chaperone central to client protein folding and maturation in eukaryotic cells. During its chaperone cycle, Hsp90 undergoes ATPase-coupled large-scale conformational changes between open and closed states, where the N-terminal and middle domains of the protein form a compact dimerized conformation. However, the molecular principles of the switching motion between the open and closed states remain poorly understood. Here we show by integrating atomistic and coarse-grained molecular simulations with small-angle X-ray scattering experiments and nuclear magnetic resonance spectroscopy data that Hsp90 exhibits rich conformational dynamics modulated by the charged linker, which connects the N-terminal with the middle domain of the protein. We show that the dissociation of these domains is crucial for the conformational flexibility of the open state, with the separation distance controlled by a beta-sheet motif next to the linker region. Taken together, our results suggest that the conformational ensemble of Hsp90 comprises highly extended states, which could be functionally crucial for client processing. AU - Jussupow, A.* AU - Lopez, A. AU - Baumgart, M.* AU - Mader, S.L.* AU - Sattler, M. AU - Kaila, V.R.I.* C1 - 65462 C2 - 52691 TI - Extended conformational states dominate the Hsp90 chaperone dynamics. JO - J. Biol. Chem. VL - 298 IS - 7 PY - 2022 SN - 0021-9258 ER - TY - JOUR AB - Integrin-linked kinase (ILK), a central component of the intracellular ILK–pinch–parvin complex, localizes together with paxillin to focal adhesions and regulates integrin-mediated cell functions. ILK was initially misclassified as a kinase based on phenotypical characterization of cells expressing ILK mutated in the “kinase” domain, such as the E359K and K220M mutants and a V386G/T387G mutation in the paxillin-binding site (PBS). ILK is now known to be a pseudokinase, and mechanisms of action of these mutants are not clear. We selectively induced expression of only the E359K, PBS, and K220M ILK mutations in the developing kidney collecting system and kidney collecting duct (CD) cells and analyzed their impact on structural integrity using molecular dynamics (MD) simulations. Mice or CD cells carrying the E359K mutation had a severe phenotype that is indistinguishable from ILK-null mice or ILK-null CD cells. The K220M mutant mice developed normally, and K220M-CD cells had a mild adhesion, migration, and tubulogenesis defect. The PBS mutant mice had a subtle developmental defect, and PBS-CD cells had moderate functional abnormalities. Consistent with these observed phenotypes, MD studies suggest that the E359K mutant produces the most structurally perturbed, and K220M the most WT-like ILK molecules. Although all three mutations disrupted ILK binding to parvin and paxillin in vitro, only the E359K mutation decreased ILK binding to pinch suggesting that it increases ILK misfolding. Thus, point mutations in the ILK pseudokinase domain cause functional abnormalities by altering the ILK structure, leading to increased turnover and destabilization of ILK–parvin and (sometimes) ILK–pinch interactions. The integrin-linked kinase (ILK)–pinch–parvin (IPP) complex is a critical component of focal adhesions that binds to the cytoplasmic tail of the integrin β subunits. Integrins, composed of an α and a β subunit, are the principal receptors that mediate cell–extracellular matrix interactions and regulate many cell functions, including adhesion, spreading, migration, polarization, and tubulogenesis. ILK is a 450 amino acid multidomain. AU - Bulus, N.* AU - Brown, K.L.* AU - Mernaugh, G.* AU - Böttcher, A. AU - Dong, X.* AU - Sanders, C.R.* AU - Pozzi, A.* AU - Fässler, R.* AU - Zent, R.* C1 - 61666 C2 - 50376 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Disruption of the integrin-linked kinase (ILK) pseudokinase domain affects kidney development in mice. JO - J. Biol. Chem. VL - 296 PB - Elsevier PY - 2021 SN - 0021-9258 ER - TY - JOUR AB - Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein-protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsis mdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene, implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid; however, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems. AU - Gruner, K.* AU - Leissing, F.* AU - Sinitski, D.* AU - Thieron, H.* AU - Axstmann, C.* AU - Baumgarten, K.* AU - Reinstädler, A.* AU - Winkler, P.* AU - Altmann, M. AU - Flatley, A. AU - Jaouannet, M.* AU - Zienkiewicz, K.* AU - Feussner, I.* AU - Keller, H.* AU - Coustau, C.* AU - Falter-Braun, P. AU - Feederle, R. AU - Bernhagen, J.* AU - Panstruga, R.* C1 - 61744 C2 - 50434 CY - Radarweg 29, 1043 Nx Amsterdam, Netherlands TI - Chemokine-like MDL proteins modulate flowering time and innate immunity in plants. JO - J. Biol. Chem. VL - 296 PB - Elsevier PY - 2021 SN - 0021-9258 ER - TY - JOUR AB - Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt b-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Ca chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure. AU - Pradhan, T. AU - Annamalai, K.* AU - Sarkar, R. AU - Huhn, S.* AU - Hegenbart, U.* AU - Schönland, S.* AU - Fändrich, M.* AU - Reif, B. C1 - 60893 C2 - 49615 CY - 11200 Rockville Pike, Suite 302, Rockville, Md, United States SP - 18474-18484 TI - Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability. JO - J. Biol. Chem. VL - 295 IS - 52 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2020 SN - 0021-9258 ER - TY - JOUR AB - The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor ? (TNF?), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane?spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface?biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow?derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow?derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17. AU - Weskamp, G.* AU - Tüshaus, J.* AU - Li, D.* AU - Feederle, R. AU - Maretzky, T.* AU - Swendemann, S.* AU - Falck-Pedersen, E.* AU - McIlwain, D.R.* AU - Mak, T.W.* AU - Salmon, J.E.* AU - Lichtenthaler, S.F.* AU - Blobel, C.P.* C1 - 58771 C2 - 48312 CY - 11200 Rockville Pike, Suite 302, Rockville, Md, United States SP - 4350-4358 TI - ADAM17 stabilizes its interacting partner inactive Rhomboid 2 (iRhom2) but not inactive Rhomboid 1 (iRhom1). JO - J. Biol. Chem. VL - 295 IS - 13 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2020 SN - 0021-9258 ER - TY - JOUR AB - The Ser/Thr protein kinase ataxia telangiectasia mutated (ATM) plays an important role in the DNA damage response, signaling in response to redox signals, the control of metabolic processes, and mitochondrial homeostasis. ATM localizes to the nucleus and at the plasma membrane, mitochondria, peroxisomes, and other cytoplasmic vesicular structures. It has been shown that the C-terminal FATC domain of human ATM (hATMfatc) can interact with a range of membrane mimetics and may thereby act as a membrane-anchoring unit. Here, NMR structural and N-15 relaxation data, NMR data using spin-labeled micelles, and MD simulations of micelle-associated hATMfatc revealed that it binds the micelle by a dynamic assembly of three helices with many residues of hATMfatc located in the headgroup region. We observed that none of the three helices penetrates the micelle deeply or makes significant tertiary contacts to the other helices. NMR-monitored interaction experiments with hATMfatc variants in which two conserved aromatic residues (Phe(3049) and Trp(3052)) were either individually or both replaced by alanine disclosed that the double substitution does not abrogate the interaction with micelles and bicelles at the high concentrations at which these aggregates are typically used, but impairs interactions with small unilamellar vesicles, usually used at much lower lipid concentrations and considered a better mimetic for natural membranes. We conclude that the observed dynamic structure of micelle-associated hATMfatc may enable it to interact with differently composed membranes or membrane-associated interaction partners and thereby regulate ATM's kinase activity. Moreover, the FATC domain of ATM may function as a membrane-anchoring unit for other biomolecules. AU - Abd Rahim, M.S.* AU - Cherniavskyi, Y.K.* AU - Tieleman, D.P.* AU - Dames, S.A. C1 - 56019 C2 - 46739 CY - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa SP - 7098-7112 TI - NMR- and MD simulation-based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated. JO - J. Biol. Chem. VL - 294 IS - 17 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2019 SN - 0021-9258 ER - TY - JOUR AB - Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by -secretase (ADAM) and -secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting -secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-A-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of -secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that -secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by -secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by -secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells. AU - Huang, Y.* AU - Chanou, A.* AU - Kranz, G.* AU - Pan, M.* AU - Kohlbauer, V.* AU - Ettinger, A. AU - Gires, O. C1 - 55797 C2 - 46574 CY - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa SP - 3051-3064 TI - Membrane-associated epithelial cell adhesion molecule is slowly cleaved by -secretase prior to efficient proteasomal degradation of its intracellular domain. JO - J. Biol. Chem. VL - 294 IS - 9 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2019 SN - 0021-9258 ER - TY - JOUR AB - Type 1 diabetes islet cell autoantigen 512 (ICA512/IA-2) is a tyrosine phosphatase-like intrinsic membrane protein involved in the biogenesis and turnover of insulin secretory granules (SGs) in pancreatic islet -cells. Whereas its membrane-proximal and cytoplasmic domains have been functionally and structurally characterized, the role of the ICA512 N-terminal segment named regulated endocrine-specific protein 18 homology domain (RESP18HD), which encompasses residues 35-131, remains largely unknown. Here, we show that ICA512 RESP18HD residues 91-131 encode for an intrinsically disordered region (IDR), which in vitro acts as a condensing factor for the reversible aggregation of insulin and other -cell proteins in a pH and Zn2+-regulated fashion. At variance with what has been shown for other granule cargoes with aggregating properties, the condensing activity of ICA512 RESP18HD is displayed at a pH close to neutral, i.e. in the pH range found in the early secretory pathway, whereas it is resolved at acidic pH and Zn2+ concentrations resembling those present in mature SGs. Moreover, we show that ICA512 RESP18HD residues 35-90, preceding the IDR, inhibit insulin fibrillation in vitro. Finally, we found that glucose-stimulated secretion of RESP18HD upon exocytosis of SGs from insulinoma INS-1 cells is associated with cleavage of its IDR, conceivably to prevent its aggregation upon exposure to neutral pH in the extracellular milieu. Taken together, these findings point to ICA512 RESP18HD being a condensing factor for protein sorting and granulogenesis early in the secretory pathway and for prevention of amyloidogenesis. AU - Toledo, P.L.* AU - Torkko, J.M. AU - Müller, A. AU - Wegbrod, C. AU - Sönmez, A. AU - Solimena, M. AU - Ermácora, M.R.* C1 - 56292 C2 - 46967 CY - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa SP - 8564-8576 TI - ICA512 RESP18 homology domain is a protein-condensing factor and insulin fibrillation inhibitor. JO - J. Biol. Chem. VL - 294 IS - 21 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2019 SN - 0021-9258 ER - TY - JOUR AB - alpha-Melanocyte-stimulating hormone (-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that -MSH suppresses the transient outward A-type K+ current (I-A) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to -MSH concentration-dependently decreased I-A. This -MSH-induced I-A decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated I-A response. Blocking G(i/o)-protein signaling with pertussis toxin or by dialysis of TG neurons with the G-blocking synthetic peptide QEHA abolished the -MSH-mediated decrease in I-A. Further, -MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38 abrogated the -MSH-induced I-A response. Additionally, -MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by I-A blockade. Taken together, our findings suggest that -MSH suppresses I-A by activating MC4R, which is coupled sequentially to the G complex of the G(i/o)-protein and downstream class I PI3K-dependent p38 signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats. AU - Zhang, Y.* AU - Jiang, D. AU - Li, H.* AU - Sun, Y.* AU - Jiang, X.* AU - Gong, S.* AU - Qian, Z.* AU - Tao, J.* C1 - 55488 C2 - 46370 CY - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa SP - 5496-5507 TI - Melanocortin type 4 receptor-mediated inhibition of A-type K+ current enhances sensory neuronal excitability and mechanical pain sensitivity in rats. JO - J. Biol. Chem. VL - 294 IS - 14 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2019 SN - 0021-9258 ER - TY - JOUR AB - Constitutive NE-kappa B signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-kappa B pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NE-kappa B activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-kappa B signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6 -Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-kappa B activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases. AU - Brenke, J.K. AU - Popowicz, G.M. AU - Schorpp, K.K. AU - Rothenaigner, I. AU - Roesner, M.* AU - Meininger, I. AU - Kalinski, C. AU - Ringelstetter, L. AU - R'kyek, O. AU - Jürjens, G. AU - Vincendeau, M. AU - Plettenburg, O. AU - Sattler, M. AU - Krappmann, D. AU - Hadian, K. C1 - 53730 C2 - 44989 CY - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa SP - 13191-13203 TI - Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity. JO - J. Biol. Chem. VL - 293 IS - 34 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2018 SN - 0021-9258 ER - TY - JOUR AB - The enzyme AICAR-transformylase/IMP cyclohydrolase (ATIC) catalyzes the last two steps of purine de novo synthesis. It metabolizes 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is an AMP analogue, leading to activation of AMP-activated kinase (AMPK). We investigated whether the AICAR-ATIC pathway plays a role in the high glucose (HG)-mediated DNA damage response and AICAR-mediated AMPK activation, explaining the detrimental effects of glucose on neuronal damage and shortening of the lifespan. HG up-regulated the expression and activity of the Caenorhabditis elegans homologue of ATIC, C55F2.1 (atic-1), and increased the levels of reactive oxygen species and methylglyoxal-derived advanced glycation end products. Overexpression of atic-1 decreased the lifespan and head motility and increased neuronal damage under both standard and HG conditions. Inhibition of atic-1 expression, by RNAi, under HG was associated with increased lifespan and head motility and reduced neuronal damage, reactive oxygen species, and methylglyoxal-derived advanced glycation end product accumulation. This effect was independent of an effect on DNA damage or antioxidant defense pathways, such as superoxide dismutase (sod-3) or glyoxalase-1 (glod-4), but was dependent on AMPK and accumulation of AICAR. Through AMPK, AICAR treatment also reduced the negative effects of HG. The mitochondrial inhibitor rotenone abolished the AICAR/AMPK-induced amelioration of HG effects, pointing to mitochondria as a prime target of the glucotoxic effects in C. elegans. We conclude that atic-1 is involved in glucotoxic effects under HG conditions, either by blocked atic-1 expression or via AICAR and AMPK induction. AU - Riedinger, C.* AU - Mendler, M.* AU - Schlotterer, A.* AU - Fleming, T.* AU - Okun, J.G.* AU - Hammes, H.P.* AU - Herzig, S. AU - Nawroth, P.P.* C1 - 52904 C2 - 44489 CY - Bethesda SP - 4845-4859 TI - High-glucose toxicity is mediated by AICAR-transformylase/ IMP cyclohydrolase and mitigated by AMP-activated protein kinase in Caenorhabditis elegans. JO - J. Biol. Chem. VL - 293 IS - 13 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2018 SN - 0021-9258 ER - TY - JOUR AB - Membrane-bound proteinase 3 (PR3(m)) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3(m) triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3(m) and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefevre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3(m) expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3(m) on PLS neutrophils, whereas the total amount of PR3(m) on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34(+) hematopoietic stem cell model. Human CD34(+) hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3(m), cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised. AU - Seren, S.* AU - Rashed Abouzaid, M.* AU - Eulenberg-Gustavus, C.* AU - Hirschfeld, J.* AU - Soliman, H.* AU - Jerke, U.* AU - N'Guessan, K.* AU - Dallet-Choisy, S.* AU - Lesner, A.* AU - Lauritzen, C.* AU - Schacher, B.* AU - Eickholz, P.* AU - Nagy, N.* AU - Széll, M.* AU - Croix, C.* AU - Viaud-Massuard, M.C.* AU - Al Farraj Aldosari, A.* AU - Ragunatha, S.* AU - Ibrahim Mostafa, M.* AU - Giampieri, F.* AU - Battino, M.* AU - Cornillier, H.* AU - Lorette, G.* AU - Stephan, J.L.* AU - Goizet, C.* AU - Pedersen, J.* AU - Gauthier, F.* AU - Jenne, D. AU - Marchand-Adam, S.* AU - Chapple, I.L.* AU - Kettritz, R.* AU - Korkmaz, B.* C1 - 53685 C2 - 44950 CY - 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa SP - 12415-12428 TI - Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis. JO - J. Biol. Chem. VL - 293 IS - 32 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2018 SN - 0021-9258 ER - TY - JOUR AB - Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and -secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM’s proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein–tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways. AU - Tretter, J. AU - Schorpp, K.K. AU - Luxenburger, E.* AU - Trambauer, J.* AU - Steiner, H.* AU - Hadian, K. AU - Gires, O.* AU - Niessing, D. C1 - 53458 C2 - 44875 CY - Elsevier House, Brookvale Plaza, East Park Shannon, Co, Clare, 00000, Ireland SP - 8994-9005 TI - A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor. JO - J. Biol. Chem. VL - 293 IS - 23 PB - Elsevier Ireland Ltd PY - 2018 SN - 0021-9258 ER - TY - JOUR AB - Methylglyoxal (MG) is a reactive metabolite that forms adducts on cysteine, lysine and arginine residues of proteins, thereby affecting their function. Methylglyoxal is detoxified by the Glyoxalase system, consisting of two enzymes, Glo1 and Glo2, that act sequentially to convert MG into D-lactate. Recently, the Parkinsonism-associated protein DJ-1 was described in vitro to have glyoxalase activity, thereby detoxifying the MG metabolite, or deglycase activity, thereby removing the adduct formed by MG on proteins. Since Drosophila is an established model system to study signaling, neurodegeneration, and metabolic regulation in vivo, we asked whether DJ-1 contributes to MG detoxification in vivo. Using both DJ-1 knockdown in Drosophila cells in culture, and DJ-1 β knock-out flies, we could detect no contribution of DJ-1 to survival to MG challenge or to accumulation of MG protein adducts. Furthermore, we provide data suggesting that the previously reported deglycation activity of DJ- 1 can be ascribed to a TRIS buffer artifact. AU - Pfaff, D.H.* AU - Fleming, T.* AU - Nawroth, P.P. AU - Teleman, A.A.* C1 - 50409 C2 - 42295 SP - 685-690 TI - Evidence against a role for the parkinsonism-associated protein DJ-1 in methylglyoxal detoxification. JO - J. Biol. Chem. VL - 292 IS - 2 PY - 2017 SN - 0021-9258 ER - TY - JOUR AU - Pfaff, D.H.* AU - Fleming, T.