TY - JOUR AB - Lagunamide A is a biologically active natural product with a yet unidentified molecular mode of action. Cellular studies revealed that lagunamide A is a potent inhibitor of cancer cell proliferation, promotes apoptosis and causes mitochondrial dysfunction. To decipher the cellular mechanism responsible for these effects, we utilized thermal protein profiling (TPP) and identified EYA3 as a stabilized protein in cells upon lagunamide A treatment. EYA3, involved in the DNA damage repair process, was functionally investigated via siRNA based knockdown studies and corresponding effects of lagunamide A on DNA repair were confirmed. Furthermore, we showed that lagunamide A sensitized tumor cells to treatment with the drug doxorubicin highlighting a putative therapeutic strategy. AU - Hu, Y.* AU - Mostert, D.* AU - Orgler, C.* AU - Andler, O.* AU - Zischka, H. AU - Kazmaier, U.* AU - Vollmar, A.* AU - Braig, S.* AU - Sieber, S.* AU - Zahler, S.* C1 - 70664 C2 - 55810 CY - Postfach 101161, 69451 Weinheim, Germany TI - Thermal proteome profiling reveals insight to antiproliferative and pro-apoptotic effects of Lagunamide A in the modulation of DNA damage repair. JO - ChemBioChem PB - Wiley-v C H Verlag Gmbh PY - 2024 SN - 1439-4227 ER - TY - JOUR AB - Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414 ). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance. AU - Mötzing, M.* AU - Blüher, M. AU - Grunwald, T.* AU - Hoffmann, R.* C1 - 68704 C2 - 54913 CY - Postfach 101161, 69451 Weinheim, Germany TI - Immunological quantitation of the glycation site lysine-414 in serum albumin in human plasma samples by indirect ELISA using highly specific monoclonal antibodies. JO - ChemBioChem PB - Wiley-v C H Verlag Gmbh PY - 2023 SN - 1439-4227 ER - TY - JOUR AB - G protein-coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein-stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope-labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist-induced activity of the receptor. Furthermore, this assay can be used as a readout when re-introducing agonist-dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively F-19-labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis. AU - Goba, I. AU - Goricanec, D.* AU - Schum, D.* AU - Hillenbrand, M.* AU - Plückthun, A.* AU - Hagn, F. C1 - 60558 C2 - 49419 CY - Postfach 101161, 69451 Weinheim, Germany SP - 139-146 TI - Probing the conformation states of neurotensin receptor 1 variants by NMR site-directed methyl labeling. JO - ChemBioChem VL - 22 IS - 5 PB - Wiley-v C H Verlag Gmbh PY - 2021 SN - 1439-4227 ER - TY - JOUR AB - The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP-driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N-terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD−MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD−MD arrangement with the accessibility of a client binding site on the NTD. AU - Lopez, A. AU - Elimelech, A.R.* AU - Klimm, K.* AU - Sattler, M. C1 - 60752 C2 - 49515 CY - Postfach 101161, 69451 Weinheim, Germany TI - The charged linker modulates the conformations and molecular interactions of Hsp90. JO - ChemBioChem PB - Wiley-v C H Verlag Gmbh PY - 2021 SN - 1439-4227 ER - TY - JOUR AB - Positron emission tomography (PET) tracer molecules like thioflavin T specifically recognize amyloid deposition in brain tissue by selective binding to hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The molecular basis of this interaction is, however, not well understood. We have employed magic angle spinning (MAS) solid-state NMR spectroscopy to characterize Aβ-PET tracer complexes at atomic resolution. We established a titration protocol by using bovine serum albumin as a carrier to transfer hydrophobic small molecules to Aβ(1-40) fibrillar aggregates. The same Aβ(1-40) amyloid fibril sample was employed in subsequent titrations to minimize systematic errors that potentially arise from sample preparation. In the experiments, the small molecules 13C-methylated Pittsburgh compound B (PiB) as well as a novel Aβ tracer based on a diarylbithiazole (DABTA) scaffold were employed. Classical 13C-detected as well as proton-detected spectra of protonated and perdeuterated samples with back-substituted protons, respectively, were acquired and analyzed. After titration of the tracers, chemical-shift perturbations were observed in the loop region involving residues Gly25-Lys28 and Ile32-Gly33, thus suggesting that the PET tracer molecules interact with the loop region connecting β-sheets β1 and β2 in Aβ fibrils. We found that titration of the PiB derivatives suppressed fibril polymorphism and stabilized the amyloid fibril structure. AU - Niu, Z. AU - Sarkar, R. AU - Aichler, M. AU - Wester, H.* AU - Yousefi, B.H.* AU - Reif, B. C1 - 59213 C2 - 48729 CY - Postfach 101161, 69451 Weinheim, Germany SP - 2495-2502 TI - Mapping of the binding interface of PET tracer molecules and Alzheimer Disease Aβ fibrils using MAS solid-state NMR spectroscopy. JO - ChemBioChem VL - 21 IS - 17 PB - Wiley-v C H Verlag Gmbh PY - 2020 SN - 1439-4227 ER - TY - JOUR AB - Chi-Huey Wong turned 70 in August 2018: This special issue is dedicated to that event. It can be seen from the variety of topics covered that he influenced the thinking of many of his former co-workers, who then transformed and developed these thoughts into fascinating, creative and independent research areas, enriching the science of bioorganic chemistry. AU - Plettenburg, O. C1 - 55122 C2 - 46210 CY - Postfach 101161, 69451 Weinheim, Germany SP - 129-130 TI - Milestones in bioorganic chemistry. JO - ChemBioChem VL - 20 IS - 2 PB - Wiley-v C H Verlag Gmbh PY - 2019 SN - 1439-4227 ER - TY - JOUR AB - Sorbicillinoids