TY - JOUR AB - Abstract: Astrocytes are the main homeostatic cells in the central nervous system (CNS) that provide mechanical, metabolic, and trophic support to neurons. Disruption of their physiological role or acquisition of senescence-associated phenotype can contribute to the CNS dysfunction and pathology. However, molecular mechanisms underlying the complex physiology of astrocytes are explored insufficiently. Recent studies have shown that miRNAs are involved in the regulation of astrocyte function through different mechanisms. Although miR-21 has been reported as an astrocytic miRNA with an important role in astrogliosis, no link between this miRNA and cellular senescence of astrocytes has been identified. To address the role of miR-21 in astrocytes, with special focus on cellular senescence, we used NT2/A (astrocytes derived from NT2/D1 cells). Downregulation of miR-21 expression in both immature and mature NT2/A by the antisense technology induced the arrest of cell growth and premature cellular senescence, as indicated by senescence hallmarks such as increased expression of cell cycle inhibitors p21 and p53 and augmented senescence-associated β-galactosidase activity. Additionally, in silico analysis predicted many of the genes, previously shown to be upregulated in astrocytes with the irradiation-induced senescence, as miR-21 targets. Taken together, our results point to miR-21 as a potential regulator of astrocyte senescence. To the best of our knowledge, these are the first data showing the link between miR-21 and cellular senescence of astrocytes. Since senescent astrocytes are associated with different CNS pathologies, development of novel therapeutic strategies based on miRNA manipulation could prevent senescence and may improve the physiological outcome. AU - Balint, V.* AU - Stanisavljevic Ninkovic, D.* AU - Anastasov, N. AU - Lazic, S.* AU - Kovacevic-Grujicic, N.* AU - Stevanovic, M.* AU - Lazic, A.* C1 - 63549 C2 - 51582 CY - 233 Spring St, New York, Ny 10013-1578 Usa SP - 1434-1445 TI - Inhibition of miR-21 promotes cellular senescence in NT2-derived astrocytes. JO - Biochemistry VL - 86 IS - 11 PB - Maik Nauka/interperiodica/springer PY - 2021 SN - 0006-2960 ER - TY - JOUR AU - Hadian, K. C1 - 58198 C2 - 48155 CY - 1155 16th St, Nw, Washington, Dc 20036 Usa SP - 637-638 TI - Ferroptosis suppressor protein 1 (FSP1) and coenzyme Q(10) cooperatively suppress ferroptosis. JO - Biochemistry VL - 59 IS - 5 PB - Amer Chemical Soc PY - 2020 SN - 0006-2960 ER - TY - JOUR AB - Recent pharmacological findings regarding rimonabant, an anorectic and cannabinoid type 1 receptor (CB1R) antagonist, strongly suggest that some of its effects on the metabolic parameters and energy balance in rats are not related to the centrally mediated reduction in caloric intake. Instead, they may be associated with acute induction of glycogenolysis in the liver, in combination with transient increase in glucose oxidation and persistent increase in fat oxidation. It is possible that rimonabant produced direct short- or long-term stimulatory effect on these processes in primary and cultured rat cells. Rimonabant slightly stimulated β-oxidation of long-chain fatty acids in cultured rat myocytes overexpressing glucose transporter isoform 4, as well as activated phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK) in primary rat hepatocytes upon long-term incubation. However, short-term action of rimonabant failed to stimulate β-oxidation in myocytes, myotubes, and hepatocytes, as well as to upregulate AMPK phosphorylation, glycogenolysis, and cAMP levels in hepatocytes. As a consequence, the acute effects of rimonabant on hepatic glycogen content (reduction) and total energy expenditure (increase) in rats fed with a standard diet cannot be explained by direct stimulation of glycogenolysis and fatty acid oxidation in muscles and liver. Rather, these effects seem to be centrally mediated. AU - Müller, G. AU - Wied, S.