* AU - Nawroth, P.P. AU - Teleman, A.A.* C1 - 51710 C2 - 43430 SP - 12784-12785 TI - Reply to Richarme: Evidence against a role of DJ-1 in methylglyoxal detoxification. JO - J. Biol. Chem. VL - 292 IS - 31 PY - 2017 SN - 0021-9258 ER - TY - JOUR AB - Members of the IL-12 family perform essential functions in immunoregulation by connecting innate and adaptive immunity and are emerging therapeutic targets. They are unique among other interleukins in forming heterodimers that arise from extensive subunit sharing within the family, leading to the production of at least four functionally distinct heterodimers from only five subunits. This raises important questions about how the assembly of IL-12 family members is regulated and controlled in the cell. Here, using cell-biological approaches, we have dissected basic principles that underlie the biogenesis of the founding member of the family, IL-12. Within the native IL-12 heterodimer, composed of IL-12 alpha and IL-12 alpha, IL-12 alpha possesses three intramolecular and one intermolecular disulfide bridges. We show that, in isolation, IL-12 alpha fails to form its native structure but, instead, misfolds, forming incorrect disulfide bonds. Co-expression of its beta subunit inhibits misfolding and thus allows secretion of biologically active heterodimeric IL-12. On the basis of these findings, we identified the disulfide bonds in IL-12 alpha that are critical for assembly-induced secretion and biological activity of IL-12 versus misfolding and degradation of IL-12 alpha. Surprisingly, two of the three disulfide bridges in IL-12 alpha are dispensable for IL-12 secretion, stability, and biological activity. Extending our findings, we show that misfolding also occurs for IL-23 alpha, another IL-12 family protein. Our results indicate that assembly-induced folding is key in IL-12 family biogenesis and secretion. The identification of essential disulfide bonds that underlie this process lays the basis for a simplified yet functional IL-12 cytokine. AU - Reitberger, S.* AU - Hammerl, P. AU - Aschenbrenner, I.* AU - Esser-von Bieren, J. AU - Feige, M.J.* C1 - 51216 C2 - 42925 CY - Bethesda SP - 8073-8081 TI - Assembly-induced folding regulates interleukin 12 biogenesis and secretion. JO - J. Biol. Chem. VL - 292 IS - 19 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2017 SN - 0021-9258 ER - TY - JOUR AB - The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90 – co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1–Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90 –Aha1-dependent client proteins in vivo. We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90. AU - Stiegler, S.C.* AU - Rübbelke, M. AU - Korotkov, V.S.* AU - Weiwad, M.* AU - John, C.* AU - Fischer, G.* AU - Sieber, S.A.* AU - Sattler, M. AU - Buchner, J.* C1 - 52166 C2 - 43747 CY - Bethesda SP - 17073-17083 TI - A chemical compound inhibiting the Aha1–Hsp90 chaperone complex. JO - J. Biol. Chem. VL - 292 IS - 41 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2017 SN - 0021-9258 ER - TY - JOUR AB - Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16 as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed β-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nM, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable to that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal β-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs. AU - Täschner, M.* AU - Mourao, A. AU - Awasthi, M.* AU - Basquin, J.* AU - Lorentzen, E.* C1 - 50728 C2 - 42870 CY - Bethesda SP - 7462-7473 TI - Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46. JO - J. Biol. Chem. VL - 292 IS - 18 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2017 SN - 0021-9258 ER - TY - JOUR AB - Numerous membrane-bound proteins undergo regulated intramembrane proteolysis (RIP). RIP is initiated by shedding and the remaining stubs are further processed by intramembrane cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site APP cleaving enzyme 1; BACE1). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Due to the hairpin nature of NRG1 type III two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1 oriented stub is further cleaved by γ-secretase at an ε-like site 5 amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ε-cleavage site is only 1 amino acid N-terminal to a V/L substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer's disease associated mutations within the Amyloid precursor protein. The type 2 oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with (Z-LL)2 ketone inhibits formation of a N-terminal ICD and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate, which is not only cleaved by multiple sheddases but also processed by three different I-CLiPs. AU - Fleck, D.* AU - Voss, M.* AU - Brankatschk, B.* AU - Giudici, C.* AU - Hampel, H.* AU - Schwenk, B.M.* AU - Edbauer, D.* AU - Fukumori, A.* AU - Steiner, H.* AU - Kremmer, E. AU - Haug-Kröeper, M.* AU - Rossner, M.J.* AU - Fluhrer, R.* AU - Willem, M.* AU - Haass, C.* C1 - 47314 C2 - 40574 SP - 318-333 TI - Proteolytic processing of Neuregulin 1 type III by three intramembrane cleaving proteases. JO - J. Biol. Chem. VL - 291 IS - 1 PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - Monoglyceride lipase (MGL) is required for efficient hydrolysis of the endocannabinoid 2-arachidonoylglyerol (2-AG) in the brain generating arachidonic acid (AA) and glycerol. This metabolic function makes MGL an interesting target for the treatment of neuroinflammation, since 2-AG exhibits anti-inflammatory properties and AA is a precursor for pro-inflammatory prostaglandins. Astrocytes are an important source of AA and 2-AG, and highly express MGL. In the present study, we dissected the distinct contribution of MGL in astrocytes on brain 2-AG and AA metabolism by generating a mouse model with genetic deletion of MGL specifically in astrocytes (MKO(GFAP)). MKO(GFAP) mice exhibit moderately increased 2-AG and reduced AA levels in brain. Minor accumulation of 2-AG in the brain of MKO(GFAP) mice does not cause cannabinoid receptor desensitization as previously observed in mice globally lacking MGL. Importantly, MKO(GFAP) mice exhibit reduced brain prostaglandin E2 and pro-inflammatory cytokines levels upon peripheral lipopolysaccharide (LPS) administration. These observations indicate that MGL-mediated degradation of 2-AG in astrocytes provides AA for prostaglandin synthesis promoting LPS-induced neuroinflammation. The beneficial effect of astrocyte-specific MGL-deficiency is not fully abrogated by the inverse cannabinoid receptor 1 agonist SR141716 (Rimonabant) suggesting that the anti-inflammatory effects are rather caused by reduced prostaglandin synthesis than by activation of cannabinoid receptors. In conclusion, our data demonstrate that MGL in astrocytes is an important regulator of 2-AG levels, AA availability, and neuroinflammation. AU - Grabner, G.F.* AU - Eichmann, T.O.* AU - Wagner, B.* AU - Gao, Y. AU - Farzi, A.* AU - Taschler, U.* AU - Radner, F.P.* AU - Schweiger, M.* AU - Lass, A.* AU - Holzer, P.* AU - Zinser, E.* AU - Tschöp, M.H. AU - Yi, C.-X. AU - Zimmermann, R.* C1 - 47299 C2 - 40587 SP - 913-923 TI - Deletion of monoglyceride lpase in astrocytes attenuates lipopolysaccharide-induced neuroinflammation. JO - J. Biol. Chem. VL - 291 IS - 2 PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases. AU - Hamon, Y. AU - Legowska, M.* AU - Herve, V.* AU - Dallet-Choisy, S.* AU - Marchand-Adam, S.* AU - Vanderlynden, L.* AU - Demonte, M.* AU - Williams, R.* AU - Scott, C.J.* AU - Si-Tahar, M.* AU - Heuze-Vourc'h, N.* AU - Lalmanach, G.* AU - Jenne, D. AU - Lesner, A.* AU - Gauthier, F.* AU - Korkmaz, B.* C1 - 50301 C2 - 42078 SP - 8486-8499 TI - Neutrophilic cathepsin C is maturated by a multistep proteolytic process and secreted by activated cells during inflammatory lung diseases. JO - J. Biol. Chem. VL - 291 IS - 16 PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - The glyoxalase system is a highly specific enzyme system existing in all mammalian cells which is responsible for the detoxification of dicarbonyl species, primarily methylglyoxal (MG). It has been implicated to play an essential role in preventing the increased formation of advanced glycation endproducts under certain pathological conditions. We have established the first glyoxalase 1 knock-out model (GLO1-/-) in mammalian Schwann cells using CRISPR/Cas9 technique to investigate compensatory mechanisms. Neither elevated concentrations of MG nor associated protein modifications were observed in GLO1-/- cells. Alternative detoxification of MG in GLO1-/- is achieved by increased catalytic efficiency of aldose reductase towards hemithioacetal (product of glutathione and MG), most likely caused by S-nitrosylation of aldose reductase. The hemithioacetal is mainly converted into lactaldehyde, which is paralleled by a loss of reduced glutathione. Inhibition of aldose reductase in GLO1-/- cells is associated with an increased sensitivity against MG, elevated intracellular MG levels and associated modifications, as well as increased oxidative stress. Our data suggest that aldose reductase can compensate the loss of GLO1. This might be of clinical importance within the context of neuronal diseases caused by an impaired glyoxalase system and elevated levels of dicarbonyl species, such as MG. AU - Morgenstern, J.* AU - Fleming, T.* AU - Schumacher, D.* AU - Eckstein, V.* AU - Freichel, M.* AU - Herzig, S. AU - Nawroth, P.P. C1 - 50366 C2 - 42172 CY - Bethesda SP - 3224-3238 TI - Loss of glyoxalase 1 induces compensatory mechanism to achieve dicarbonyl detoxification in mammalian Schwann cells. JO - J. Biol. Chem. VL - 292 IS - 8 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - The human genome contains 25 genes coding for selenocysteine- containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein- encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP),SELENOT(selenoprotein T, SELT),SELENOV(selenoprotein V, SELV), andSELENOW(selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates. AU - Regina, B.F.* AU - Gladyshev, V.N.* AU - Arner, E.S.J.* AU - Berry, M.J.* AU - Bruford, E.A.* AU - Burk, R.F.* AU - Carlson, B.A.* AU - Castellano, S.* AU - Chavatte, L.* AU - Conrad, M. AU - Copeland, P.R.* AU - Diamond, A.M.* AU - Driscoll, D.M.* AU - Ferreiro, A.* AU - Flohé, L.* AU - Green, F.R.* AU - Guigo, R.* AU - Handy, D.E.* AU - Hatfield, D.L.* AU - Hesketh, J.* AU - Hoffmann, P.R.* AU - Holmgren, A.* AU - Hondal, R.J.* AU - Howard, M.T.* AU - Huang, K.M.* AU - Kim, H.Y.* AU - Kim, I.Y.* AU - Köhrle, J.* AU - Krol, A.* AU - Kryukov, G.V.* AU - Lee, B.J.* AU - Lee, B.C.* AU - Lei, X.G.* AU - Liu, Q.* AU - Lescure, A.* AU - Lobanov, A.V.* AU - Loscalzo, J.* AU - Maiorino, M.* AU - Mariotti, M.* AU - Prabhu, K.S.* AU - Rayman, M.P.* AU - Rozovsky, S.* AU - Salinas, G.* AU - Schmidt, E.E.* AU - Schomburg, L.* AU - Schweizer, U.* AU - Simonović, M.* AU - Sunde, R.A.* AU - Tsuji, P.A.* AU - Tweedie, S.* AU - Ursini, F.F.* AU - Whanger, P.D.* AU - Zhang, Y.* C1 - 50036 C2 - 41980 CY - Bethesda SP - 24036-24040 TI - Selenoprotein gene nomenclature. JO - J. Biol. Chem. VL - 291 IS - 46 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28αβ, PA28γ, PA200 and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28γ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28γ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of β5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA mediated knockdown of the 19S subunit Rpn6 induced recruitment of only PA200 to 20S proteasomes whereas PA28γ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28γsensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis. AU - Welk, V. AU - Coux, O.* AU - Kleene, V. AU - Abeza, C.* AU - Trümbach, D. AU - Eickelberg, O. AU - Meiners, S. C1 - 48503 C2 - 41124 CY - Bethesda SP - 13147-13159 TI - Inhibition of proteasome activity induces formation of alternative proteasome complexes. JO - J. Biol. Chem. VL - 291 IS - 25 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1-75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74-147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148-420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages. AU - Wittwer, M.* AU - Luo, Q.* AU - Kaila, V.R.I.* AU - Dames, S.A. C1 - 50248 C2 - 42249 CY - Bethesda SP - 27062-27072 TI - Oxidative unfolding of the rubredoxin domain and the natively disordered N-terminal region regulate the catalytic activity of Mycobacterium tuberculosis protein kinase G. JO - J. Biol. Chem. VL - 291 IS - 53 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2016 SN - 0021-9258 ER - TY - JOUR AB - TET proteins oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC) and 5-carboxylcytosine (caC) and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions - the N-terminus and the low complexity insert between the two parts of the dioxygenase domains - is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of PTMs that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The glycosyltransferase OGT, which we identified as a strong interactor of all three TET proteins, catalyzes the addition of an N-acetylglucosamine (GlcNAc) group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites as well as the site occupancy. Interestingly, the different TET proteins display unique PTM patterns and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N-terminus and the low complexity insert region. Our data suggest strong crosstalk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli. AU - Bauer, C.* AU - Gobel, K.* AU - Nagaraj, N.* AU - Colantuoni, C.* AU - Wang, M.* AU - Müller, U.* AU - Kremmer, E. AU - Rottach, A.* AU - Leonhardt, H.* C1 - 43073 C2 - 35995 CY - Bethesda SP - 4801-4812 TI - Phosphorylation of TET proteins is regulated via O-GlcNAcylation by the glycosyltransferase OGT. JO - J. Biol. Chem. VL - 290 IS - 8 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/ Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signalregulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37.Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery. AU - Eckl, J.M.* AU - Scherr, M.J.* AU - Freiburger, L. AU - Daake, M.A.* AU - Sattler, M. AU - Richter, K.* C1 - 47622 C2 - 39439 SP - 30843-30854 TI - Hsp90.Cdc37 complexes with protein kinases form cooperatively with multiple distinct interaction sites. JO - J. Biol. Chem. VL - 290 IS - 52 PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - Many Gram-positive bacteria produce lantibiotics, genetically encoded and posttranslationally modified peptide antibiotics, which inhibit the growth of other Gram-positive bacteria. To protect themselves against their own lantibiotics these bacteria express a variety of immunity proteins including the LanI lipoproteins. The structural and mechanistic basis for LanI-mediated lantibiotic immunity is not yet understood. Lactococcus lactis produces the lantibiotic nisin, which is widely used as a food preservative. Its LanI protein NisI provides immunity against nisin but not against structurally very similar lantibiotics from other species such as subtilin from Bacillus subtilis. To understand the structural basis for LanI-mediated immunity and their specificity we investigated the structure of NisI. We found that NisI is a two-domain protein. Surprisingly, each of the two NisI domains has the same structure as the LanI protein from B. subtilis, SpaI, despite the lack of significant sequence homology. The two NisI domains and SpaI differ strongly in their surface properties and function. Additionally, SpaI-mediated lantibiotic immunity depends on the presence of a basic unstructured N-terminal region that tethers SpaI to the membrane. Such a region is absent from NisI. Instead, the N-terminal domain of NisI interacts with membranes but not with nisin. In contrast, the C-terminal domain specifically binds nisin and modulates the membrane affinity of the N-terminal domain. Thus, our results reveal an unexpected structural relationship between NisI and SpaI and shed light on the structural basis for LanI mediated lantibiotic immunity. AU - Hacker, C.* AU - Christ, N.A.* AU - Duchardt-Ferner, E.* AU - Korn, S.* AU - Göbl, C. AU - Berninger, L.* AU - Düsterhus, S.* AU - Hellmich, U.A.* AU - Madl, T. AU - Kötter, P.* AU - Entian, K.D.* AU - Wöhnert, J.* C1 - 47435 C2 - 39335 SP - 28869-28886 TI - The solution structure of the lantibiotic immunity protein NisI and its interactions with Nisin. JO - J. Biol. Chem. VL - 290 IS - 48 PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 (mGpx4) was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with targeted mutation of the active site selenocysteine (Sec) of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4-/- embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breedings and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoan midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mGpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared to Sec at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Since the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and being a structural protein, tightly controlled expression of functional Gpx4 emerges being key for full male fertility. AU - Ingold, I. AU - Aichler, M. AU - Yefremova, E. AU - Roveri, A.* AU - Buday, K. AU - Doll, S. AU - Tasdemir, A. AU - Hoffard, N. AU - Wurst, W. AU - Walch, A.K. AU - Ursini, F.* AU - Friedmann Angeli, J.P.F. AU - Conrad, M. C1 - 44736 C2 - 36981 CY - Bethesda SP - 14668-14678 TI - Expression of a catalytically inactive mutant form of Glutathione peroxidase 4 (Gpx4) confers a dominant-negative effect in male fertility. JO - J. Biol. Chem. VL - 290 IS - 23 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - The cystine/glutamate transporter, designated as system xc-, is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc-, xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues such as thymus and spleen. While cystine and glutamate are the well-established substrates of system xc- and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analysed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged to be absent specifically in thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knockout cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc-, and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc-. AU - Kobayashi, S. AU - Sato, M.* AU - Kasakoshi, T.* AU - Tsutsui, T.* AU - Sugimoto, M.* AU - Osaki, M.* AU - Okada, F.* AU - Igarashi, K.* AU - Hiratake, J.* AU - Homma, T.* AU - Conrad, M. AU - Fujii, J.* AU - Soga, T.* AU - Bannai, S.* AU - Sato, H.* C1 - 43511 C2 - 36657 CY - Bethesda SP - 8778-8788 TI - Cystathionine is a novel substrate of cystine/glutamate transporter: Implications for immune function. JO - J. Biol. Chem. VL - 290 IS - 14 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - The Patched1 (Ptch)-mediated inhibition of Smoothened (Smo) is still an open question. However, a direct Ptch/Smo interaction has been excluded, Smo modulators were identified, but the endogenous signal transmitting molecule remains undiscovered. Here, we demonstrate that calcitriol, the hormonally active form of vitamin D3, is an excellent candidate for transmission of Ptch/Smo interaction. Our study reveals that Ptch expression is sufficient to release calcitriol from the cell and that calcitriol inhibits Smo action and ciliary translocation by acting on a site distinct from the 7-transmembrane-domain or the cysteine-rich-domain. Moreover calcitriol strongly synergizes with itraconazole (ITZ) in Smo inhibition which not results from elevated calcitriol bio-availability due to ITZ-mediated 24-hydroxylase inhibition but rather from a direct interaction of the compounds at the level of Smo. Together, we suggest that calcitriol represents a possible endogenous transmitter of Ptch/Smo interaction. Moreover calcitriol or calcitriol derivatives combined with ITZ might be a treatment option of Hedgehog-associated cancers. AU - Linder, B.