are fungal polyketides characterized by highly complex and diverse molecular structures, with considerable stereochemical intricacy combined with a high degree of oxygenation. Many sorbicillinoids possess promising biological activities. An interesting member of this natural product family is sorbicatecholA, which is reported to have antiviral activity, particularly against influenzaA virus (H1N1). Through a straightforward, one-pot chemoenzymatic approach with recently developed oxidoreductase SorbC, the characteristic bicyclo[2.2.2]octane core of sorbicatechol is structurally diversified by variation of its natural 2-methoxyphenol substituent. This facilitates the preparation of a focused library of structural analogues bearing substituted aromatic systems, alkanes, heterocycles, and ethers. Fast access to this structural diversity provides an opportunity to explore the antiviral potential of the sorbicatechol family. AU - Sib, A.* AU - Milzarek, T.M.* AU - Herrmann, A. AU - Oubraham, L.* AU - Müller, J.I.* AU - Pichlmair, A.* AU - Brack-Werner, R. AU - Gulder, T.A.M.* C1 - 57392 C2 - 47776 CY - Postfach 101161, 69451 Weinheim, Germany SP - 492-495 TI - Chemoenzymatic total synthesis of sorbicatechol structural analogues and evaluation of their antiviral potential. JO - ChemBioChem VL - 21 IS - 4 PB - Wiley-v C H Verlag Gmbh PY - 2019 SN - 1439-4227 ER - TY - JOUR AB - Phospholipid nanodiscs are a native-like membrane mimetic that is suitable for structural studies of membrane proteins. Although nanodiscs of different sizes exist for various structural applications, their thermal and long-term stability can vary considerably. Covalently circularized nanodiscs are a perfect tool to overcome these limitations. Existing methods for the production of circularized nanodiscs can be time-consuming and technically demanding. Therefore, an easy in vivo approach, in which circularized membrane scaffold proteins (MSPs) can be directly obtained from Escherichia coli culture, is reported herein. Nostoc punctiforme DnaE split-intein fusions with MSPs of various lengths are used and consistently provide circularized nanodiscs in high yields. With this approach, a large variety of circularized nanodiscs, ranging from 7 to 26 nm in diameter, that are suitable for NMR spectroscopy and electron microscopy (EM) applications can be prepared. These nanodiscs are superior to those of the corresponding linear versions in terms of stability and size homogeneity, which affects the quality of NMR spectroscopy data and EM experiments. Due to their long-term stability and homogeneity, the presented small circular nanodiscs are suited for high-resolution NMR spectroscopy studies, as demonstrated with two membrane proteins of 17 or 32 kDa in size. The presented method will provide easy access to circularized nanodiscs for structural studies of membrane proteins and for applications in which a defined and stable nanodisc size is required. AU - Miehling, J. AU - Goricanec, D. AU - Hagn, F. C1 - 54129 C2 - 45370 CY - Postfach 101161, 69451 Weinheim, Germany SP - 1927-1933 TI - A split-intein-based method for the efficient production of circularized nanodiscs for structural studies of membrane proteins. JO - ChemBioChem VL - 19 IS - 18 PB - Wiley-v C H Verlag Gmbh PY - 2018 SN - 1439-4227 ER - TY - JOUR AB - Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono- and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild-type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity. A mono- and diglyceride lipase was engineered to a triacylglyceride lipase by introducing a single point mutation (Q282L). The variant has broad substrate specificity on triacylglycerides. The results indicate that the main reason that the wild-type enzyme does not accept triacylglycerides is not their bulkiness, but specific hydrophobic interactions. AU - Lan, D.* AU - Popowicz, G.M. AU - Pavlidis, I.V.* AU - Zhou, P.* AU - Bornscheuer, U.T.* AU - Wang, Y.* C1 - 45144 C2 - 37278 CY - Weinheim SP - 1431-1434 TI - Conversion of a mono- and diacylglycerol lipase into a triacylglycerol lipase by protein engineering. JO - ChemBioChem VL - 16 IS - 10 PB - Wiley-v C H Verlag Gmbh PY - 2015 SN - 1439-4227 ER - TY - JOUR AB - The structures of oligomeric intermediate states in the aggregation process of Alzheimer's disease β-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimer's disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus mimics the conditions encountered in the cell under stress and allows the structures of Aβ aggregation intermediate states to be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all the requirements to be classified as amyloids: they seed fibril growth, are Congo red positive and show characteristic β-sheet-rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-β-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18-F19-F20 adopt β-sheet conformations, whereas the C termini adopt α-helical coiled-coil structures. In the past, an α-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which Aβ oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than Aβ42 fibrils. E. coli inclusion bodies formed by the Alzheimer's disease β-amyloid peptides Aβ40 and Aβ42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment. AU - Dasari, M.* AU - Espargaro, A.* AU - Sabate, R.* AU - Lopez del Amo, J.M.* AU - Fink, U.* AU - Grelle, G.* AU - Bieschke, J.* AU - Ventura, S.* AU - Reif, B. C1 - 3759 C2 - 28882 CY - Weinheim, Germany SP - 407-423 TI - Bacterial inclusion bodies of Alzheimer's disease: β-amyloid peptides can be employed to study native-like aggregation intermediate states. JO - ChemBioChem VL - 12 IS - 3 PB - Wiley-VCH PY - 2011 SN - 1439-4227 ER -