* AU - Herling, A.W.* C1 - 56778 C2 - 47345 CY - 233 Spring St, New York, Ny 10013-1578 Usa SP - 954-962 TI - Analysis of direct effects of the CB1 receptor antagonist rimonabant on fatty acid oxidation and glycogenolysis in liver and muscle cells in vitro. JO - Biochemistry VL - 84 IS - 8 PB - Maik Nauka/interperiodica/springer PY - 2019 SN - 0006-2960 ER - TY - JOUR AB - Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged beta 2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling. AU - Perkins, L.A.* AU - Yan, Q.* AU - Schmidt, B.F.* AU - Kolodieznyi, D.* AU - Saurabh, S.* AU - Larsen, M.B.* AU - Watkins, S.C.* AU - Kremer, L.S. AU - Bruchez, M.P.* C1 - 52973 C2 - 44395 CY - Washington SP - 861-871 TI - Genetically targeted ratiometric and activated pH indicator complexes (TRApHIC) for receptor trafficking. JO - Biochemistry VL - 57 IS - 5 PB - Amer Chemical Soc PY - 2018 SN - 0006-2960 ER - TY - JOUR AB - The heat shock protein 90 (Hsp90) family plays a critical role in maintaining the homeostasis of the intracellular environment for human and prokaryotic cells. Hsp90 orthologues were identified as important target proteins for cancer and plant disease therapies. It was shown that gambogic acid (GBA) has the potential to inhibit human Hsp90. However, it is unknown whether it is also able to act on the bacterial high-temperature protein (HtpG) analogue. In this work, we screened GBA and nine other novel potential Hsp90 inhibitors using a miniaturized high-throughput protein microarray-based assay and found that GBA shows an inhibitory effect on different Hsp90s after dissimilarity analysis of the protein sequence alignment. The dissociation constant of GBA and HtpG Xanthomonas (XcHtpG) computed from microscale thermophoresis is 682.2 ± 408 μM in the presence of ATP, which is indispensable for the binding of GBA to XcHtpG. Our results demonstrate that GBA is a promising Hsp90/HtpG inhibitor. The work further demonstrates that our assay concept has great potential for finding new potent Hsp/HtpG inhibitors. AU - Yue, Q.* AU - Stahl, F.R.* AU - Plettenburg, O. AU - Kirschning, A.* AU - Warnecke, A.* AU - Zeilinger, C.* C1 - 53567 C2 - 44751 SP - 2601-2605 TI - The noncompetitive effect of gambogic acid displaces fluorescence-labeled ATP but requires ATP for binding to Hsp90/HtpG. JO - Biochemistry VL - 57 IS - 18 PY - 2018 SN - 0006-2960 ER - TY - JOUR AB - Alzheimer's disease is characterized by deposition of the amyloid β-peptide (Aβ) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the Non-Steroidal Anti-Inflammatory Drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aβ peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aβ solution. We find that sulindac sulfide induced aggregates are structurally homogeneous. The C-terminal part of the peptide adopts β-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in the wild type fibril structure. 13C-19F TEDOR experiments suggest that sulindac sulfide co-localizes with the Aβ peptide in the aggregate. AU - Prade, E.* AU - Barucker, C.* AU - Sarkar, R.* AU - Althoff-Ospelt, G.* AU - Lopez del Amo, J.M.* AU - Hossain, S.* AU - Zhong, Y.* AU - Multhaup, G.* AU - Reif, B. C1 - 48007 C2 - 39839 CY - Washington SP - 1839-1849 TI - Sulindac sulfide induces the formation of large oligomeric aggregates of the Alzheimer's disease amyloid-β peptide which exhibit reduced neurotoxicity. JO - Biochemistry VL - 55 IS - 12 PB - Amer Chemical Soc PY - 2016 SN - 0006-2960 ER - TY - JOUR AB - More than 100 distinct mutations in the gene CuZnSOD encoding human copper-zinc superoxide dismutase (CuZnSOD) have been associated with familial amyotrophic lateral sclerosis (fALS), a fatal neuronal disease. Many studies of different mutant proteins have found effects on protein stability, catalytic