* AU - Weber, S. AU - Dittmann, K.H.* AU - Adamski, J. AU - Hahn, H.* AU - Uhmann, A.* C1 - 45676 C2 - 37411 CY - Bethesda SP - 19614-19628 TI - A functional and putative physiological role of calcitriol in Patched1/Smoothened interaction. JO - J. Biol. Chem. VL - 290 IS - 32 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - IPSE/alpha-1, the major secretory product of eggs from the parasitic worm Schistosoma mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/alpha-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEδNLS, a monomeric IPSE/alpha-1 mutant, revealed that IPSE/alpha-1 is a new member of the β&]gamma]-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEδNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/alpha-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/alpha-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism. AU - Meyer, N.H. AU - Mayerhofer, H.* AU - Tripsianes, K. AU - Blindow, S.* AU - Barths, D.* AU - Mewes, A.* AU - Weimar, T.* AU - Köhli, T.* AU - Bade, S.* AU - Madl, T. AU - Frey, A.* AU - Haas, H.* AU - Müller-Dieckmann, J.* AU - Sattler, M. AU - Schramm, G.* C1 - 46483 C2 - 37586 SP - 22111-22126 TI - A crystallin fold in the interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE/alpha-1) mediates IgE binding for antigen-independent basophil activation. JO - J. Biol. Chem. VL - 290 IS - 36 PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - Alzheimer's disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation have been suggested. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators (GSMs). It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. We present here a magic angle spinning (MAS) solid-state NMR study that shows that the NSAID sulindac sulfide interacts specifically with Alzheimer's disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure, but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic D23-K28 salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue G33, a residue involved in M35 oxidation. The results presented here could be useful in the search for pharmacologically active molecules which can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer's disease. AU - Prade, E.* AU - Bittner, H.J.* AU - Sarkar, R.* AU - Lopez del Amo, J.M.* AU - Althoff-Ospelt, G.* AU - Multhaup, G.* AU - Hildebrand, P.W.* AU - Reif, B. C1 - 47105 C2 - 39191 SP - 28737-28745 TI - Structural mechanism of the interaction of Alzheimer's disease Aβ fibrils with the NSAID sulindac sulfide. JO - J. Biol. Chem. VL - 290 IS - 48 PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - In response to fasting or hyperglycemia, the pancreatic β-cell alters its output of secreted insulin; however the pathways governing this adaptive response are not entirely established. While the precise role of microRNAs (miRNAs) is also unclear, a recurring theme emphasizes their function in cellular stress responses. We recently showed that miR-184, an abundant miRNA in the β-cell, regulates compensatory proliferation and secretion during insulin resistance. Consistent with previous studies showing miR-184 suppresses insulin release, expression of this miRNA was increased in islets after fasting, demonstrating an active role in the β-cell as glucose levels lower and the insulin demand ceases. Additionally, miR-184 was negatively regulated upon administration of a sucrose-rich diet in Drosophila demonstrating strong conservation of this pathway through evolution. Furthermore, miR-184 and its target Argonaute2 (Ago2) remained inversely correlated as concentrations of extracellular glucose increased, underlining a functional relationship between this miRNA and its targets. Lastly, restoration of Ago2 in the presence of miR-184 rescued suppression of miR-375-targeted genes suggesting these genes act in a coordinated manner during changes in the metabolic context. Together, these results highlight the adaptive role of miR-184 according to glucose metabolism and suggest the regulatory role of this miRNA in energy homeostasis is highly conserved. AU - Tattikota, S.G.* AU - Rathjen, T.* AU - Hausser, J.* AU - Khedkar, A.* AU - Kabra, U.D. AU - Pandey, V.K.* AU - Sury, M.* AU - Wessels, H.H.* AU - Mollet, I.G.* AU - Eliasson, L.* AU - Selbach, M.* AU - Zinzen, R.P.* AU - Zavolan, M.* AU - Kadener, S.* AU - Tschöp, M.H. AU - Jastroch, M. AU - Friedländer, M.R.* AU - Poy, M.N.* C1 - 46481 C2 - 37584 CY - Bethesda SP - 20284-20294 TI - MiR-184 regulates pancreatic β-cell function according to glucose metabolism. JO - J. Biol. Chem. VL - 290 IS - 33 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - Alto-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17β-hydroxysteroid dehydrogenase activity) as well as 3-ketoacyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues. AU - Weber, S. AU - Salabei, J.K.* AU - Möller, G. AU - Kremmer, E. AU - Bhatnagar, A.* AU - Adamski, J. AU - Barski, O.A.* C1 - 43958 C2 - 36717 CY - Bethesda SP - 6531-6545 TI - Aldo-Keto Reductase 1B15 (AKR1B15): A mitochondrial human aldo-keto reductase with activity toward steroids and 3-keto-acyl-CoA conjugates. JO - J. Biol. Chem. VL - 290 IS - 10 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2015 SN - 0021-9258 ER - TY - JOUR AB - Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multi-domain architecture that includes an N-terminal trans-membrane tetrameric ion channel followed by a cytoplasmic "C-linker", which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, while cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V1/2 voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavoured. The steric clashes of the apo tetramer are eliminated either by cAMP-binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo HCN to monomers or dimer of dimers, in which the C-linker becomes less structured and channel opening is not facilitated. AU - Akimoto, M.* AU - Zhang, Z.* AU - Boulton, S.* AU - Selvaratnam, R.* AU - van Schouwen, B.* AU - Gloyd, M.* AU - Accili, E.A.* AU - Lange, O.F. AU - Melacini, G.* C1 - 31673 C2 - 34640 CY - Bethesda SP - 22205-22220 TI - A mechanism for the auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel opening and its relief by cAMP. JO - J. Biol. Chem. VL - 289 IS - 32 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AU - Floß, T. C1 - 31818 C2 - 34794 CY - Bethesda SP - 18594 TI - Reply to Jawaid et al.: Mitochondrial dysfunction and decrease in body weight of transgenic knock-in mouse model for TDP-43: The question of glucose?. JO - J. Biol. Chem. VL - 289 IS - 26 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure: Ac-peptidylP(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl-phosphonates to identify PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils, despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect and control PR3 activity in a variety of inflammatory diseases. AU - Guarino, C.* AU - Legowska, M.* AU - Epinette, C.* AU - Kellenberger, C.* AU - Dallet-Choisy, S.* AU - Sienczyk, M.* AU - Gabant, G.* AU - Cadène, M.* AU - Zoidakis, J.* AU - Vlahou, A.* AU - Wysocka, M.* AU - Marchand-Adam, S.* AU - Jenne, D. AU - Lesner, A.* AU - Gauthier, F.* AU - Korkmaz, B.* C1 - 32488 C2 - 35112 SP - 31777-31791 TI - New selective peptidyl di(chlorophenyl)-phosphonate esters to visualize and block neutrophil proteinase 3 in human diseases. JO - J. Biol. Chem. VL - 289 IS - 46 PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - Uromodulin (UMOD)-associated kidney disease (UAKD) belongs to the hereditary progressive ER storage diseases caused by maturation defects of mutant UMOD protein. Current treatments of UAKD patients are symptomatic and cannot prevent disease progression. Two in vitro studies reported a positive effect of the chemical chaperone sodium-4-phenylbutyrate (4-PBA) on mutant UMOD maturation. Thus, 4-PBA was suggested as potential treatment for UAKD. This study evaluated the effects of 4-PBA in two mouse models of UAKD. In contrast to previous in vitro studies, treatment with 4-PBA did not increase HSP70 expression or improve maturation and trafficking of mutant UMOD in vivo. Kidney function of UAKD mice was actually deteriorated by 4-PBA-treatment. In transfected tubular epithelial cells, 4-PBA did not improve maturation, but increased the expression level of both mutant and wild-type UMOD protein. Activation of NF-κB pathway in thick ascending limb of Henle's loop cells of UAKD mice was detected by increased abundance of RelB and phospho-IKKα/β, an indirect activator of NF-κB. Further, the abundance of NF-κB1 p105/p50, NF-κB2 p100/p52 and TRAF2 was increased in UAKD. NF-κB activation was identified as a novel disease mechanism of UAKD and might be a target for therapeutic intervention. AU - Kemter, E.* AU - Sklenak, S.* AU - Rathkolb, B. AU - Hrabě de Angelis, M. AU - Wolf, E.* AU - Aigner, B.* AU - Wanke, R.* C1 - 30663 C2 - 33779 CY - Bethesda SP - 10715-10726 TI - No amelioration of uromodulin maturation and trafficking defect by sodium-4-phenylbutyrate in vivo: Studies in mouse models of uromodulin-associated kidney disease. JO - J. Biol. Chem. VL - 289 IS - 15 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57-71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner. AU - Neuhaus, A.* AU - Kooshapur, H. AU - Wolf, J.* AU - Meyer, N.H. AU - Madl, T. AU - Saidowsky, J.* AU - Hambruch, E.* AU - Lazam, A.* AU - Jung, M.* AU - Sattler, M. AU - Schliebs, W.* AU - Erdmann, R.* C1 - 28863 C2 - 33549 SP - 437-448 TI - A novel Pex14 protein-interacting site of human Pex5 is critical for matrix protein import into peroxisomes. JO - J. Biol. Chem. VL - 289 IS - 1 PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - The cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cell membrane receptors constitute an elaborate signaling system fulfilling important functions in immune regulation and tumor surveillance. Activation of the death receptors TRAILR1 and TRAILR2 can lead to apoptosis, whereas TRAILR3 and TRAILR4 are generally referred to as decoy receptors, which have been shown to inhibit TRAIL-induced apoptosis. The underlying molecular mechanisms, however, remain unclear. Alike other members of the TNF receptor superfamily, TRAIL receptors contain a pre-ligand binding assembly domain (PLAD) mediating receptor oligomerization. Still, the stoichiometry of TRAIL receptor oligomers as well as the issue whether the PLAD mediates only homotypic or also heterotypic interactions, remained inconclusive until now. Performing acceptor-photobleaching FRET studies with TRAIL receptors 1, 2 and 4 we demonstrate interactions in all possible combinations. Formation of dimers is shown by chemical crosslinking experiments for homophilic and heterophilic interactions of these receptors. Implications of the demonstrated receptor-receptor interactions on signaling were investigated next. Both, apoptosis induction and activation of the transcription factor NFκB were significantly reduced in presence of TRAILR4. Our experimental data, highly consistent with results from a mathematical model, show that the inhibitory capacity of TRAILR4 is attributable to signaling-independent mechanisms, strongly suggesting a reduction of signaling-competent death receptors through formation of heteromeric receptor complexes. In summary, we propose a model of TRAIL receptor interference driven by PLAD-mediated formation of receptor heterodimers on the cell membrane. AU - Neumann, S.* AU - Hasenauer, J. AU - Pollak, N.* AU - Scheurich, P.* C1 - 31218 C2 - 34208 CY - Bethesda SP - 16576-16587 TI - Dominant negative effects of TNF-Related Apoptosis-Inducing Ligand (TRAIL) receptor 4 on TRAIL receptor 1 signaling by formation of heteromeric complexes. JO - J. Biol. Chem. VL - 289 IS - 23 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16. AU - Niedzielska, M.* AU - Bodendorfer, B.* AU - Münch, S.* AU - Eichner, A.* AU - Derigs, M.* AU - da Costa, O.P.* AU - Schweizer, A.* AU - Neff, F. AU - Nitschke, L.* AU - Sparwasser, T.* AU - Keyse, S.M.* AU - Lang, R.* C1 - 30557 C2 - 33688 CY - Bethesda SP - 2112-2126 TI - Gene trap mice reveal an essential function of dual specificity phosphatase Dusp16/MKP-7 in perinatal survival and regulation of Toll-Like Receptor (TLR)-induced cytokine production. JO - J. Biol. Chem. VL - 289 IS - 4 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - Background: Activation of SplB protease requires precise N-terminal processing, but the molecular mechanism remains unknown. Results: The new N-terminal Glu-1 forms a distinctive H-bond network essential for full catalytic activity. Conclusion: Changes in protein dynamics rather than a direct effect on the active site are of crucial importance in SplB activation. Significance: A novel serine protease activation mechanism was uncovered. AU - Pustelny, K.* AU - Zdzalik, M.* AU - Stach, N.* AU - Stec-Niemczyk, J.* AU - Cichon, P.* AU - Czarna, A.* AU - Popowicz, G.M. AU - Mak, P.* AU - Dra̧g, M.* AU - Salvesen, G.S.* AU - Władyka, B.* AU - Potempa, J.S.* AU - Dubin, A.* AU - Dubin, G.* C1 - 31604 C2 - 34654 CY - Bethesda SP - 15544-15553 TI - Staphylococcal SplB serine protease utilizes a novel molecular mechanism of activation. JO - J. Biol. Chem. VL - 289 IS - 22 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - The majority of Amyotrophic Lateral Sklerosis (ALS) cases as well as many patients suffering from Frontotemporal Lobar Dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR-DNA binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange (RMCE) to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes while the endogenous mouse Tdp-43 was decreased to 20% of wildtype levels as a result of disturbed feedback regulation. Heterozygous TDP-43A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes, as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol and glucose in the blood. As seen in Transmission Electron Microscopy, neuronal cells in motor cortices of TDP-43A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90% but only slight motoric impairment was detected. The observed phenotype was interpreted as a pre-disease model which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration. AU - Stribl, C.B. AU - Samara, A. AU - Trümbach, D. AU - Augustin, R. AU - Neumann, M.* AU - Fuchs, H. AU - Gailus-Durner, V. AU - Hrabě de Angelis, M. AU - Rathkolb, B. AU - Wolf, E.* AU - Beckers, J. AU - Horsch, M. AU - Neff, F. AU - Kremmer, E. AU - Koob, S.* AU - Reichert, A.S.* AU - Hans, W. AU - Rozman, J. AU - Klingenspor, M. AU - Aichler, M. AU - Walch, A.K. AU - Becker, L. AU - Klopstock, T.* AU - Glasl, L. AU - Hölter, S.M. AU - Wurst, W. AU - Floß, T. C1 - 29354 C2 - 33686 CY - Bethesda SP - 10769-10784 TI - Mitochondrial dysfunction and decrease in body weight of a transgenic knock-in mouse model for TDP-43. JO - J. Biol. Chem. VL - 289 IS - 15 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - The REtention and Splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and to promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total eighteen residues of Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp232 in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that the Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RNA recognition motif (RRM)-protein interactions, analogous to the one seen in for nucleic acid interactions. AU - Tripsianes, K.* AU - Friberg, A. AU - Barrandon, C.* AU - Brooks, M.* AU - van Tilbeurgh, H.* AU - Séraphin, B.* AU - Sattler, M. C1 - 32042 C2 - 34954 CY - Bethesda SP - 28640-28650 TI - A novel protein-protein interaction in the RES (REtention and Splicing) complex. JO - J. Biol. Chem. VL - 289 IS - 41 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2014 SN - 0021-9258 ER - TY - JOUR AB - Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing. AU - Burger, K. AU - Mühl, B. AU - Rohrmoser, M. AU - Coordes, B.* AU - Heidemann, M. AU - Kellner, M. AU - Gruber-Eber, A. AU - Heissmeyer, V. AU - Strässer, K.* AU - Eick, D. C1 - 26324 C2 - 32174 SP - 21173-21183 TI - Cyclin-dependent kinase 9 links RNA polymerase II transcription to processing of ribosomal RNA. JO - J. Biol. Chem. VL - 288 IS - 29 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - Small conductance calcium-activated potassium (SK2/K(Ca)2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K+ currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction. AU - Dolga, A.M.* AU - Netter, M.F.* AU - Perocchi, F.* AU - Doti, N.* AU - Meissner, L.* AU - Tobaben, S.* AU - Grohm, J.* AU - Zischka, H. AU - Plesnila, N.* AU - Decher, N.* AU - Culmsee, C.* C1 - 24542 C2 - 31580 SP - 10792-10804 TI - Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction. JO - J. Biol. Chem. VL - 288 IS - 15 PB - Amer. Soc. Biochemistry Molecular Biology PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1' pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis. AU - Hinkofer, L.C. AU - Seidel, S.A.* AU - Korkmaz, B. AU - Silva, F.* AU - Hummel, A.M.* AU - Braun, D.* AU - Jenne, D. AU - Specks, U.* C1 - 28893 C2 - 33563 CY - Bethesda SP - 26635-26648 TI - A monoclonal antibody (MCPR3-7) interfering with the activity of proteinase 3 by an allosteric mechanism. JO - J. Biol. Chem. VL - 288 IS - 37 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - GBA1 and GBA2 are both beta-glucosidases, which cleave glucosylceramide (GlcCer) to glucose and ceramide. GlcCer is a main precursor for higher order glycosphingolipids but might also serve as intracellular messenger. Mutations in the lysosomal GBA1 underlie Gaucher disease, the most common lysosomal storage disease in humans. Knocking out the non-lysosomal GBA2 in mice results in accumulation of GlcCer outside the lysosomes in various tissues (e. g. testis and liver) and impairs sperm development and liver regeneration. However, the underlying mechanisms are not well understood. To reveal the physiological function of GBA2 and, thereby, of the non-lysosomal GlcCer pool, it is important to characterize the localization of GBA2 and its activity in different tissues. Thus, we generated GBA2-specific antibodies and developed an assay that discriminates between GBA1 and GBA2 without the use of detergent. We show that GBA2 is not, as previously thought, an integral membrane protein but rather a cytosolic protein that tightly associates with cellular membranes. The interaction with the membrane, in particular with phospholipids, is important for its activity. GBA2 is localized at the ER and Golgi, which puts GBA2 in a key position for a lysosome-independent route of GlcCer-dependent signaling. Furthermore, our results suggest that GBA2 might affect the phenotype of Gaucher disease, because GBA2 activity is reduced in Gba1 knock-out fibroblasts and fibroblasts from a Gaucher patient. Our results provide the basis to understand the mechanism for GBA2 function in vivo and might help to unravel the role of GBA2 during pathogenesis of Gaucher disease. AU - Korschen, H.G.* AU - Yildiz, Y.* AU - Raju, D.N.* AU - Schonauer, S.* AU - Bonigk, W.* AU - Jansen, V.* AU - Kremmer, E. AU - Kaupp, U.B.* AU - Wachten, D.* C1 - 22952 C2 - 30982 SP - 3381-3393 TI - The non-lysosomal β-glucosidase GBA2 is a non-integral membrane-associated protein at the Endoplasmic Reticulum (ER) and golgi. JO - J. Biol. Chem. VL - 288 IS - 5 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - The nuclei of most vertebrate cells contain members of the high mobility group N (HMGN) protein family which bind specifically to nucleosome core particles and affect the structure and function of chromatin, including transcription. Here we study the biological role of this protein family by systematic analysis of phenotypes and tissue transcription profiles in mice lacking functional HMGN variants. Phenotypic analysis of Hmgn1-/-, Hmgn3-/-, and Hmgn5-/- mice and their wild type littermates with a battery of standardized tests uncovered variant-specific abnormalities. Gene expression analysis of four different tissues in each of the Hmgn-/- lines reveals very little overlap between the genes affected by the specific variants in the different tissues. Pathway analysis revels that loss of an HMGN variant affects subtly the expression of numerous genes in specific biological processes. We conclude that within the biological framework of an entire organism, HMGNs modulate the fidelity of the cellular transcriptional profile in a tissue- and HMGN variant-specific manner. AU - Kugler, J.E.* AU - Horsch, M. AU - Huang, D.* AU - Furusawa, T.* AU - Rochman, M.* AU - Garrett, L. AU - Becker, L. AU - Bohla, A. AU - Hölter, S.M. AU - Prehn, C. AU - Rathkolb, B. AU - Rácz, I.* AU - Aguilar-Pimentel, J.A. AU - Adler, T. AU - Adamski, J. AU - Beckers, J. AU - Busch, D.H.* AU - Eickelberg, O. AU - Klopstock, T.* AU - Ollert, M.* AU - Stöger, T. AU - Wolf, E.* AU - Wurst, W. AU - Yildirim, A.Ö. AU - Zimmer, A.* AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Garfinkel, B.* AU - Orly, Y.* AU - Ovcharenko, I.* AU - Bustin, M.* C1 - 24229 C2 - 31346 SP - 16690-16703 TI - High mobility group N proteins modulate the fidelity of the cellular transcriptional profile in a tissue- and variant-specific manner. JO - J. Biol. Chem. VL - 288 IS - 23 PB - Amer. Soc. Biochemistry Molecular Biology PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, beta-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or R-p-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N-6-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability. AU - Neukamm, S.S. AU - Ott, J.* AU - Dammeier, S.* AU - Lehmann, R. AU - Häring, H.-U. AU - Schleicher, E.D. AU - Weigert, C. C1 - 25975 C2 - 32036 SP - 16403-16415 TI - Phosphorylation of Serine 1137/1138 of mouse Insulin Receptor Substrate (IRS) 2 regulates cAMP-dependent binding to 14-3-3 proteins and IRS2 protein degradation. JO - J. Biol. Chem. VL - 288 IS - 23 PB - Amer Soc. Biochemistry Molecular Biology Inc. PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - The vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, has come into focus as an attractive target in cancer invasion. However, little is known about the role of V-ATPase in cell death, and especially the underlying mechanisms remain mostly unknown. We used the myxobacterial macrolide archazolid B, a potent inhibitor of the V-ATPase, as an experimental drug as well as a chemical tool to decipher V-ATPase-related cell death signaling. We found that archazolid induced apoptosis in highly invasive tumor cells at nanomolar concentrations which was executed by the mitochondrial pathway. Prior to apoptosis induction archazolid led to the activation of a cellular stress response including activation of the hypoxia-inducible factor-1α (HIF1α) and autophagy. Autophagy, which was demonstrated by degradation of p62 or fusion of autophagosomes with lysosomes, was induced at low concentrations of archazolid that not yet increase pH in lysosomes. HIF1α was induced due to energy stress shown by a decline of the ATP level and followed by a shutdown of energy-consuming processes. As silencing HIF1α increases apoptosis, the cellular stress response was suggested to be a survival mechanism. We conclude that archazolid leads to energy stress which activates adaptive mechanisms like autophagy mediated by HIF1α and finally leads to apoptosis. We propose V-ATPase as a promising drugable target in cancer therapy caught up at the interplay of apoptosis, autophagy, and cellular/metabolic stress. AU - von Schwarzenberg, K.* AU - Wiedmann, R.M.* AU - Oak, P.* AU - Schulz, S. AU - Zischka, H. AU - Wanner, G.* AU - Efferth, T.* AU - Trauner, D.* AU - Vollmar, A.M.* C1 - 11849 C2 - 30832 SP - 1385-1396 TI - Mode of cell death induction by pharmacological Vacuolar H+-ATPase (V-ATPase) inhibition. JO - J. Biol. Chem. VL - 288 IS - 2 PB - Amer. Soc. Biochemistry Molecular Biology PY - 2013 SN - 0021-9258 ER - TY - JOUR AB - Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-alpha (ProT alpha), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by co-immunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProT alpha remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProT alpha expression in the cells enhanced mHtt-caused cell death. Deletion of the central acidic domain of ProT alpha abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProT alpha inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProT alpha protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProT alpha might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders. AU - Dong, G.* AU - Callegari, E.A.* AU - Gloeckner, C.J. AU - Ueffing, M. AU - Wang, H.M.* C1 - 8016 C2 - 29977 SP - 1279-1289 TI - Prothymosin-α interacts with mutant huntingtin and suppresses its cytotoxicity in cell culture. JO - J. Biol. Chem. VL - 287 IS - 2 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress. AU - Guler, G.D.* AU - Liu, H.* AU - Vaithiyalingam, S.* AU - Arnett, D.R.* AU - Kremmer, E. AU - Chazin, W.J.* AU - Fanning, E.* C1 - 7261 C2 - 29621 SP - 6469-6481 TI - Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress. JO - J. Biol. Chem. VL - 287 IS - 9 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes. AU - Hochstatter, J.* AU - Hölzel, M. AU - Rohrmoser, M. AU - Schermelleh, L.* AU - Leonhardt, H.* AU - Keough, R.* AU - Gonda, T.J.* AU - Imhof, A.* AU - Eick, D. AU - Längst, G.* AU - Németh, A.* C1 - 8210 C2 - 30045 SP - 24365-24377 TI - Myb-binding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA. JO - J. Biol. Chem. VL - 287 IS - 29 PB - American Society for Biochemistry and Molecular Biology PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Mitosis begins with the tethering of chromosomes to the mitotic spindle and their orientation perpendicular to the axis of cell division. In budding yeast, mitotic spindle orientation and the subsequent chromosome segregation are two independent processes. Early spindle orientation is driven by the actin-bound myosin Myo2p, which interacts with the adapter Kar9p. The latter also binds to microtubule-associated Bim1p, thereby connecting both types of cytoskeleton. This study focuses on the interaction between Kar9p and Bim1p and its regulation. We solved the crystal structure of the previously reported Kar9p-binding motif of Bim1p and identified a second, novel Kar9p interaction domain. We further show that two independent post-translational modification events regulate their interaction. Whereas Kar9p sumoylation is required for efficient complex formation with Bim1p, Aurora B/Ipl1p-dependent phosphorylation of Bim1p down-regulates their interaction. The observed effects of these modifications allow us to propose a novel regulatory framework for the assembly and disassembly of the early spindle orientation complex. AU - Hüls, D. AU - Storchova, Z.* AU - Niessing, D. C1 - 8012 C2 - 29981 SP - 16238-16245 TI - Post-translational modifications regulate assembly of early spindle orientation complex in yeast. JO - J. Biol. Chem. VL - 287 IS - 20 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E. AU - Jacso, T. AU - Schneider, E.* AU - Rupp, B.* AU - Reif, B. C1 - 8445 C2 - 30149 SP - 17040-17049 TI - Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli. JO - J. Biol. Chem. VL - 287 IS - 21 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - TMEM106B was identified as a major risk factor in a genome-wide association study for frontotemporal lobar degeneration (FTLD) with TAR DNA-binding protein (TDP)-43 pathology. The most significant association of TMEM106B single nucleotide polymorphisms with risk of FTLD-TDP was observed in patients with progranulin (GRN) mutations. Subsequent studies suggested an inverse correlation between TMEM106B expression and GRN levels in patient serum. However, in this study, this was not confirmed as we failed to detect a significant alteration of GRN levels upon knockdown or exogenous expression of TMEM106B in heterologous cells. To provide a basis for understanding TMEM106B function in health and disease, we investigated the membrane orientation and subcellular localization of this completely uncharacterized protein. By differential membrane extraction and sequential mutagenesis of potential N-glycosylation sites, we identified TMEM106B as a type 2 integral membrane protein with a highly glycosylated luminal domain. Glycosylation is partially required for the transport of TMEM106B beyond the endoplasmic reticulum to late cellular compartments. Endogenous as well as overexpressed TMEM106B localizes to late endosomes and lysosomes. Interestingly, the inhibition of vacuolar H+-ATPases significantly increased the levels of TMEM106B, a finding that may provide an unexpected biochemical link to GRN, because this protein is also strongly increased under the same conditions. Our findings provide a biochemical and cell biological basis for the understanding of the pathological role of TMEM106B in FTLD, an incurable neurodegenerative disorder. AU - Lang, C.M.* AU - Fellerer, K.* AU - Schwenk, B.M.* AU - Kuhn, P.-H.* AU - Kremmer, E. AU - Edbauer, D.* AU - Capell, A.* AU - Haass, C.* C1 - 8303 C2 - 30107 SP - 19355-19365 TI - Membrane orientation and subcellular localization of transmembrane protein 106B (TMEM106B), a major risk factor for frontotemporal lobar degeneration. JO - J. Biol. Chem. VL - 287 IS - 23 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Lamin B receptor (LBR) is a polytopic protein of the nuclear envelope thought to connect the inner nuclear membrane with the underlying nuclear lamina and peripheral heterochromatin. To better understand the function of this protein, we have examined in detail its nucleoplasmic region, which is predicted to harbor a Tudor domain (LBR-TD). Structural analysis by multidimensional NMR spectroscopy establishes that LBR-TD indeed adopts a classical β-barrel Tudor fold in solution, which, however, features an incomplete aromatic cage. Removal of LBR-TD renders LBR more mobile at the plane of the nuclear envelope, but the isolated module does not bind to nuclear lamins, heterochromatin proteins (MeCP2), and nucleosomes, nor does it associate with methylated Arg/Lys residues through its aromatic cage. Instead, LBR-TD exhibits tight and stoichiometric binding to the "histone-fold" region of unassembled, free histone H3, suggesting an interesting role in histone assembly. Consistent with such a role, robust binding to native nucleosomes is observed when LBR-TD is extended toward its carboxyl terminus, to include an area rich in Ser-Arg residues. The Ser-Arg region, alone or in combination with LBR-TD, binds both unassembled and assembled H3/H4 histones, suggesting that the TD/RS interface may operate as a "histone chaperone-like platform." AU - Liokatis, S.* AU - Edlich, C.* AU - Soupsana, K.* AU - Giannios, I.* AU - Panagiotidou, P.* AU - Tripsianes, K. AU - Sattler, M. AU - Georgatos, S.D.* AU - Politou, A.S.* C1 - 6824 C2 - 29318 SP - 1032-1042 TI - Solution structure and molecular interactions of lamin B receptor Tudor domain. JO - J. Biol. Chem. VL - 287 IS - 2 PB - American Society for Biochemistry and Molecular Biology PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB. AU - Mainz, A. AU - Bardiaux, B.* AU - Kuppler, F.* AU - Multhaup, G.* AU - Felli, I.C.* AU - Pierattelli, R.* AU - Reif, B. C1 - 7124 C2 - 29518 SP - 1128-1138 TI - Structural and mechanistic implications of metal binding in the small heat-shock protein αB-crystallin. JO - J. Biol. Chem. VL - 287 IS - 2 PB - American Society for Biochemistry and Molecular Biology, Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Although TGF-beta acts as a tumor suppressor in normal tissues and in early carcinogenesis, these tumor suppressor effects are lost in advanced malignancies. Single cell migration and epithelial-mesenchymal transition (EMT), both of which are regulated by TGF-beta, are critical steps in mediating cancer progression. Here, we sought to identify novel direct targets of TGF-beta signaling in lung cancer cells and have indentified the zyxin gene as a target of Smad3-mediated TGF-beta 1 signaling. Zyxin concentrates at focal adhesions and along the actin cytoskeleton; as such, we hypothesized that cytoskeletal organization, motility, and EMT in response to TGF-beta 1 might be regulated by zyxin expression. We show that TGF-beta 1 treatment of lung cancer cells caused rapid phospho-Smad3-dependent expression of zyxin. Zyxin expression was critical for the formation and integrity of cell adherens junctions. Silencing of zyxin decreased expression of the focal adhesion protein vasodilator-activated phosphoprotein (VASP), although the formation and morphology of focal adhesions remained unchanged. Zyxin-depleted cells displayed significantly increased integrin alpha 5 beta 1 levels, accompanied by enhanced adhesion to fibronectin and acquisition of a mesenchymal phenotype in response to TGF-beta 1. Zyxin silencing led to elevated integrin alpha 5 beta 1-dependent single cell motility. Importantly, these features are mirrored in the K-ras-driven mouse model of lung cancer. Here, lung tumors revealed decreased levels of both zyxin and phospho-Smad3 when compared with normal tissues. Our data thus demonstrate that zyxin is a novel functional target and effector of TGF-beta signaling in lung cancer. By regulating cell-cell junctions, integrin alpha 5 beta 1 expression, and cell-extracellular matrix adhesion, zyxin may regulate cancer cell motility and EMT during lung cancer development and progression. AU - Mise, N. AU - Savai, R.* AU - Yu, H. AU - Schwarz, J. AU - Kaminski, N.* AU - Eickelberg, O. C1 - 10628 C2 - 30464 SP - 31393-31405 TI - Zyxin is a Transforming Growth Factor-β (TGF-β)/Smad3 target gene that regulates lung cancer cell motility via integrin α5β1. JO - J. Biol. Chem. VL - 287 IS - 37 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins. AU - Ohlig, S.* AU - Pickhinke, U.* AU - Sirko, S. AU - Bandari, S.* AU - Hoffmann, D.* AU - Dreier, R.* AU - Farshi, P.* AU - Götz, M. AU - Grobe, K.* C1 - 11884 C2 - 30839 SP - 43708-43719 TI - An emerging role of sonic hedgehog shedding as a modulator of heparan sulfate interactions. JO - J. Biol. Chem. VL - 287 IS - 52 PB - Amer. Soc. Biochemistry Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases. AU - Voss, M.* AU - Fukumori, A.* AU - Kuhn, P.H.* AU - Künzel, U.* AU - Klier, B.* AU - Grammer, G.* AU - Haug-Kröper, M.* AU - Kremmer, E. AU - Lichtenthaler, S.F.* AU - Steiner, H.* AU - Schröder, B.* AU - Haass, C.* AU - Fluhrer, R.* C1 - 11589 C2 - 30703 SP - 43401-43409 TI - Foamy virus envelope protein is a substrate for Signal Peptide Peptidase-Like 3 (SPPL3). JO - J. Biol. Chem. VL - 287 IS - 52 PB - American Society of Biochemistry and Molecular Biology Inc. PY - 2012 SN - 0021-9258 ER - TY - JOUR AB - The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation. We show here that the NEMO C terminus, comprising the ubiquitin binding region and a zinc finger, has a high preference for binding to linear ubiquitin chains. However, immobilization of NEMO, which may be reminiscent of cellular oligomerization, facilitates the interaction with Lys-63 ubiquitin chains. Moreover, selective mutations in NEMO that abolish association with linear ubiquitin but do not affect binding to Lys-63 ubiquitin are only partially compromising NF-κB signaling in response to TNFα stimulation in fibroblasts and T cells. In line with this, TNFα-triggered expression of NF-κB target genes and induction of apoptosis was partially compromised by NEMO mutations that selectively impair the binding to linear ubiquitin chains. Thus, in vivo NEMO interaction with linear and Lys-63 ubiquitin chains is required for optimal IKK activation, suggesting that both type of chains are cooperating in triggering canonical NF-κB signaling. AU - Hadian, K. AU - Griesbach, R.A. AU - Dornauer, S. AU - Wanger, T.M. AU - Nagel, D. AU - Metlitzky, M. AU - Beisker, W. AU - Schmidt-Supprian, M.* AU - Krappmann, D. C1 - 6111 C2 - 28998 SP - 26107-26117 TI - NF-κB essential modulator (NEMO) interaction with linear and Lys-63 ubiquitin chains contributes to NF-κB activation. JO - J. Biol. Chem. VL - 286 IS - 29 PB - American Society for Biochemistry and Molecular Biology PY - 2011 SN - 0021-9258 ER - TY - JOUR AB - ADAR2, an RNA editing enzyme that converts specific adenosines to inosines in certain pre-mRNAs, often leading to amino acid substitutions in the encoded proteins, is mainly expressed in brain. Of all ADAR2-mediated edits, a single one in the pre-mRNA of the AMPA receptor subunit GluA2 is essential for survival. Hence, early postnatal death of mice lacking ADAR2 is averted when the critical edit is engineered into both GluA2 encoding Gria2 alleles. Adar2(-/-)/Gria2(R/R) mice display normal appearance and life span, but the general phenotypic effects of global lack of ADAR2 have remained unexplored. Here we have employed the Adar2(-/-)/Gria2(R/R) mouse line, and Gria2(R/R) mice as controls, to study the phenotypic consequences of loss of all ADAR2-mediated edits except the critical one in GluA2. Our extended phenotypic analysis covering ∼320 parameters identified significant changes related to absence of ADAR2 in behavior, hearing ability, allergy parameters and transcript profiles of brain. AU - Horsch, M. AU - Seeburg, PH.* AU - Adler, T.* AU - Aguilar-Pimentel, J.A. AU - Becker, L.* AU - Calzada-Wack, J.* AU - Garrett, L. AU - Götz, A. AU - Hans, W. AU - Higuchi, M.* AU - Hölter, S.M. AU - Naton, B. AU - Prehn, C. AU - Puk, O. AU - Rácz, I.* AU - Rathkolb, B.* AU - Rozman, J.* AU - Schrewe, A. AU - Adamski, J. AU - Busch, D.H.* AU - Esposito, I.* AU - Graw, J. AU - Ivandic, B.* AU - Klingenspor, M.* AU - Klopstock, T.* AU - Mempel, M. AU - Ollert, M.* AU - Schulz, S. AU - Wolf, E.* AU - Wurst, W. AU - Zimmer, A.* AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Beckers, J. C1 - 6375 C2 - 28131 SP - 18614-18622 TI - Requirement of the RNA-editing enzyme ADAR2 for normal physiology in mice. JO - J. Biol. Chem. VL - 286 IS - 21 PB - American Society for Biochemistry and Molecular Biology PY - 2011 SN - 0021-9258 ER - TY - JOUR AB - T cell receptor (TCR) ligation induces increased diacylglycerol and Ca(2+) levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-κB signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-κB signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-κB activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCR-induced NF-κB activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-κB, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA + ionomycin- or anti-CD3 + CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-κB activity. AU - Palkowitsch, L.* AU - Marienfeld, U.* AU - Brunner, C.* AU - Eitelhuber, A. AU - Krappmann, D. AU - Marienfeld, R.B.* C1 - 4841 C2 - 28661 SP - 7522-7534 TI - The Ca²⁺-dependent phosphatase calcineurin controls the formation of the Carma1-Bcl10-Malt1 complex during T cell receptor-induced NF-κB activation. JO - J. Biol. Chem. VL - 286 IS - 9 PB - Amer Soc Biochemistry Molecular Biology Inc. PY - 2011 SN - 0021-9258 ER - TY - JOUR AB - La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signalling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as upregulation and serine 15 phosphorylation of p53, or serine 1524 phosphorylation of BRCA1, were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shut down of RNA polymerase II transcription, and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits. AU - Verbruggen, P.* AU - Ruf, M.* AU - Blakqori, G.* AU - Oeverby, A.K.* AU - Heidemann, M. AU - Eick, D. AU - Weber, F.