activity, and metal binding, but without a common pattern. Notably, these studies were often performed under conditions far from physiological. Here, we have used experimental conditions of pH 7 and 37 °C and at an ionic strength of 0.2 M to mimic physiological conditions as close as possible in a sample of pure protein. Thus, by using NMR spectroscopy, we have analyzed amide hydrogen exchange of the fALS-associated I113T CuZnSOD variant in its fully metalated state, both at 25 and 37 °C, where 15N relaxation data, as expected, reveals that CuZnSOD I113T exists as a dimer under these conditions. The local dynamics at 82% of all residues have been analyzed in detail. When compared to the wild-type protein, it was found that I113T CuZnSOD is particularly destabilized locally at the ion binding sites of loop 4, the zinc binding loop, which results in frequent exposure of the aggregation prone outer β-strands I and VI of the β-barrel, possibly enabling fibril or aggregate formation. A similar study (Museth, A. K., et al. (2009) Biochemistry, 48, 8817-8829) of amide hydrogen exchange at pH 7 and 25 °C on the G93A variant also revealed a selective destabilization of the zinc binding loop. Thus, a possible scenario in ALS is that elevated local dynamics at the metal binding region can result in toxic species from formation of new interactions at local β-strands. AU - Hennig, J. AU - Andrésen, C.* AU - Museth, A.K.* AU - Lundström, P.* AU - Tibell, L.A.E.* AU - Jönsson, B.H.* C1 - 43379 C2 - 36624 CY - Washington SP - 323-333 TI - Local destabilization of the metal-binding region in human copper-zinc superoxide dismutase by remote mutations is a possible determinant for progression of ALS. JO - Biochemistry VL - 54 IS - 2 PB - Amer Chemical Soc PY - 2015 SN - 0006-2960 ER - TY - JOUR AB - Wilson's disease is a human genetic disorder which results in copper accumulation in liver and brain. Treatments such as copper chelation therapy or dietary supplementation with zinc can ameliorate the effects of the disease, but if left untreated, it results in hepatitis, neurological complications, and death. Tetrathiomolybdate (TTM) is a promising new treatment for Wilson's disease which has been demonstrated both in an animal model and in clinical trials. X-ray absorption spectroscopy suggests that TTM acts as a novel copper chelator, forming a complex with accumulated copper in liver. We have used X-ray absorption spectroscopy and X-ray fluorescence imaging to trace the molecular form and distribution of the complex in liver and kidney of an animal model of human Wilson's disease. Our work allows new insights into metabolism of the metal complex in the diseased state. AU - Zhang, L.M.* AU - Lichtmannegger, J. AU - Summer, K.H. AU - Webb, S.* AU - Pickering, I.J.* AU - George, G.N.* C1 - 1510 C2 - 25965 SP - 891-897 TI - Tracing copper-thiomolybdate complexes in a prospective treatment for Wilson's disease. JO - Biochemistry VL - 48 IS - 5 PB - Amer Chemical Soc PY - 2009 SN - 0006-2960 ER - TY - JOUR AB - The nonspecific lipid transfer protein sterol carrier protein 2 (SCP2) is involved in organellar fatty acid metabolism. A hydrophobic cavity in the structure of SCP2 accommodates a wide variety of apolar ligands such as cholesterol derivatives or fatty acyl-coenzyme A (CoA) conjugates. The properties of this nonspecific lipid binding pocket are explored using NMR chemical shift perturbations, paramagnetic relaxation enhancement, amide hydrogen exchange, and 15N relaxation measurements. A common binding cavity shared by different physiological ligands is identified. NMR relaxation measurements reveal that residues in the three C-terminal alpha-helices within the lipid binding region exhibit mobility at fast (picosecond to nanosecond) and slow (microsecond to millisecond) time scales. Ligand binding is associated with a considerable loss of peptide