* C1 - 6064 C2 - 27792 SP - 3681-3692 TI - Interferon antagonist NSs of La Crosse virus triggers a DNA-damage response-like degradation of transcribing RNA polymerase II. JO - J. Biol. Chem. VL - 286 IS - 5 PB - American Society for Biochemistry and Molecular Biology PY - 2011 SN - 0021-9258 ER - TY - JOUR AB - IL-6 gene expression is controlled by a promoter-region containing multiple regulatory elements such as NF-kB, NF-IL6, CRE, GRE and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knockout mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defence and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes. AU - Wruck, C.J.* AU - Streetz, K.* AU - Pavic, G.* AU - Götz, M.E.* AU - Tohidnezhad, M.* AU - Brandenburg, L.O.* AU - Varoga, D.* AU - Eickelberg, O. AU - Herdegen, T.* AU - Trautwein, C.* AU - Cha, K.* AU - Kan, Y.W.* AU - Pufe, T.* C1 - 2091 C2 - 28078 SP - 4493-4499 TI - Nrf2 induces interleukin-6 (IL-6) expression via an antioxidant response element within the IL-6 promoter. JO - J. Biol. Chem. VL - 286 IS - 6 PB - American Society for Biochemistry and Molecular Biology, Inc. PY - 2011 SN - 0021-9258 ER - TY - JOUR AB - The unfolded protein response (UPR) or endoplasmic reticulum (ER) stress response is a physiological process enabling cells to cope with altered protein synthesis demands. However, under conditions of obesity, prolonged activation of the UPR has been shown to have deteriorating effects on different metabolic pathways. Here we identify Bax inhibitor-1 (BI-1), an evolutionary conserved ER-membrane protein, as a novel modulator of the obesity-associated alteration of the UPR. BI-1 partially inhibits the UPR by interacting with IRE1alpha and inhibiting IRE1alpha endonuclease activity as seen on the splicing of the transcription factor Xbp-1. Because we observed a down-regulation of BI-1 expression in liver and muscle of genetically obese ob/ob and db/db mice as well as in mice with diet-induced obesity in vivo, we investigated the effect of restoring BI-1 expression on metabolic processes in these mice. Importantly, BI-1 overexpression by adenoviral gene transfer dramatically improved glucose metabolism in both standard diet-fed mice as well as in mice with diet-induced obesity and, critically, reversed hyperglycemia in db/db mice. This improvement in whole body glucose metabolism and insulin sensitivity was due to dramatically reduced gluconeogenesis as shown by reduction of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase expression. Taken together, these results identify BI-1 as a critical regulator of ER stress responses in the development of obesity-associated insulin resistance and provide proof of concept evidence that gene transfer-mediated elevations in hepatic BI-1 may represent a promising approach for the treatment of type 2 diabetes. AU - Bailly-Maitre, B.* AU - Belgardt, B.F.* AU - Jordan, S.D.* AU - Coornaert, B.* AU - von Freyend, M.J.* AU - Kleinridders, A.* AU - Mauer, J.* AU - Cuddy, M.* AU - Kress, C.L.* AU - Willmes, D.* AU - Essig, M.* AU - Hampel, B.* AU - Protzer, U. AU - Reed, J.C.* AU - Brüning, J.C.* C1 - 5572 C2 - 27772 SP - 6198-6207 TI - Hepatic Bax inhibitor-1 inhibits IRE1α and protects from obesity-associated insulin resistance and glucose intolerance. JO - J. Biol. Chem. VL - 285 IS - 9 PB - ASBMB PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - The largest subunit of RNA polymerase II (RNAPII) C-terminal heptarepeat domain (CTD) is subject to phosphorylation during initiation and elongation of transcription by RNA polymerase II. Here we study the molecular mechanisms leading to phosphorylation of Ser-7 in the human enzyme. Ser-7 becomes phosphorylated before initiation of transcription at promoter regions. We identify cyclin-dependent kinase 7 (CDK7) as one responsible kinase. Phosphorylation of both Ser-5 and Ser-7 is fully dependent on the cofactor complex Mediator. A subform of Mediator associated with an active RNAPII is critical for preinitiation complex formation and CTD phosphorylation. The Mediator-RNAPII complex independently recruits TFIIB and CDK7 to core promoter regions. CDK7 phosphorylates Ser-7 selectively in the context of an intact preinitiation complex. CDK7 is not the only kinase that can modify Ser-7 of the CTD. ChIP experiments with chemical inhibitors provide evidence that other yet to be identified kinases further phosphorylate Ser-7 in coding regions. AU - Boeing, S.* AU - Rigault, C.* AU - Heidemann, M. AU - Eick, D. AU - Meisterernst, M.* C1 - 507 C2 - 26812 SP - 188-196 TI - RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion. JO - J. Biol. Chem. VL - 285 IS - 1 PB - American Society for Biochemistry and Molecular Biology PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. oxaliplatin, doxorubicin, mitoxantrone, methotrexate), (ii) early rRNA processing (e.g. camptothecin, flavopiridol, roscovitine), or (iii) late rRNA processing (e.g. 5-fluorouracil, MG-132, homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, whereas blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens. AU - Burger, K. AU - Mühl, B. AU - Harasim, T. AU - Rohrmoser, M. AU - Malamoussi, A. AU - Orban, M. AU - Kellner, M. AU - Gruber-Eber, A. AU - Kremmer, E. AU - Hölzel, M. AU - Eick, D. C1 - 4784 C2 - 27627 SP - 12416-12425 TI - Chemotherapeutic drugs inhibit ribosome biogenesis at various levels. JO - J. Biol. Chem. VL - 285 IS - 16 PB - American Society Biochemistry Molecular Biology Inc. PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase. AU - Dettmer, U.* AU - Kuhn, P.H.* AU - Abou-Ajram, C.* AU - Lichtenthaler, S.F.* AU - Krüger, M.* AU - Kremmer, E. AU - Haass, C.* AU - Haffner, C.* C1 - 5420 C2 - 27489 SP - 26174-26181 TI - Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex. JO - J. Biol. Chem. VL - 285 IS - 34 PB - American Society for Biochemistry and Molecular Biology Inc. PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - Calcium oxalate stone formation occurs under pathological conditions and accounts for more than 80% of all types of kidney stones. In the current study, we show for the first time that calcium oxalate stones are formed in the mouse inner ear of a genetic model for hearing loss and vestibular dysfunction in humans. The vestibular system within the inner ear is dependent on extracellular tiny calcium carbonate minerals for proper function. Thousands of these biominerals, known as otoconia, are associated with the utricle and saccule sensory maculae and are vital for mechanical stimulation of the sensory hair cells. We show that a missense mutation within the Slc26a4 gene abolishes the transport activity of its encoded protein, pendrin. As a consequence, dramatic changes in mineral composition, size, and shape occur within the utricle and saccule in a differential manner. Although abnormal giant carbonate minerals reside in the utricle at all ages, in the saccule, a gradual change in mineral composition leads to a formation of calcium oxalate in adult mice. By combining imaging and spectroscopy tools, we determined the profile of mineral composition and morphology at different time points. We propose a novel mechanism for the accumulation and aggregation of oxalate crystals in the inner ear. AU - Dror, A.A.* AU - Politi, Y.* AU - Shahin, H.* AU - Lenz, D.R.* AU - Dossena, S.* AU - Nofziger, C.* AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Paulmichl, M.* AU - Weiner, S.* AU - Avraham, K.B.* C1 - 458 C2 - 27227 SP - 21724-21735 TI - Calcium oxalate stone formation in the inner ear as a result of an Slc26a4 mutation. JO - J. Biol. Chem. VL - 285 IS - 28 PB - American Society for Biochemistry and Molecular Biology PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - The p53 tumor suppressor pathway is activated by defective ribosome synthesis. Ribosomal proteins are released from the nucleolus and block human double minute-2 (Hdm2) that targets p53 for degradation. However, it remained elusive how abrogation of individual rRNA processing pathways contributes to p53 stabilization. Here, we show that selective inhibition of 18 S rRNA processing provokes accumulation of p53 as efficiently as abrogated 28 S rRNA maturation. We describe hUTP18 as a novel mammalian rRNA processing factor that is specifically involved in 18 S rRNA production. hUTP18 was essential for the cleavage of the 5'-external transcribed spacer leader sequence from the primary polymerase I transcript, but was dispensable for rRNA transcription. Because maturation of the 28 S rRNA was unaffected in hUTP18-depleted cells, our results suggest that the integrity of both the 18 S and 28 S rRNA synthesis pathways can be monitored independently by the p53 pathway. Interestingly, accumulation of p53 after hUTP18 knock down required the ribosomal protein L11. Therefore, cells survey the maturation of the small and large ribosomal subunits by separate molecular routes, which may merge in an L11-dependent signaling pathway for p53 stabilization. AU - Hölzel, M.* AU - Orban, M.* AU - Hochstatter, J.* AU - Rohrmoser, M.* AU - Harasim, T.* AU - Malamoussi, A.* AU - Kremmer, E. AU - Längst, G.* AU - Eick, D. C1 - 615 C2 - 27188 SP - 6364-6370 TI - Defects in 18 S or 28 S rRNA processing activate the p53 pathway. JO - J. Biol. Chem. VL - 285 IS - 9 PB - American Society for Biochemistry and Molecular Biology PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - GSH is the major antioxidant and detoxifier of xenobiotics in mammalian cells. A strong decrease of intracellular GSH has been frequently linked to pathological conditions like ischemia/reperfusion injury and degenerative diseases including diabetes, atherosclerosis, and neurodegeneration. Although GSH is essential for survival, the deleterious effects of GSH deficiency can often be compensated by thiol-containing antioxidants. Using three genetically defined cellular systems, we show here that forced expression of xCT, the substrate-specific subunit of the cystine/glutamate antiporter, in γ-glutamylcysteine synthetase knock-out cells rescues GSH deficiency by increasing cellular cystine uptake, leading to augmented intracellular and surprisingly high extracellular cysteine levels. Moreover, we provide evidence that under GSH deprivation, the cytosolic thioredoxin/thioredoxin reductase system plays an essential role for the cells to deal with the excess amount of intracellular cystine. Our studies provide first evidence that GSH deficiency can be rescued by an intrinsic genetic mechanism to be considered when designing therapeutic rationales targeting specific redox enzymes to combat diseases linked to GSH deprivation. AU - Mandal, P.K. AU - Seiler, A. AU - Perisic, T. AU - Kölle, P.* AU - Canak, A.B. AU - Förster, H. AU - Weiss, N.* AU - Kremmer, E. AU - Liebermann, M.W.* AU - Bannai, S.* AU - Kuhlencordt, P.* AU - Sato, H.* AU - Bornkamm, G.W. AU - Conrad, M. C1 - 4997 C2 - 27743 SP - 22244-22253 TI - System xc− and thioredoxin reductase 1 cooperatively rescue glutathione deficiency. JO - J. Biol. Chem. VL - 285 IS - 29 PB - American Society for Biochemistry and Molecular Biology PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - The CARMA1, Bcl10, and MALT1 proteins together constitute a signaling complex (CBM signalosome) that mediates antigen-dependent activation of NF-kappaB in lymphocytes, thereby representing a cornerstone of the adaptive immune response. Although CARMA1 is restricted to cells of the immune system, the analogous CARMA3 protein has a much wider expression pattern. Emerging evidence suggests that CARMA3 can substitute for CARMA1 in non-immune cells to assemble a CARMA3-Bcl10-MALT1 signalosome and mediate G protein-coupled receptor activation of NF-kappaB. Here we show that one G protein-coupled receptor, the type 1 receptor for angiotensin II, utilizes this mechanism for activation of NF-kappaB in endothelial and vascular smooth muscle cells, thereby inducing pro-inflammatory signals within the vasculature, a key factor in atherogenesis. Further, we demonstrate that Bcl10-deficient mice are protected from developing angiotensin-dependent atherosclerosis and aortic aneurysms. By uncovering a novel vascular role for the CBM signalosome, these findings illustrate that CBM-dependent signaling has functions outside the realm of adaptive immunity and impacts pathobiology more broadly than previously known. AU - McAllister-Lucas, L.M.* AU - Jin, X.H.* AU - Gu, S.F.* AU - Siu, K.* AU - McDonnell, S.* AU - Ruland, J. AU - Delekta, P.C.* AU - van Beek, M.* AU - Lucas, P.C.* C1 - 2765 C2 - 27521 SP - 25880-25884 TI - The CARMA3-Bcl10-MALT1 signalosome promotes angiotensin II-dependent vascular inflammation and atherogenesis. JO - J. Biol. Chem. VL - 285 IS - 34 PB - American Society Biochemistry Molecular Biology Inc. PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - Sam68 (Src-associated during mitosis, 68 kDa) is a prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. STAR proteins bind mRNA targets and modulate cellular processes such as cell cycle regulation and tissue development in response to extracellular signals. Sam68 has been shown to modulate alternative splicing of the pre-mRNAs of CD44 and Bcl-xL, which are linked to tumor progression and apoptosis. Sam68 and other STAR proteins recognize bipartite RNA sequences and are thought to function as homodimers. However, the structural and functional roles of the self-association are not known. Here, we present the solution structure of the Sam68 Qua1 homodimerization domain. Each monomer consists of two antiparallel alpha-helices connected by a short loop. The two subunits are arranged perpendicular to each other in an unusual four-helix topology. Mutational analysis of Sam68 in vitro and in a cell-based assay revealed that the Qua1 domain and residues within the dimerization interface are essential for alternative splicing of a CD44 minigene. Together, our results indicate that the Qua1 homodimerization domain is required for regulation of alternative splicing by Sam68. AU - Meyer, N.H. AU - Tripsianes, K. AU - Vincendeau, M. AU - Madl, T. AU - Kateb, F. AU - Brack-Werner, R. AU - Sattler, M. C1 - 5949 C2 - 27637 SP - 28893-28901 TI - Structural basis for homodimerization of the Src-associated during mitosis, 68-kDa protein (Sam68) Qua1 domain. JO - J. Biol. Chem. VL - 285 IS - 37 PB - American Society for Biochemistry and Molecular Biology, Inc. PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I.C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-kappaB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I.C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses. AU - Neerincx, A.* AU - Lautz, K.* AU - Menning, M.* AU - Kremmer, E. AU - Zigrino, P.* AU - Hösel, M.* AU - Buning, H.* AU - Schwarzenbacher, R.* AU - Kufer, T.A.* C1 - 5419 C2 - 27488 SP - 26223-26232 TI - A role for the human nucleotide-binding domain, leucine-rich repeat-containing family member NLRC5 in antiviral responses. JO - J. Biol. Chem. VL - 285 IS - 34 PB - American Society for Biochemistry and Molecular Biology Inc. PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). At present, little is known about the cellular mechanisms that control APP shedding and Abeta generation. Here, we identified a novel protein, transmembrane protein 59 (TMEM59), as a new modulator of APP shedding. TMEM59 was found to be a ubiquitously expressed, Golgi-localized protein. TMEM59 transfection inhibited complex N- and O-glycosylation of APP in cultured cells. Additionally, TMEM59 induced APP retention in the Golgi and inhibited Abeta generation as well as APP cleavage by alpha- and beta-secretase cleavage, which occur at the plasma membrane and in the endosomes, respectively. Moreover, TMEM59 inhibited the complex N-glycosylation of the prion protein, suggesting a more general modulation of Golgi glycosylation reactions. Importantly, TMEM59 did not affect the secretion of soluble proteins or the alpha-secretase like shedding of tumor necrosis factor alpha, demonstrating that TMEM59 did not disturb the general Golgi function. The phenotype of TMEM59 transfection on APP glycosylation and shedding was similar to the one observed in cells lacking conserved oligomeric Golgi (COG) proteins COG1 and COG2. Both proteins are required for normal localization and activity of Golgi glycosylation enzymes. In summary, this study shows that TMEM59 expression modulates complex N- and O-glycosylation and suggests that TMEM59 affects APP shedding by reducing access of APP to the cellular compartments, where it is normally cleaved by alpha- and beta-secretase. AU - Ullrich, S.* AU - Münch, A.* AU - Neumann, S.* AU - Kremmer, E. AU - Tatzelt, J.* AU - Lichtenthaler, S.F.* C1 - 5924 C2 - 27496 SP - 20664-20674 TI - The novel membrane protein TMEM59 modulates complex glycosylation, cell surface expression, and secretion of the amyloid precursor protein. JO - J. Biol. Chem. VL - 285 IS - 27 PB - American Society for Biochemistry and Molecular Biology Inc. PY - 2010 SN - 0021-9258 ER - TY - JOUR AB - The role of endogenous inducers of inflammation is poorly understood. To produce the proinflammatory master cytokine interleukin (IL)-1beta, macrophages need double stimulation with ligands to both Toll-like receptors (TLRs) for IL-1beta gene transcription and nucleotide-binding oligomerization domain-like receptors for activation of the inflammasome. It is particularly intriguing to define how this complex regulation is mediated in the absence of an infectious trigger. Biglycan, a ubiquitous leucine-rich repeat proteoglycan of the extracellular matrix, interacts with TLR2/4 on macrophages. The objective of this study was to define the role of biglycan in the synthesis and activation of IL-1beta. Here we show that in macrophages, soluble biglycan induces the NLRP3/ASC inflammasome, activating caspase-1 and releasing mature IL-1beta without the need for additional costimulatory factors. This is brought about by the interaction of biglycan with TLR2/4 and purinergic P2X(4)/P2X(7) receptors, which induces receptor cooperativity. Furthermore, reactive oxygen species formation is involved in biglycan-mediated activation of the inflammasome. By signaling through TLR2/4, biglycan stimulates the expression of NLRP3 and pro-IL-1beta mRNA. Both in a model of non-infectious inflammatory renal injury (unilateral ureteral obstruction) and in lipopolysaccharide-induced sepsis, biglycan-deficient mice displayed lower levels of active caspase-1 and mature IL-1beta in the kidney, lung, and circulation. Our results provide evidence for direct activation of the NLRP3 inflammasome by biglycan and describe a fundamental paradigm of how tissue stress or injury is monitored by innate immune receptors detecting the release of the extracellular matrix components and turning such a signal into a robust inflammatory response. AU - Babelova, A.* AU - Moreth, K.* AU - Tsalastra-Greul, W.* AU - Zeng-Brouwers, J.* AU - Eickelberg, O. AU - Young, M.F.* AU - Bruckner, P.* AU - Pfeilschifter, J.* AU - Schaefer, R.M.* AU - Gröne, H.J.* AU - Schaefer, L.* C1 - 6464 C2 - 26810 SP - 24035-24048 TI - Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors. JO - J. Biol. Chem. VL - 284 IS - 36 PB - Amer Soc Biochemistry Molecular Biology Inc. PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - Prion diseases are neurodegenerative diseases associated with the accumulation of a pathogenic isoform of the host-encoded prion protein. The cellular responses to prion infection are not well defined. By performing microarray analysis on cultured neuronal cells infected with prion strain 22L, in the group of up-regulated genes we observed predominantly genes of the cholesterol pathway. Increased transcript levels of at least nine enzymes involved in cholesterol synthesis, including the gene for the rate-limiting hydroxymethylglutaryl-CoA reductase, were detected. Up-regulation of cholesterogenic genes was attributable to a prion-dependent increase in the amount and activity of the sterol regulatory element-binding protein Srebp2, resulting in elevated levels of total and free cellular cholesterol. The up-regulation of cholesterol biosynthesis appeared to be a characteristic response of neurons to prion challenge, as cholesterogenic transcripts were also elevated in persistently infected GT-1 cells and prion-exposed primary hippocampal neurons but not in microglial cells and primary astrocytes. These results convincingly demonstrate that prion propagation not only depends on the availability of cholesterol but that neuronal cells themselves respond to prions with specific up-regulation of cholesterol biosynthesis. AU - Bach, C.E. AU - Gilch, S.* AU - Rost, R.* AU - Greenwood, A.D. AU - Horsch, M. AU - Hajj,G.N.M.* AU - Brodesser, S.* AU - Facius, A. AU - Schädler, S. AU - Sandhoff, K.* AU - Beckers, J. AU - Leib-Mösch, C. AU - Schatzl, H.M.* AU - Vorberg, I.* C1 - 696 C2 - 26597 CY - Bethesda SP - 31260-31269 TI - Prion-induced activation of cholesterogenic gene expression by Srebp2 in neuronal cells. JO - J. Biol. Chem. VL - 284 IS - 45 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstrom resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF(65), and SF3b155. Compared with U2AF(65)-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF(65) and Puf60 may involve simultaneous interactions of the two proteins with SF3b155. AU - Corsini, L.* AU - Hothorn, M.* AU - Stier, G.* AU - Rybin, V.* AU - Scheffzek, K.* AU - Gibson, T.J.* AU - Sattler, M. C1 - 452 C2 - 26083 SP - 630-639 TI - Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60. JO - J. Biol. Chem. VL - 284 IS - 1 PB - Amer Soc Biochemistry Molecular Biology PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state. AU - Everett, K.L.* AU - Bunney, T.D.* AU - Yoon, Y.D.* AU - Rodrigues-Lima, F.* AU - Harris, R.* AU - Driscoll, P.C.* AU - Abe, K. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Yu, P.* AU - Cho, W.W.* AU - Katan, M.* C1 - 576 C2 - 26421 SP - 23083-23093 TI - Characterization of phospholipase Cγ enzymes with gain-of-function mutations. JO - J. Biol. Chem. VL - 284 IS - 34 PB - Amer Soc Biochemistry Molecular Biology PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - The Rev protein is a key regulator of human immunodeficiency virus type 1 (HIV-1) gene expression. Rev is primarily known as an adaptor protein for nuclear export of HIV RNAs. However, Rev also contributes to numerous other processes by less well known mechanisms. Understanding the functional nature of Rev requires extensive knowledge of its cellular interaction partners. Here we demonstrate that Rev interacts with members of a large family of multifunctional host cell factors called hnRNPs. Rev employs amino acids 9-14 for specific binding to the heterogeneous nuclear ribonucleoproteins (hnRNP) A1, Q, K, R, and U. In addition, Rev interacts with hnRNP E1 and E2 by a different mechanism. The set of hnRNPs recognized by the N terminus of Rev feature RGG boxes. Exemplary testing of hnRNP A1 revealed a critical role of arginine residues within the RGG box for interaction with Rev. Finally, we demonstrate that expression levels of hnRNP A1, Q, K, R, and U influence HIV-1 production by persistently infected astrocytes, linking these hnRNPs to HIV replication. The novel interaction of HIV-1 Rev with functionally diverse hnRNPs lends further support to the idea that Rev is a multifunctional protein and may be involved in coupling HIV replication to diverse cellular processes and promoting virus-host cell interactions. AU - Hadian, K. AU - Vincendeau, M. AU - Mäusbacher, N. AU - Nagel, D. AU - Hauck, S.M. AU - Ueffing, M. AU - Loyter, A.* AU - Werner, T.* AU - Wolff, H. AU - Brack-Werner, R. C1 - 2714 C2 - 26857 SP - 33384-33391 TI - Identification of a heterogeneous nuclear ribonucleoprotein-recognition region in the HIV Rev protein. JO - J. Biol. Chem. VL - 284 IS - 48 PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1 gamma) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1 gamma is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1 Delta DAD), indicating that deregulation of autoinhibition is effective in FMNL1 gamma. Expression of both FMNL1 gamma and FMNL1 Delta DAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells. AU - Han, Y. AU - Eppinger, E. AU - Schuster, I.G. AU - Weigand, L.U. AU - Liang, X. AU - Kremmer, E. AU - Peschel, C.* AU - Krackhardt, A.M. C1 - 2709 C2 - 26544 SP - 33409-33417 TI - Formin-like 1 (FMNL1) is regulated by N-terminal myristoylation and induces polarized membrane blebbing. JO - J. Biol. Chem. VL - 284 IS - 48 PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1- deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria. AU - Lutz, A.K.* AU - Exner, N.* AU - Fett, M.E.* AU - Schlehe, J.S.* AU - Kloos, K. AU - Lämmermann, K.* AU - Brunner, B.* AU - Kurz-Drexler, A. AU - Vogel, F.* AU - Reichert, AS.* AU - Bouman, L.* AU - Vogt Weisenhorn, D.M. AU - Wurst, W. AU - Tatzelt, J.* AU - Haass, C.* AU - Winklhofer, K.F.* C1 - 1344 C2 - 26290 SP - 2108-2111 TI - Loss of parkin or PINK1 function increases Drp1-dependent mitochondrial fragmentation. JO - J. Biol. Chem. VL - 284 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulinlike domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes. AU - Meyer, D.* AU - Seth, S.* AU - Albrecht, J.* AU - Maier, M.K.* AU - du Pasquier, L.* AU - Ravens, I.* AU - Dreyer, L.* AU - Burger, R.* AU - Gramatzki, M.* AU - Schwinzer, R.* AU - Kremmer, E. AU - Foerster, R.* AU - Bernhardt, G.* C1 - 985 C2 - 26511 SP - 2235-2244 TI - CD96 interaction with CD155 via its first Ig-like domain is modulated by alternative splicing or mutations in distal Ig-like domains. JO - J. Biol. Chem. VL - 284 IS - 4 PB - Amer Soc Biochemistry Molecular Biology Inc PY - 2009 SN - 0021-9258 ER - TY - JOUR AB - Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation. AU - Bendz, H. AU - Marincek, B.C.* AU - Momburg, F.* AU - Ellwart, J.W. AU - Issels, R.D. AU - Nelson, P.J.* AU - Nößner, E. C1 - 176 C2 - 25752 SP - 26477-26483 TI - Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation. JO - J. Biol. Chem. VL - 283 IS - 39 PB - ASBMB PY - 2008 SN - 0021-9258 ER - TY - JOUR AB - More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40. AU - Fluhrer, R.* AU - Fukumori, A.* AU - Martin, L.* AU - Grammer, G.* AU - Haug-Kröper, M.* AU - Klier, B.* AU - Winkler, E.* AU - Kremmer, E. AU - Condron, M.M.* AU - Teplow, D.B.* AU - Steiner, H.* AU - Haass, C.* C1 - 3634 C2 - 25782 SP - 30121-30128 TI - Intramembrane proteolysis of GXGD-type aspartyl proteases is slowed by a familial Alzheimer disease-like mutation. JO - J. Biol. Chem. VL - 283 IS - 44 PB - American Society for Biochemistry and Molecular Biology PY - 2008 SN - 0021-9258 ER - TY - JOUR AB - Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle. AU - Jasiak, A.J.* AU - Hartmann, H.* AU - Karakasili, E.* AU - Kalocsay, M.* AU - Flatley, A. AU - Kremmer, E. AU - Strässer, K.* AU - Martin, D.E.* AU - Söding, J.* AU - Cramer, P.* C1 - 177 C2 - 25780 SP - 26423-26427 TI - Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex. JO - J. Biol. Chem. VL - 283 IS - 39 PB - ASBMB PY - 2008 SN - 0021-9258 ER - TY - JOUR AB - Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment. AU - Martin, L.* AU - Fluhrer, R.* AU - Reiss, K.* AU - Kremmer, E. AU - Saftig, P.* AU - Haass, C.* C1 - 676 C2 - 25699 SP - 1644-1652 TI - Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b. JO - J. Biol. Chem. VL - 283 IS - 3 PB - ASBMB PY - 2008 SN - 0021-9258 ER - TY - JOUR AB - Erythroid precursor cells undergo nuclear extrusion and degradation of mitochondria when they mature to erythrocytes. It has been suggested before that the reticulocyte 15-lipoxygenase (r15-LOX) plays an important role in initiating the breakdown of mitochondria in rabbit reticulocytes. The expression of rabbit r15-LOX is regulated by the heterogeneous nuclear ribonucleoproteins (hnRNP) K and E1 at the translational level. However, this mechanism has never been confirmed in human erythropoiesis. Based on K562 cells we have set up an inducible human erythroid cell system. We show that, during induction, K562 cells exhibit changes in morphology and protein expression that are characteristic for terminal erythroid maturation: nuclear exclusion, expression of endogenous human r15-LOX regulated by hnRNP K and hnRNP E1, and loss of mitochondria. Importantly, induction of terminal erythroid maturation in primary human CD34(+) cells recapitulated the results obtained in K562 cells. Employing the physiologically relevant K562 cell system we uncovered a new mechanism of interdependent post-transcriptional regulation of gene expression. The timely expression of the tyrosine kinase c-Src, which phosphorylates hnRNP K in later stages, is controlled by hnRNP K in early stages of erythroid maturation. hnRNP K binds to the 3'-untranslated region of the c-Src mRNA and inhibits its translation by blocking 80 S ribosome formation. In premature erythroid cells, small interfering RNA-mediated knockdown of hnRNP K, but not of hnRNP E1, leads to the de-repression of c-Src synthesis. AU - Naarmann, I.S.* AU - Harnisch, C.* AU - Flach, N.* AU - Kremmer, E. AU - Kühn, H.* AU - Ostareck, D.H.* AU - Ostareck-Lederer, A.* C1 - 2429 C2 - 25700 SP - 18461-18472 TI - mRNA silencing in human erythroid cell maturation: Heterogeneous nuclear ribonucleoprotein K controls the expression of its regulator c-Src.. JO - J. Biol. Chem. VL - 283 IS - 26 PB - ASBMB PY - 2008 SN - 0021-9258 ER - TY - JOUR AB - Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). beta-Secretase catalyzes the first step in Abeta generation, whereas alpha-secretase cleaves within the Abeta domain, prevents Abeta generation, and generates a secreted form of APP with neuroprotective properties. At present, little is known about the cellular mechanisms that control APP alpha-secretase cleavage and Abeta generation. To explore the contributory pathways, we carried out an expression cloning screen. We identified a novel member of the sorting nexin (SNX) family of endosomal trafficking proteins, called SNX33, as a new activator of APP alpha-secretase cleavage. SNX33 is a homolog of SNX9 and was found to be a ubiquitously expressed phosphoprotein. Exogenous expression of SNX33 in cultured cells increased APP alpha-secretase cleavage 4-fold but surprisingly had little effect on beta-secretase cleavage. This effect was similar to the expression of the dominant negative dynamin-1 mutant K44A. SNX33 bound the endocytic GTPase dynamin and reduced the rate of APP endocytosis in a dynamin-dependent manner. This led to an increase of APP at the plasma membrane, where alpha-secretase cleavage mostly occurs. In summary, our study identifies SNX33 as a new endocytic protein, which modulates APP endocytosis and APP alpha-secretase cleavage, and demonstrates that the rate of APP endocytosis is a major control factor for APP alpha-secretase cleavage. AU - Schöbel, S.* AU - Neumann, S.* AU - Hertweck, M.* AU - Dislich, B.* AU - Kuhn, P.H.* AU - Kremmer, E. AU - Seed, B.* AU - Baumeister, R.* AU - Haass, C.* AU - Lichtenthaler, S.F.* C1 - 3324 C2 - 25705 SP - 14257-14268 TI - A novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage of the amyloid precursor protein. JO - J. Biol. Chem. VL - 283 IS - 21 PB - ASBMB PY - 2008 SN - 0021-9258 ER - TY - JOUR AB - Heat shock proteins (HSPs) have shown promise for the optimization of protein-based vaccines because they can transfer exogenous antigens to dendritic cells and at the same time induce their maturation. Great care must be exercised in interpretating HSP-driven studies, as by-products linked to the recombinant generation of these proteins have been shown to mediate immunological effects. We generated highly purified human recombinant Hsp70 and demonstrated that it strongly enhances the cross-presentation of exogenous antigens resulting in better antigen-specific T cell stimulation. Augmentation of T cell stimulation was a direct function of the degree of complex formation between Hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. The Hsp70 activity was independent of TAP proteins and was not inhibited by exotoxin A or endosomal acidification. Consequently, Hsp70 enhanced cross-presentation of various antigenic sequences, even when they required different post-uptake processing and trafficking, as exemplified by the tumor antigens tyrosinase and Melan-A/MART-1. Furthermore, Hsp70 enhanced cross-presentation by different antigen-presenting cells (APCs), including dendritic cells and B cells. Importantly, enhanced cross-presentation and antigen-specific T cell activation were observed in the absence of innate signals transmitted by Hsp70. As Hsp70 supports the cross-presentation of different antigens and APCs and is inert to APC function, it may show efficacy in various settings of immune modulation, including induction of antigen-specific immunity or tolerance. AU - Bendz, H. AU - Ruhland, S.C. AU - Pandya, M.J.* AU - Hainzl, O.* AU - Riegelsberger, S.* AU - Bräuchle, C.* AU - Mayer, M.P.* AU - Buchner, J.* AU - Issels, R.D. AU - Nößner, E. C1 - 3633 C2 - 24769 SP - 31688-31702 TI - Human heat shock protein 70 enhances tumor antigen presentation through complex formation and intracellular antigen delivery without innate immune signaling. JO - J. Biol. Chem. VL - 282 IS - 43 PB - ASBMB PY - 2007 SN - 0021-9258 ER - TY - JOUR AB - Mutations in the PARK7 gene encoding DJ-1 cause autosomal recessive Parkinson disease. The most deleterious point mutation is the L166P substitution, which resides in a structure motif comprising two alpha-helices (G and H) separated by a kink. Here we subjected the C-terminal helix-kink-helix motif to systematic site-directed mutagenesis, introducing helix-incompatible proline residues as well as conservative substitutions into the helical interface. Furthermore, we generated deletion mutants lacking the H-helix, the kink, and the entire C terminus. When transfected into neural and nonneural cell lines, steady-state levels of G-helix breaking and kink deletion mutants were dramatically lower than wild-type DJ-1. The effects of H-helix breakers were comparably smaller, and the non-helix breaking mutants only slightly destabilized DJ-1. The decreased steady-state levels were due to accelerated protein degradation involving in part the proteasome. G-helix breaking DJ-1 mutations abolished dimer formation. These structural perturbations had functional consequences on the cytoprotective activities of DJ-1. The destabilizing mutations conferred reduced cytoprotection against H(2)O(2) in transiently retransfected DJ-1 knock-out mouse embryonic fibroblasts. The loss of survival promoting activity of the DJ-1 mutants with destabilizing C-terminal mutations correlated with impaired anti-apoptotic signaling. We found that wild-type, but not mutant DJ-1 facilitated the Akt pathway and simultaneously blocked the apoptosis signal-regulating kinase 1, with which DJ-1 interacted in a redox-dependent manner. Thus, the G-helix and kink are critical determinants of the C-terminal helix-kink-helix motif, which is absolutely required for stability and the regulation of survival-promoting redox signaling of the Parkinson disease-associated protein DJ-1. AU - Görner, K.* AU - Holtorf, E.* AU - Waak, J.* AU - Pham, T.T. AU - Vogt Weisenhorn, D.M. AU - Wurst, W. AU - Haass, C.* AU - Kahle, P.J.* C1 - 2210 C2 - 24855 SP - 13680-13691 TI - Structural determinants of the C-terminal helix-kink-helix motif essential for protein stability and survival promoting activity of DJ-1. JO - J. Biol. Chem. VL - 282 IS - 18 PB - ASBMB PY - 2007 SN - 0021-9258 ER - TY - JOUR AB - VP16 has been widely used to unravel the mechanisms underlying gene transcription. Much of the previous work has been conducted in reconstituted in vitro systems. Here we study the formation of transcription complexes at stable reporters under the control of an inducible Tet-VP16 activator in living cells. In this simplified model for gene activation VP16 recruits the general factors and the cofactors Mediator, GCN5, CBP, and PC4, within minutes to the promoter region. Activation is accompanied by only minor changes in histone acetylation and H3K4 methylation but induces a marked promoter-specific increase in H3K79 methylation. Mediated through contacts with VP16 several subunits of the cleavage and polyadenylation factor (CPSF/CstF) are concentrated at the promoter region. We provide in vitro and in vivo evidence that VP16 activates transcription through a specific MED25-associated Mediator, which is deficient in CDK8. AU - Uhlmann, T. AU - Boeing, S. AU - Lehmbacher, M. AU - Meisterernst, M. C1 - 2891 C2 - 24390 SP - 2163-2173 TI - The VP16 activation domain establishes an active mediator lacking CDK8 in vivo. JO - J. Biol. Chem. VL - 282 PB - ASBMB PY - 2007 SN - 0021-9258 ER - TY - JOUR AB - DNA damage induced by the topoisomerase I inhibitor irinotecan (CPT-11) triggers in p53WT colorectal carcinoma cells a long term cell cycle arrest and in p53MUT cells a transient arrest followed by apoptosis (Magrini, R., Bhonde, M. R., Hanski, M. L., Notter, M., Scherübl, H., Boland, C. R., Zeitz, M., and Hanski, C. (2002) Int. J. Cancer 101, 23-31; Bhonde, M. R., Hanski, M. L., Notter, M., Gillissen, B. F., Daniel, P. T., Zeitz, M., and Hanski, C. (2006) Oncogene 25, 165-175). The mechanism of the p53-independent apoptosis still remains largely unclear. Here we used five p53WT and five p53MUT established colon carcinoma cell lines to identify gene expression alterations associated with apoptosis in p53MUT cells after treatment with SN-38, the irinotecan metabolite. After treatment, 16 mitosis-related genes were found to be expressed at least 2-fold stronger in the apoptosis-executing p53MUT cells than in the cell cycle-arrested p53WT cells by oligonucleotide microarray analysis. One of the genes whose strong post-treatment expression was associated with apoptosis was the mitotic checkpoint kinase hMps1 (human ortholog of the yeast monopolar spindle 1 kinase). hMps1 mRNA and protein expression were suppressed by the treatment-induced and by the exogenous adenovirus-coded p53 protein. The direct suppression of hMps1 on RNA level or inhibition of its activity by a dominant-negative hMps1 partly suppressed apoptosis. Together, these data indicate that the high expression of mitotic genes in p53 MUT cells after SN-38 treatment contributes to DNA damage-induced apoptosis, whereas their suppression in p53WT cells acts as a safeguard mechanism preventing mitosis initiation and the subsequent apoptosis. hMps1 kinase is one of the mitotic checkpoint proteins whose expression after DNA damage in p53 MUT cells activates the checkpoint and contributes to apoptosis. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. AU - Bhonde, M.R.* AU - Hanski, M.L.* AU - Budczies, J. AU - Cao, M.* AU - Gillissen, B.* AU - Moorthy, D.* AU - Simonetta, F.* AU - Scherübl, H.* AU - Truss, M.* AU - Hagemeier, C.* AU - Mewes, H.-W. AU - Daniel, P.T.* AU - Zeitz, M.* AU - Hanski, C.* C1 - 2890 C2 - 24033 SP - 8675-8685 TI - DNA damage-induced expression of p53 suppresses mitotic checkpoint kinase hMps1. JO - J. Biol. Chem. VL - 281 IS - 13 PB - ASBMB PY - 2006 SN - 0021-9258 ER - TY - JOUR AB - Mutation in CDC48 (cdc48(S565G)), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48(S565G) yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48(S565G) cells. Mitochondrial dysfunction in the cdc48(S565G) strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis. AU - Braun, R.J. AU - Zischka, H. AU - Madeo, F.* AU - Eisenberg, T.* AU - Wissing, S.* AU - Büttner, S.* AU - Engelhardt, S.M.* AU - Büringer, D.