backbone mobility. The observed conformational dynamics in SCP2 may play a role for the access of hydrophobic ligands to an occluded binding pocket. The C-terminal peroxisomal targeting signal of SCP2 is specifically recognized by the Pex5p receptor protein, which conducts cargo proteins toward the peroxisomal organelle. Neither the C-terminal targeting signal nor the N-terminal precursor sequence interferes with lipid binding by SCP2. The alpha-helices involved in lipid binding also mediate a secondary interaction interface with the Pex5p receptor. Silencing of conformational dynamics of the peptide backbone in these helices upon either lipid or Pex5p binding might communicate the loading state of the cargo protein to the targeting receptor. AU - Filipp, F.V.* AU - Sattler, M. C1 - 2431 C2 - 24822 SP - 7980-7991 TI - Conformational plasticity of the lipid transfer protein SCP2. JO - Biochemistry VL - 46 IS - 27 PB - ACS PY - 2007 SN - 0006-2960 ER - TY - JOUR AB - This work describes the synthesis and activity of a novel backbone cyclic (BC) peptide library based on the sequence of the HIV-1 Rev arginine-rich motif (ARM). All the peptides in the library possess the same sequence but differ in their ring-moiety properties. The BC peptides were synthesized using simultaneous multiple-peptide synthesis and were fully assembled using bis(trichloromethyl)carbonate as a coupling agent. All the peptides in the library had inhibitory effects on the binding of Rev-GFP to importin β in vitro. Studies performed with one of the BC Rev-ARM analogues, Rev-13, demonstrated that, like its parental linear peptide, it is karyophilic; i.e., it is able to mediate the nuclear import of conjugated bovine serum albumin (BSA) molecules. The cell penetrating properties of the BC peptides were assessed utilizing an ELISA-based system. This assay provides a quantitative evaluation of cell penetration. Most of the peptides from the library were able to penetrate intact Colo-205 cells to varying degrees. Furthermore, these BC peptides were able to carry BSA into intact Colo-205 cells. In addition to its cell penetrating and binding properties, the BC Rev-13 analogue inhibited Rev-induced gene expression in HeLa cells by 60−70% in the low micromolar range and exhibited no cell toxicity. The potential of BC peptides bearing ARM domains as lead compounds for the production of anti-HIV drugs is discussed. AU - Hariton-Gazal, E.* AU - Rosenbluh, J.* AU - Zakai, N.* AU - Fridkin, G.* AU - Brack-Werner, R. AU - Wolff, H. AU - Devaux, C.* AU - Gilon, C.* AU - Loyter, A.* C1 - 3024 C2 - 22831 SP - 11555-11566 TI - Functional analysis of backbone cyclic peptides bearing the arm domain of the HIV-1 Rev protein: Characterization of the karyophilic and inhibition of Rev-induced gene expression. JO - Biochemistry VL - 44 IS - 34 PY - 2005 SN - 0006-2960 ER - TY - JOUR AB - Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S413. At nontoxic concentrations, fluorescently labeled S413 was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S413. The resulting peptides, which were designated as PV-S413 and RR-S413 penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S413 and the RR-S413 peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 °C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S413 remained in the cytoplasm while microinjected RR-S413 was translocated into the cells' nuclei. The new type of cell-permeable “karyophilic” peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus. AU - Hariton-Gazal, E.* AU - Feder, R.* AU - Graessmann, A.* AU - Brack-Werner, R. AU - Jans, D.* AU - Gilon, C.* AU - Loyter, A.* C1 - 21988 C2 - 20518 SP - 9208-9214 TI - Targeting of Nonkaryophilic Cell-Permeable Peptides into the Nuclei of Intact Cells by Covalently Attached Nuclear Localization Signals. JO - Biochemistry VL - 41 IS - 29 PY - 2002 SN - 0006-2960 ER -