* AU - Ueffing, M. C1 - 2039 C2 - 23915 SP - 25757-25767 TI - Crucial mitochondrial impairment upon CDC48 mutation in apoptotic yeast. JO - J. Biol. Chem. VL - 281 IS - 35 PB - ASBMB PY - 2006 SN - 0021-9258 ER - TY - JOUR AB - Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase gamma. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network. AU - Duvezin-Caubet, S.* AU - Jagasia, R. AU - Wagener, J.* AU - Hofmann, S.* AU - Trifunovic, A.* AU - Hansson, A.* AU - Chomyn, A.* AU - Bauer, M.F.* AU - Attardi, G.* AU - Larsson, N.G.* AU - Neupert, W.* AU - Reichert, A.S.* C1 - 1921 C2 - 25072 SP - 37972 TI - Proteolytic processing of OPA1 links mitochondrial dysfunction to alterations in mitochondrial morphology. JO - J. Biol. Chem. VL - 281 IS - 49 PB - ASBMB PY - 2006 SN - 0021-9258 ER - TY - JOUR AB - Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis. AU - Guo, K.* AU - Lukacik, P.* AU - Papagrigoriou, E.* AU - Meier, M. AU - Lee, W.H.* AU - Adamski, J. AU - Oppermann, U.* C1 - 2806 C2 - 23528 SP - 10291-10297 TI - Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme. JO - J. Biol. Chem. VL - 281 IS - 15 PB - ASBMB PY - 2006 SN - 0021-9258 ER - TY - JOUR AB - Mutations in the gene encoding the glycosyltransferase polypeptide GalNAc-T3, which is involved in initiation of O-glycosylation, were recently identified as a cause of the rare autosomal recessive metabolic disorder familial tumoral calcinosis (OMIM 211900). Familial tumoral calcinosis is associated with hyperphosphatemia and massive ectopic calcifications. Here, we demonstrate that the secretion of the phosphaturic factor fibroblast growth factor 23 (FGF23) requires O-glycosylation, and that GalNAc-T3 selectively directs O-glycosylation in a subtilisin-like proprotein convertase recognition sequence motif, which blocks processing of FGF23. The study suggests a novel posttranslational regulatory model of FGF23 involving competing O-glycosylation and protease processing to produce intact FGF23. AU - Kato, K.* AU - Jeanneau, C.* AU - Tarp, M.A.* AU - Benet-Pagès, A. AU - Lorenz-Depiereux, B. AU - Bennett, E.P.* AU - Mandel, U.* AU - Strom, T.M. AU - Clausen, H.* C1 - 2038 C2 - 23673 SP - 18370-18377 TI - Polypeptide GaINAc-transferase T3 and familial tumoral calcinosis. JO - J. Biol. Chem. VL - 281 IS - 27 PB - ASBMB PY - 2006 SN - 0021-9258 ER - TY - JOUR AB - In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, whereas MAT2 and MAT3 were not significantly affected. Cys-114 of MAT1 was identified as the most promising target of NO-induced inhibition of MAT1, because this residue is absent in MAT2 and MAT3. Structural analysis of MAT1 revealed that Cys-114 is located nearby the putative substrate binding site of this enzyme. Furthermore, Cys-114 is flanked by S-nitrosylation-promoting amino acids. The inhibitory effect of GSNO was drastically reduced when Cys-114 of MAT1 was replaced by arginine, and mass spectrometric analyses of Cys-114-containing peptides obtained after chymotryptic digestion demonstrated that Cys-114 of MAT1 is indeed S-nitrosylated. Because MAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling. AU - Lindermayr, C. AU - Saalbach, G.* AU - Bahnweg, G. AU - Durner, J. C1 - 2807 C2 - 23584 SP - 4285-4291 TI - Differential inhibition of Arabidopsis methionine adenosyltransferases by protein S-nitrosylation. JO - J. Biol. Chem. VL - 281 IS - 7 PB - ASBMB PY - 2006 SN - 0021-9258 ER - TY - JOUR AB - Signal-anchored proteins are a class of mitochondrial outer membrane proteins that expose a hydrophilic domain to the cytosol and are anchored to the membrane by a single transmembrane domain in the N-terminal region. Like the vast majority of mitochondrial proteins, signal-anchored proteins are synthesized on cytosolic ribosomes and are subsequently imported into the organelle. We have studied the mechanisms by which precursors of these proteins are recognized by the mitochondria and are inserted into the outer membrane. The import of signal-anchored proteins was found to be independent of the known import receptors, Tom20 and Tom70, but to require the major Tom component, Tom40. In contrast to precursors destined to internal compartments of mitochondria and those of outer membrane beta-barrel proteins, precursors of signal-anchored proteins appear not to be inserted via the general import pore. Taken together, we propose a novel pathway for insertion of these proteins into the outer membrane of mitochondria. AU - Ahting, U. AU - Waizenegger, T.* AU - Neupert, W.* AU - Rapaport, D.* C1 - 4963 C2 - 22491 SP - 48-53 TI - Signal-anchored proteins follow a unique insertion pathway into the outer membrane of mitochondria. JO - J. Biol. Chem. VL - 280 IS - 1 PB - ASBMB PY - 2005 SN - 0021-9258 ER - TY - JOUR AB - Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence. AU - Gilfillan, S. AU - Stelzer, G. AU - Piaia, E. AU - Hofmann, M.G. AU - Meisterernst, M. C1 - 628 C2 - 22893 SP - 6222-6230 TI - Efficient binding of NC2-TATA-binding protein to DNA in the absence of TATA. JO - J. Biol. Chem. VL - 280 IS - 7 PB - ASBMB PY - 2005 SN - 0021-9258 ER - TY - JOUR AB - A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis. AU - Hamimes, S.* AU - Arakawa, H. AU - Stasiak, A.Z.* AU - Kierek, A.M.* AU - Hirano, S.* AU - Yang, Y.-G.* AU - Takata, M.* AU - Buerstedde, J.-M. AU - van Dyck, E.* C1 - 4962 C2 - 22378 SP - 9225-9235 TI - RDM1, a novel RNA-recognition MOTIF (RRM)-containing protein involved in the cell response to cisplatin in vertebrates. JO - J. Biol. Chem. VL - 280 IS - 10 PB - ASBMB PY - 2005 SN - 0021-9258 ER - TY - JOUR AB - The transcription factors of the Myc/Max/Mad network play essential roles in the regulation of cellular behavior. Mad1 inhibits cell proliferation by recruiting an mSin3-corepressor complex that contains histone deacetylase activity. Here we demonstrate that Mad1 is a potent inhibitor of the G1 to S phase transition, a function that requires Mad1 to heterodimerize with Max and to bind to the corepressor complex. Cyclin E/CDK2, but not cyclin D and cyclin A complexes, fully restored S phase progression. In addition inhibition of colony formation and gene repression by Mad1 were also efficiently antagonized by cyclin E/CDK2. This was the result of cyclin E/CDK2 interfering with the interaction of Mad1 with HDAC1 and reducing HDAC activity. Our findings define a novel interplay between the cell cycle regulator cyclin E/CDK2 and Mad1 and its associated repressor complex and suggests an additional mechanism how cyclin E/CDK2 affects the G1 to S phase transition. AU - Rottmann, S.* AU - Menkel, A.R. AU - Bouchard, J.* AU - Loidl, P.* AU - Kremmer, E. AU - Eilers, M.* AU - Luscher-Firzlaff, J.* AU - Lilischkis, R.* AU - Lüscher, B.* C1 - 629 C2 - 26266 SP - 15489-15492 TI - Mad1 function in cell proliferation and transcriptional repression is antagonized by cyclin E/CDK2. JO - J. Biol. Chem. VL - 280 IS - 16 PB - ASBMB PY - 2005 SN - 0021-9258 ER - TY - JOUR AB - Transport of nuclear encoded proteins into mitochondria is mediated by multisubunit translocation machineries in the outer and inner membranes of mitochondria. The TOM complex contains receptor and pore components that facilitate the recognition of preproteins and their transfer through the outer membrane. In addition, the complex contains a set of small proteins. Tom7 and Tom6 have been found in Neurospora and yeast, Tom5 has been found so far only in the latter organism. In the present study, we identified Neurospora Tom5 and analyzed its function in comparison to yeast Tom5, which has been proposed to play a role as a receptor-like component. Neurospora Tom5 crosses the outer membrane with its carboxyl terminus facing the intermembrane space like the other small Tom components. The temperature-sensitive growth phenotype of the yeast TOM5 deletion was rescued by overexpression of Neurospora Tom5. On the other hand, Neurospora cells deficient in tom5 did not exhibit any defect in growth. The structural stability of TOM complexes from cells devoid of Tom5 was significantly altered in yeast but not in Neurospora. The efficiency of protein import in Neurospora mitochondria was not affected by deletion of tom5, whereas in yeast it was reduced as compared with wild type. We conclude that the main role of Tom5, rather than being a receptor, is maintaining the structural integrity of the TOM complex. AU - Schmitt, S.* AU - Ahting, U. AU - Eichacker, L.* AU - Granvogl, B.* AU - Go, N.E.* AU - Nargang, F.E.* AU - Neupert, W.* AU - Nussberger, S.* C1 - 23549 C2 - 31361 SP - 14499-14506 TI - Role of TOM5 in maintaining the structural stability of the TOM complex of mitochondria. JO - J. Biol. Chem. VL - 280 IS - 15 PB - AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. PY - 2005 SN - 0021-9258 ER - TY - JOUR AB - The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential. AU - Rubio-Aliaga, I.* AU - Boll, M.* AU - Vogt Weisenhorn, D.M. AU - Foltz, M.* AU - Kottra, G.* AU - Daniel, H.* C1 - 3823 C2 - 21734 SP - 2754-2760 TI - The proton/amino acid cotransporter PAT2 is expressed in neurons with a different subcellular localization than its paralog PAT1. JO - J. Biol. Chem. VL - 279 IS - 4 PB - ASBMB PY - 2004 SN - 0021-9258 ER - TY - JOUR AB - The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPAΔp14, consisting of p70 and p32. RPAΔp14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPAΔp14 complex p32 only minimally recognized the 3′-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPAΔp14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase α-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication. AU - Weisshart, K.* AU - Pestryakov, P.* AU - Smith, R.W.P.* AU - Hartmann, H.* AU - Kremmer, E. AU - Lavrik, O.* AU - Nasheuer, H.-P.* C1 - 2182 C2 - 22100 SP - 35368-35376 TI - Coordinated regulation of replication protein A activities by its subunits p14 and p32. JO - J. Biol. Chem. VL - 279 PB - ASBMB PY - 2004 SN - 0021-9258 ER - TY - JOUR AB - Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the non-receptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP alpha. PTP alpha becomes activated in vivo after TPA stimulation. Further, we show that PKC delta phosphorylates and activates only PTP alpha in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC delta have a markedly reduced PTP alpha and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP alpha by PKC delta, which in turn dephosphorylates and activates Src. AU - Brandt, D.T.* AU - Goerke, A.* AU - Heuer, M.* AU - Gimona, M.* AU - Leitges, M.* AU - Kremmer, E. AU - Lammers, R.* AU - Haller, H.* AU - Mischak, H.* C1 - 9418 C2 - 21422 SP - 34073-34078 TI - Protein kinase Cdelta Src kinase activity via activation of the protein tyrosine phosphatase PTPalpha. JO - J. Biol. Chem. VL - 278 IS - 36 PY - 2003 SN - 0021-9258 ER - TY - JOUR AB - Prion diseases are neurodegenerative infectious disorders for which no prophylactic regimens are known. In order to induce antibodies/auto-antibodies directed against surface-located PrP(c), we used a covalently linked dimer of mouse prion protein expressed recombinantly in Escherichia coli. Employing dimeric PrP as an immunogen we were able to effectively overcome autotolerance against murine PrP in PrP wild-type mice without inducing obvious side effects. Treatment of prion-infected mouse cells with polyclonal anti-PrP antibodies generated in rabbit or auto-antibodies produced in mice significantly inhibited endogenous PrP(Sc) synthesis. We show that polyclonal antibodies are binding to surface-located PrP(c), thereby interfering with prion biogenesis. This effect is much more pronounced in the presence of full IgG molecules, which, unlike Fab fragments, seem to induce a significant cross-linking of surface PrP. In addition, we found immune responses against different epitopes when comparing antibodies induced in rabbits and PrP wild-type mice. Only in the auto-antibody situation in mice an immune reaction against a region of PrP is found that was reported to be involved in the PrP(Sc) conversion process. Our data point to the possibility of developing means for an active immunoprophylaxis against prion diseases. AU - Gilch, S.* AU - Wopfner, F.* AU - Renner-Müller, I.* AU - Kremmer, E. AU - Bauer, C.* AU - Wolf, E.* AU - Brem, G.* AU - Groschup, M.H.* AU - Schatzl, H.M.* C1 - 9419 C2 - 21432 SP - 18524-18531 TI - Polyclonal anti-PrP auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion-infected cells. JO - J. Biol. Chem. VL - 278 IS - 20 PY - 2003 SN - 0021-9258 ER - TY - JOUR AB - There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-kappaB and C/EBPbeta, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production. Under these conditions neither activation of NF-kappaB nor IkappaB proteolysis was affected after TNF re-stimulation, albeit a slightly reduced IkappaB-alpha level was found in the TNF pretreated but not re-stimulated sample. Remarkably, in tolerant cells an increased binding of C/EBPbeta to its IL-8 promoter-specific DNA motif as well as an elevated association of C/EBPbeta protein with p65-containing NF-kappaB complexes was observed. Finally, overexpression of C/EBPbeta, but not p65 or Oct-1, markedly prevented TNF-induced IL-8 promoter-dependent transcription. Taken together, these data indicate that the expression of IL-8 is inhibited at the transcriptional level in TNF-tolerant cells and C/EBPbeta is involved under these conditions in mediating the negative-regulatory effects, a mechanism that may play a role in inflammatory processes such as sepsis. AU - Weber, M.* AU - Sydlik, C.* AU - Quirling, M.* AU - Nothdurfter, C.* AU - Zwergal, A.* AU - Heiss, P.* AU - Bell, S.* AU - Neumeier, D.* AU - Ziegler-Heitbrock, L. AU - Brand, K.* C1 - 8780 C2 - 21246 SP - 23586-23593 TI - Transcriptional Inhibition of Interleukin-8 Expression in Tumor Necrosis Factor-tolerant Cells. JO - J. Biol. Chem. VL - 278 IS - 26 PY - 2003 SN - 0021-9258 ER - TY - JOUR AB - Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process. AU - Hardy, S.* AU - Brand, M.* AU - Mittler, G. AU - Yanagisawa, J.* AU - Kato, S.* AU - Meisterernst, M. AU - Tora, L.* C1 - 21959 C2 - 20481 SP - 32875-32882 TI - TATA-binding Protein-free TAF-containing Complex (TFTC) and p300 Are Both Required for Efficient Transcriptional Activation. JO - J. Biol. Chem. VL - 277 IS - 36 PY - 2002 SN - 0021-9258 ER - TY - JOUR AB - Human heat-shock protein (HSP)70 activates innate immune cells and hence requires no additional adjuvants to render bound peptides immunogenic. Here we tested the assumption that endogenous HSP70 activates the Toll/IL-1 receptor signal pathway similar to HSP60 and pathogen-derived molecular patterns. We show that HSP70 induces interleukin-12 (IL-12) and endothelial cell-leukocyte adhesion molecule-1 (ELAM-1) promoters in macrophages and that this is controlled by MyD88 and TRAF6. Furthermore, HSP70 causes MyD88 relocalization and MyD88-deficient dendritic cells do not respond to HSP70 with proinflammatory cytokine production. Using the system of genetic complementation with Toll-like receptors (TLR) we found that TLR2 and TLR4 confer responsiveness to HSP70 in 293T fibroblasts. The expanding list of endogenous ligands able to activate the ancient Toll/IL-1 receptor signal pathway is in line with the danger hypothesis" proposing that the innate immune system senses danger signals even if they originate from self." AU - Vabulas, R.M.* AU - Ahmad-Nejad, P.* AU - Ghose, S.* AU - Kirschning, C.J.* AU - Issels, R.D. AU - Wagner, H.* C1 - 10107 C2 - 20273 SP - 15107-15112 TI - HSP70 as Endogenous Stimulus of the Tol/Interleukin-1 Receptor Signal Pathway. JO - J. Biol. Chem. VL - 277 IS - 17 PB - ASBMB PY - 2002 SN - 0021-9258 ER - TY - JOUR AB - Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species. AU - Latunde-Dada, A.O.* AU - Cabello-Hurtado, F.* AU - Czittrich, N.* AU - Didierjean, L.* AU - Schopfer, Ch.* AU - Hertkorn, N. AU - Werck-Reichhart, D.* AU - Ebel, J.* C1 - 10106 C2 - 19663 SP - 1688-1695 TI - Flavonoid 6-Hydroxylase from Soybean (Glycine max L.), a Novel Plant P-450 Monooxygenase. JO - J. Biol. Chem. VL - 276 IS - 3 PB - American Society for Biochemistry and Molecular Biology PY - 2001 SN - 0021-9258 ER - TY - JOUR AB - The beta(2) integrin LFA-1 is an important cell-cell adhesion receptor of the immune system. Evidence suggests that the molecule also participates in signaling and co-stimulatory function. We show here that clustering of the intracellular domain of the beta(2) chain but not of the alpha(L)- or beta(1)-cytoplasmic domains, respectively, triggers intracellular Ca(2+) mobilization in Jurkat cells. A beta(2)-specific NPXF motif, located in the C-terminal portion of the beta(2) tail, is required for Ca(2+) signaling, and we show that this motif is important for the induction of allo-specific target cell lysis by cytotoxic T cells in vitro. Significantly, the Ca(2+)-signaling capacity of the beta(2) integrin is abrogated in T cells that do not express the T cell receptor but may be reconstituted by co-expression of the T cell receptor-zeta chain. Our data suggest a specific function of the cytoplasmic domain of the beta(2) integrin chain in T cell signaling. AU - Sirim, P.* AU - Zeitlmann, L.* AU - Kellersch, B.* AU - Falk, C.S. AU - Schendel, D.J. AU - Kolanus, W.* C1 - 21753 C2 - 19949 SP - 42945-42956 TI - Calcium Signaling through the beta2-Cytoplasmic Domain of LFA-1 Requires Intracellular Elements of the T Cell Receptor Complex. JO - J. Biol. Chem. VL - 276 IS - 46 PY - 2001 SN - 0021-9258 ER - TY - JOUR AB - Membrane proteins are known to be solvated and functionally activated by a fixed number of lipid molecules whose multiple binding can be described by Adair-type binding equations. Lipophilic xenobiotics such as general anesthetics may act by competitive displacement of protein-bound lipids. A kinetic equation is now presented for various binding stoichiometries of lipid and xenobiotic, and microscopic binding constants of anesthetics and organic solvents are derived from two independent assay systems for the enhancement of agonist binding to the nicotinic acetylcholine receptor. These constants lead to the first available free energy estimate (−6.4 kcal/mol) for the binding of membrane lipid to an integral membrane protein. AU - Walcher, S. AU - Altschuh, J. AU - Sandermann, H. C1 - 21844 C2 - 20049 SP - 42191-42195 TI - The Lipid/Protein Interface as Xenobiotic Target Site : Kinetic Analysis of the Nicotinic Acetylcholine Receptor. JO - J. Biol. Chem. VL - 276 PY - 2001 SN - 0021-9258 ER - TY - JOUR AB - CD4 recruitment to T cell receptor (TCR)-peptide-major histocompatibility class II complexes is required for stabilization of low affinity antigen recognition by T lymphocytes. The cytoplasmic portion of CD4 is thought to amplify TCR-initiated signal transduction via its association with the protein tyrosine kinase p56(lck). Here we describe a novel functional determinant in the cytosolic tail of CD4 that inhibits TCR-induced T cell activation. Deletion of two conserved hydrophobic amino acids from the CD4 carboxyl terminus resulted in a pronounced enhancement of CD4-mediated T cell costimulation. This effect was observed in the presence or absence of p56(lck), implying involvement of alternative cytosolic ligands of CD4. A two-hybrid screen with the intracellular portion of CD4 identified a previously unknown 33-kDa protein, ACP33 (acidic cluster protein 33), as a novel intracellular binding partner of CD4. Since interaction with ACP33 is abolished by deletion of the hydrophobic CD4 C-terminal amino acids mediating repression of T cell activation, we propose that ACP33 modulates the stimulatory activity of CD4. Furthermore, we demonstrate that interaction with CD4 is mediated by the noncatalytic alpha/beta hydrolase fold domain of ACP33. This suggests a previously unrecognized function for alpha/beta hydrolase fold domains as a peptide binding module mediating protein-protein interactions. AU - Zeitlmann, L.* AU - Sirim, P.* AU - Kremmer, E. AU - Kolanus, W.* C1 - 22184 C2 - 20888 SP - 9123-9132 TI - Cloning of ACP33 as a Novel Intracellular Ligand of CD4. JO - J. Biol. Chem. VL - 276 IS - 12 PY - 2001 SN - 0021-9258 ER - TY - JOUR AB - The Yku heterodimer from Saccharomyces cerevisiae, comprising Yku70p and Yku80p, is involved in the maintenance of a normal telomeric DNA end structure and is an essential component of nonhomologous end joining (NHEJ). To investigate the role of the Yku70p subunit in these two different pathways, we generated C-terminal deletions of the Yku70 protein and examined their ability to complement the phenotypes of a yku70(-) strain. Deleting only the 30 C-terminal amino acids of Yku70p abolishes Yku DNA binding activity and causes a yku(-) phenotype; telomeres are shortened, and NHEJ is impaired. Using conditions in which at least as much mutant protein as full-length protein is normally detectable in cell extracts, deleting only 25 C-terminal amino acids of Yku70p results in no measurable effect on DNA binding of the Yku protein, and the cells are fully proficient for NHEJ. Nevertheless, these cells display considerably shortened telomeres, and significant amounts of single-stranded overhangs of the telomeric guanosine-rich strands are observed. Co-overexpression of this protein with Yku80p could rescue some but not all of the telomere-related phenotypes. Therefore, the C-terminal domain in Yku70p defines at least one domain that is especially involved in telomere maintenance but not in NHEJ. AU - Driller, L.* AU - Wellinger, R.J.* AU - Larrivee, M.* AU - Kremmer, E. AU - Jaklin, S.* AU - Feldmann, H.M.* C1 - 21603 C2 - 19734 SP - 24921-24927 TI - A short C-terminal domain of Yku70p is essential for telomere maintenance. JO - J. Biol. Chem. VL - 275 IS - 32 PY - 2000 SN - 0021-9258 ER - TY - JOUR AB - DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively. AU - Weisshart, K.* AU - Förster, H.* AU - Kremmer, E. AU - Schlott, B.* AU - Grosse, F.* AU - Nasheuer, H.-P.* C1 - 21609 C2 - 19740 SP - 17328-17337 TI - Protein-Protein interactions of the Primase Subunits p58 and p48 with Simian Virus 40 T Antigen are Required for Efficient Primer Synthesis in a Cell-free System. JO - J. Biol. Chem. VL - 275 IS - 23 PY - 2000 SN - 0021-9258 ER - TY - JOUR AB - The Epstein-Barr virus-encoded nuclear antigens EBNA2 and EBNA3C both interact with the cellular transcription factor RBP-Jkappa and modulate the expression of several shared target genes, suggesting a tight cooperation in latently infected cells. In a survey for additional cellular factors that bind to EBNA2 as well as EBNA3C, we have isolated and characterized DP103, a novel human member of the DEAD box family of putative ATP-dependent RNA helicases. The interaction with DP103 is mediated by amino acids (aa) 121-213 of EBNA2 and aa 534-778 of EBNA3C, regions that are not involved in binding of the viral proteins to RBP-Jkappa. The DP103-cDNA encodes a protein of 824 aa that harbors all of the common DEAD box motifs. Monoclonal antibodies raised against DP103 detect a protein of 103 kDa in mammalian cells that resides in high molecular weight complexes in vivo. We have detected an ATPase activity intrinsic to or closely associated with DP103. By subcellular fractionation, we find DP103 in both a soluble nuclear fraction as well as in the insoluble skeletal fraction. Whereas the protein and its mRNA are uniformly expressed in all tested cell lines, we observed differential expression of the mRNA in normal human tissues. AU - Grundhoff, A.T.* AU - Kremmer, E. AU - Türeci, Ö.* AU - Glieden, A.* AU - Gindorf, C.* AU - Atz, J.* AU - Müller-Lantsch, N.* AU - Schubach, W.H.* AU - Grässer, F.A.* C1 - 21236 C2 - 19344 SP - 19136-19144 TI - Characterization of DP103, a Novel DEAD Box Protein that Binds to the Epstein-Barr Virus Nuclear Proteins EBNA2 and EBNA3C. JO - J. Biol. Chem. VL - 274 IS - 27 PY - 1999 SN - 0021-9258 ER - TY - JOUR AB - The NF-kappaB transcription factor is activated by a wide variety of stimuli, including phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate. In its inactive state, NF-kappaB is sequestered in the cytoplasm tethered to an inhibitor protein, IkappaB. Activation comprises the rapid phosphorylation of IkappaB-alpha at N-terminal sites, which presumably marks IkappaB-alpha for proteolytic degradation and leads to release of NF-kappaB into the nucleus. In addition, IkappaB-alpha is constitutively phosphorylated at the C terminus, which may be a prerequisite for proper IkappaB function. Protein kinase C (PKC) is activated by 12-O-tetradecanoylphorbol-13-acetate and has been previously reported to phosphorylate IkappaB-alpha in vitro. As PKC has turned out to constitute a multigene family encoding isozymes with different biological functions, we have reinvestigated IkappaB-alpha phosphorylation by PKC using recombinant PKC isozymes expressed in insect cells. While crude PKC preparations were efficient IkappaB-alpha kinases, highly purified PKC isozymes completely failed to phosphorylate IkappaB-alpha. Biochemical separation of porcine spleen yielded at least two fractions with IkappaB-alpha kinase activity, both of which were devoid of detectable PKC isozymes. One peak contained both Raf-1 and casein kinase II (CKII). Purified Raf-1 does not phosphorylate IkappaB-alpha directly, but associates with CKII, which efficiently phosphorylates the C terminus of IkappaB-alpha. Two-dimensional phosphopeptide mapping and high pressure liquid chromatography-mass spectroscopy analysis showed that all IkappaB-alpha kinases induced phosphorylation at the same prominent sites in the C terminus. Our results clearly indicate that PKC isozymes alpha, beta, gamma, delta, epsilon, eta, and zeta as well as Raf-1 are not IkappaB-alpha kinases. They furthermore demonstrate that IkappaB-alpha is targeted by several kinases, one of which appears to be CKII. AU - Janosch, P. AU - Schellerer, M. AU - Seitz, T. AU - Reim, P. AU - Eulitz, M. AU - Brielmeier, M. AU - Kolch, W. AU - Sedivy, J.M.* AU - Mischak, H. C1 - 20749 C2 - 17289 SP - 13868-13874 TI - Characterization of IkappaB kinases - IkappaB-a is not phosphorylated by Raf-1 or protein kinase C isozymes, but is a casein kinase II substrate. JO - J. Biol. Chem. VL - 271 IS - 23 PY - 1996 SN - 0021-9258 ER - TY - JOUR AB - It is generally accepted that the multiple, similar protein kinase C (PKC) isoq mes are responsible for different specialized physiol < gical processes, but evidence that directly assigns specific functions to specific isozymes is scarce. To test whether specific PKC isozymes are involved in myeloid differentiation, we have studied the effect of overexpression of PKC-a, -011, -6, -6, -( and -q in 32D, a mouse myeloid progenitor cell line that does not differentiate in response to 12-0-tetradecanoylphorbol-13-acetate (TPA). No significant morphological or phenotypic changes could be observed in unstimulated cells that overexpress any of these isozymes. However, the cell lines that overexpressed PKC-a or -6 had acquired the ability to become mature macrophages 2-6 h after TPA stimulation. The overexpression of PKC-BII, -6, -f, or -q, in contrast, did not permit TPA-induced differentiation. These results indicate that only these two members of the PKC gene family can participate in TPA-induced myeloid differentiation AU - Mischak, H. AU - Pierce, J.H. AU - Goodnight, J. AU - Kazanietz, M.G. AU - Blumberg, P.M. AU - Mushinski, J.F. C1 - 20612 C2 - 13823 SP - 20110-20115 TI - Phorbol Ester-induced Myeloid Differentiation is Mediated by Protein Kinase C-Alpha and-Delta and not by Protein Kinase C-BetaII, -Epsilon, -Zeta, and -Eta. JO - J. Biol. Chem. VL - 268 IS - 27 PY - 1993 SN - 0021-9258 ER - TY - JOUR AB - It is generally accepted that the multiple, similar protein kinase C (PKC) isozymes are responsible for different specialized physiological processes, but evidence that directly assigns specific functions to specific isozymes is scarce. To test whether specific PKC isozymes are involved in myeloid differentiation, we have studied the effect of overexpression of PKC-α, -βII, -δ, -ε, -ζ and -η in 32D, a mouse myeloid progenitor cell line that does not differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). No significant morphological or phenotypic changes could be observed in unstimulated cells that overexpress any of these isozymes. However, the cell lines that overexpressed PKC-α or -δ had acquired the ability to become mature macrophages 2-6 h after TPA stimulation. The overexpression of PKC-βII, -ε, -ζ, or -η, in contrast, did not permit TPA-induced differentiation. These results indicate that only these two members of the PKC gene family can participate in TPA-induced myeloid differentiation. AU - Mischak, H. AU - Pierce, J.H.* AU - Goodnight, J.* AU - Kazanietz, M.G.* AU - Blumberg, P.M.* AU - Mushinski, J.F.* C1 - 33562 C2 - 38971 SP - 20110-20115 TI - Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-α and -δ and not by protein kinase C-βII, -ε, -ζ, and -η. JO - J. Biol. Chem. VL - 268 IS - 27 PY - 1993 SN - 0021-9258 ER - TY - JOUR AB - Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis. AU - Ozawa, K. AU - Szallasi, Z. AU - Kazanietz, M.G. AU - Blumberg, P.M. AU - Mischak, H. AU - Mushinski, J.F. AU - Beaven, M.A. C1 - 20557 C2 - 13763 SP - 1749-1756 TI - CA(2+)-Dependent and Ca(2+)-Independent Isozymes of Protein Kinase C Mediate Exocytosis in Antigen-Stimulated Rat Basophilic RBL-2H3 Cells. JO - J. Biol. Chem. VL - 268 IS - 3 PY - 1993 SN - 0021-9258 ER - TY - JOUR AB - Mouse bone marrow-derived mast cells (BMMC) store and release serotonin whose synthesis is initiated by tryptophan 5-monooxygenase. (6R)-H4biopterin serves as the natural cofactor for this reaction. GTP cyclohydrolase I catalyzes the first and rate-limiting step of its synthesis. In this study we demonstrate that among a panel of growth-promoting cytokines including kit ligand (KL), interleukin 3 (IL-3), IL-4, IL-9, and nerve growth factor, KL selectively enhances the synthesis of H4biopterin through up-regulation of GTP cyclohydrolase I activity to 6.2-fold levels. The activities of the subsequent enzymes 6-pyruvoyl-H4pterin synthase and sepiapterin reductase remain unaffected. The activity of tryptophan 5-monooxygenase was selectively enhanced 4.5-fold by the combination of IL-3 with KL. All other factors could not substitute for KL. The constitutive high activity of aromatic L-amino acid decarboxylase is not different in cells cultured in IL-3 and/or KL. In consequence, the concerted action of IL-3 and KL on the GTP cyclohydrolase I and the tryptophan 5-monooxygenase reaction enhances the production of serotonin to about 20-fold levels. Additionally, KL specifically causes the release of about half of total serotonin produced. Hence, our data demonstrate a novel role of these cytokines for the function of mouse BMMC and provide a coherent view of the regulation of serotonin synthesis in this cell type. AU - Ziegler, I. AU - Hültner, L. AU - Egger, D. AU - Kempkes, B. AU - Kailhammer, R. AU - Gillis, S. AU - Rödl, W. C1 - 20464 C2 - 13672 SP - 12544-12551 TI - In a Concerted Action kit Ligand and Interleukin 3 Control the Synthesis of Serotonin in Murine Bone Marrow-derived Mast Cells. JO - J. Biol. Chem. VL - 268 IS - 17 PY - 1993 SN - 0021-9258 ER - TY - JOUR AB - Mouse bone marrow-derived mast cells (BMMC) store and release serotonin whose synthesis is initiated by tryptophan 5-monooxygenase. (6R)-H4biopterin serves as the natural cofactor for this reaction. GTP cyclohydrolase I catalyzes the first and rate-limiting step of its synthesis. In this study we demonstrate that among a panel of growth-promoting cytokines including kit ligand (KL), interleukin 3 (IL-3), IL-4, IL-9, and nerve growth factor, KL selectively enhances the synthesis of H4biopterin through up-regulation of GTP cyclohydrolase I activity to 6.2-fold levels. The activities of the subsequent enzymes 6-pyruvoyl-H4pterin synthase and sepiapterin reductase remain unaffected. The activity of tryptophan 5-monooxygenase was selectively enhanced 4.5-fold by the combination of IL-3 with KL. All other factors could not substitute for KL. The constitutive high activity of aromatic L-amino acid decarboxylase is not different in cells cultured in IL-3 and/or KL. In consequence, the concerted action of IL-3 and KL on the GTP cyclohydrolase I and the tryptophan 5-monooxygenase reaction enhances the production of serotonin to about 20-fold levels. Additionally, KL specifically causes the release of about half of total serotonin produced. Hence, our data demonstrate a novel role of these cytokines for the function of mouse BMMC and provide a coherent view of the regulation of serotonin synthesis in this cell type. AU - Ziegler, I. AU - Hültner, L. AU - Egger, D. AU - Kempkes, B. AU - Mailhammer, R. AU - Gillis, S.* AU - Rödl, W. C1 - 40431 C2 - 40052 SP - 12544-12551 TI - In a concerted action kit ligand and interleukin 3 control the synthesis of serotonin in murine bone marrow-derived mast cells: Up-regulation of GTP cyclohydrolase I and tryptophan 5-monooxygenase activity by the kit ligand. JO - J. Biol. Chem. VL - 268 IS - 17 PY - 1993 SN - 0021-9258 ER - TY - JOUR AB - We have characterized and analyzed IGF-I- and insulin-stimulated cell growth, receptor binding, and autophosphorylation in the human leukemic cell line HL-60. IGF-I-stimulated cell growth occurred at low (5 ng/ml) and insulin stimulated only at high (500 ng/ml) concentrations. Binding of 125I-IGF-I to partially purified plasma membrane proteins followed the characteristics of IGF-I receptor binding. 125I-IGF-I binding, as determined by chemical cross-linking, occurred to a 145-kDa protein. IGF-I, as well as insulin, stimulated the autophosphorylation of a 105-kDa band (pp105), but we could not detect a 95-kDa band corresponding to the known molecular mass of the IGF-I and insulin receptor β-subunits. Phosphorylation of pp105 followed the dose-response characteristics of the IGF-I receptor. The phosphorylation of pp105 occurred at tyrosine and threonine, and the pattern of HPLC tryptic peptide maps showed marked differences when compared with that of a phosphorylated insulin receptor β-subunit. Enzymatic deglycosylation of pp105 resulted only in a slight reduction of the molecular weight. These data suggest that pp105 is the β-subunit of an IGF-I receptor variant with a higher molecular weight, similar to that found in fetal tissue. The HL-60 cell may aquire, at least in part, malignant growth characteristics through reexpression of the fetal version of the IGF-I receptor. AU - Kellerer, M.* AU - Obermaier-Kusser, B.* AU - Ermel, B.* AU - Wallner, U. AU - Häring, H.-U.* AU - Petridest, P.E. C1 - 42172 C2 - 40150 SP - 9340-9345 TI - An altered IGF-I receptor is present in human leukemic cells. JO - J. Biol. Chem. VL - 265 IS - 16 PY - 1990 SN - 0021-9258 ER - TY - JOUR AB - The control of (6A)-5,6,7,8-tetrahydrobiopterin (H4biopterin) synthesis in primed T cells was analyzed by using the human T cell leukemia virus type I (HTLV-I)-transformed T cell line MT-2. In contrast to the slowly progressing induction of H4biopterin synthesis during activation of resting T cells, it is completed during a 59-h period and is directed by a synergism of interferon-γ (IFN-γ) and interleukin-2 (IL-2). Both GTP cyclohydrolase and (6A)-(1′,2′-dioxopropyl)-5,6,7,8-tetrahydropterin synthase activities are induced by IFN-γ. They are further enhanced by combined treatment with IL-2, which per se is ineffective. Furthermore, the combined treatment synchronizes the time periods of both maximum activities, now extending from 33 to 44 h. This period correlates with high cellular H4biopterin levels. It is preceded by a fast and transient period of H4biopterin increase which depends on the synergistic action of both IFN-γ and IL-2. It coincides with a transient increase in sepiapterin reductase activity. In contrast to MT-2 cells, HTLV-I-transformed HUT 102 cells constitutively secrete IFN-γ and express IFN-γ mRNA. The accumulation of H4biopterin is suppressed by anti-IFN-γ polyclonal antibody and correlates with constitutive expression of all H4 biopterin-synthesizing enzymes. AU - Ziegler, I. AU - Schott, K. AU - Lübbert, M.* AU - Herrmann, F.* AU - Schwulera, U.* AU - Bacher, A.* C1 - 42035 C2 - 40140 SP - 17026-17030 TI - Control of tetrahydrobiopterin synthesis in T lymphocytes by synergistic action of interferon-γ and interleukin-2. JO - J. Biol. Chem. VL - 265 IS - 28 PY - 1990 SN - 0021-9258 ER - TY - JOUR AB - Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent K(m) for trehalose of 4.7 ± 0.1 mM and a V(max) of 99 μmol of trehalose min-1 x mg-1 at 37°C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum. AU - Mittenbühler, K. AU - Holzer, H. C1 - 41623 C2 - 36160 SP - 8537-8543 TI - Purification and characterization of acid trehalase from the yeast suc2 mutant. JO - J. Biol. Chem. VL - 263 IS - 17 PY - 1988 SN - 0021-9258 ER - TY - JOUR AB - Light-dark-cycled rats were fed a 3% cholesterol-supplemented diet at the beginning of the dark phase. Cholesterol-fed and control animals were taken at intervals throughout the following 12 h and the microsomal and solubilized hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated. Immunotitrations of this microsomal and solubilized enzyme were performed with a monospecific antibody to 3-hydroxy-3-methylglutaryl coenzyme A reductase. In contrast to the specific activity of the enzyme, which differs extremely during the diurnal cycle, the immunotitrations obtained from cholesterol-fed and control animals, yielded in identical antisera equivalence points. On the other hand, when the enzyme was phosphorylated in vitro, the antisera equivalence points corresponded to the alterations of the specific activity. In contrast to the results published by Higgins and Rudney, our data prove that even the in vivo short term changes in enzyme activity are due to changes in the quantity of enzyme rather than to a modulation of the catalytic activity. AU - Jenke, H.S. AU - Loewel, M. AU - Berndt, J. C1 - 33292 C2 - 38607 SP - 9622-9625 TI - In vivo effect of cholesterol feeding on the short term regulation of hepatic hydroxymethylglutaryl coenzyme A reductase during the diurnal cycle. JO - J. Biol. Chem. VL - 256 IS - 18 PY - 1981 SN - 0021-9258 ER - TY - JOUR AB - A heat-stable protein has been detected in Saccharomyces cerevisiae which inhibits mitochondrial ATPase activity. The protein inhibitor has been isolated from extracts prepared by brief heat treatment of unbroken cell suspensions. The isolated inhibitor is a small basic protein (molecular weight close to 7000, isoelectric proint 9.05) devoid of tryptophan, tyrosine, and cysteine as well as proline. The NHP2-terminal amino acid is serine. The ultraviolet absorption spectrum shows the vibrational fine structure of the phenyl-alanine band. Like the ATPase inhibitor from bovine heart mitochondria the yeast inhibitor is rapidly destroyed by trypsin. It is also inactivated by the yeast proteinases A and B. Radioimmunological analysis indicates that the inhibitor is synthesized on cytoplasmic ribosomes. Its accumulation seems to be connected to the formation of the mitochondrial ATPase complex, since its specific activity is greatly reduced both in extracts obtained from the F1-ATPase-deficient nuclear mutant pet 936 and from the cytoplasmic petite mutant D 273-10B-1. AU - Ebner, E. AU - Maier, K.L. C1 - 33079 C2 - 35279 SP - 671-676 TI - A protein inhibitor of mitochondrial adenosine triphosphatase (F1) from Saccharomyces cerevisiae. JO - J. Biol. Chem. VL - 252 IS - 2 PY - 1977 SN - 0021-9258 ER -