المؤلفون: Samuel D. McCalpin, Lucie Khemtemourian, Saba Suladze, Magdalena I. Ivanova, Bernd Reif, Ayyalusamy Ramamoorthy

المصدر: Communications Biology, Vol 7, Iss 1, Pp 1-12 (2024)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: Abstract Aggregation of the human islet amyloid polypeptide (hIAPP) contributes to the development and progression of Type 2 Diabetes (T2D). hIAPP aggregates within a few hours at few micromolar concentration in vitro but exists at millimolar concentrations in vivo. Natively occurring inhibitors of hIAPP aggregation might therefore provide a model for drug design against amyloid formation associated with T2D. Here, we describe the combined ability of low pH, zinc, and insulin to inhibit hIAPP fibrillation. Insulin dose-dependently slows hIAPP aggregation near neutral pH but had less effect on the aggregation kinetics at acidic pH. We determine that insulin alters hIAPP aggregation in two manners. First, insulin diverts the aggregation pathway to large nonfibrillar aggregates with ThT-positive molecular structure, rather than to amyloid fibrils. Second, soluble insulin suppresses hIAPP dimer formation, which is an important early aggregation event. Further, we observe that zinc significantly modulates the inhibition of hIAPP aggregation by insulin. We hypothesize that this effect arose from controlling the oligomeric state of insulin and show that hIAPP interacts more strongly with monomeric than oligomeric insulin.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2399-3642Test

الوصول الحر: https://doaj.org/article/76196303c8fc41bb85c71773c058e15dTest

المؤلفون: Jan Blahut, Matthias J. Brandl, Riddhiman Sarkar, Bernd Reif, Zdeněk Tošner

المصدر: Journal of Magnetic Resonance Open, Vol 16, Iss , Pp 100122- (2023)

مصطلحات موضوعية: Medical physics. Medical radiology. Nuclear medicine, R895-920, Physics, QC1-999

الوصف: We have recently introduced optimal-control derived pulse sequences for sensitivity-enhanced heteronuclear correlation NMR experiments of solid proteins. Preservation of equivalent coherence transfer pathways using transverse-mixing pulses (TROP) in multidimensional pulse schemes allows to increase the sensitivity of the experiments by more than a factor of 2 per each indirect dimension. In this article, we present homonuclear CA-CO transverse-mixing elements (homoTROP) that are based on dipolar interactions and achieve similar gains as the heteronuclear TROP pulses described previously. Both transfer elements were subsequently implemented in 3D se-hCAcoNH and se-hCOcaNH, that together with the previously introduced 3D se-hCANH and se-hCONH experiments yield a complete set of sensitivity-enhanced protein backbone assignment experiments. In contrast to the J-coupling based methods that are used at fast (60 kHz) and ultrafast MAS (>100 kHz), the homoTROP experiments employ about 10-times shorter mixing times making use of the larger magnitude of the dipolar coupling in comparison to the J couplings. The experiments are demonstrated using a microcrystalline, perdeuterated sample of the chicken alpha-spectrin SH3 domain in which all exchangeable sites are fully back-substituted with protons. We evaluated the gains in efficiency in all experiments site-specifically observing that the se-hCAcoNH and se-hCOcaNH experiments yield an increase in sensitivity by a factor of 1.36±0.09 and at least a factor of 1.8 with respect to the conventional hcoCAcoNH and hCOcaNH J-based experiments.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2666441023000304Test; https://doaj.org/toc/2666-4410Test

الوصول الحر: https://doaj.org/article/c6b2ec91dcf24b0a82744cef1367d4a3Test

المؤلفون: Tejaswini Pradhan, Riddhiman Sarkar, Kevin M. Meighen-Berger, Matthias J. Feige, Martin Zacharias, Bernd Reif

المصدر: Nature Communications, Vol 14, Iss 1, Pp 1-14 (2023)

مصطلحات موضوعية: Science

الوصف: Abstract Systemic antibody light chain (AL) amyloidosis is characterized by deposition of amyloid fibrils. Prior to fibril formation, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes. We focus on the patient derived λ-III AL variable domain FOR005 which is mutated at five positions with respect to the closest germline protein. Using solution-state NMR spectroscopy, we follow the individual steps involved in protein misfolding from the native to the amyloid fibril state. Unfavorable mutations in the complementary determining regions introduce a strain in the native protein structure which yields partial unfolding. Driven by electrostatic interactions, the protein converts into a high molecular weight, oligomeric, molten globule. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into fibrils. The topology is determined by balanced electrostatic interactions in the fibril core implying a 180° rotational switch of the beta-sheets around the conserved disulfide bond.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2041-1723Test

الوصول الحر: https://doaj.org/article/16a00071844d4eef9149de55112ae2ccTest

المؤلفون: Moritz Mühlhofer, Carsten Peters, Thomas Kriehuber, Marina Kreuzeder, Pamina Kazman, Natalia Rodina, Bernd Reif, Martin Haslbeck, Sevil Weinkauf, Johannes Buchner

المصدر: Nature Communications, Vol 12, Iss 1, Pp 1-14 (2021)

مصطلحات موضوعية: Science

الوصف: Small heat shock proteins (sHsps) form large spherical assemblies and their regulation is not well understood. Here, the authors provide insights into the mechanism of Hsp26 activation by characterising phospho-mimetic mutants of yeast Hsp26. They present cryo-EM structures of the wild-type Hsp26 40mer and its phospho-mimetic mutants that reveal the location of the thermosensor in the oligomer, and the authors also show that the thermosensor domain is targeted by phosphorylation, which relieves the intrinsic inhibition of chaperone activity.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2041-1723Test

الوصول الحر: https://doaj.org/article/ba936da34061418da4605d6f906d2c8aTest

المؤلفون: Arpita Sundaria, Falk Liberta, Dilan Savran, Riddhiman Sarkar, Natalia Rodina, Carsten Peters, Nadine Schwierz, Christian Haupt, Matthias Schmidt, Bernd Reif

المصدر: Journal of Structural Biology: X, Vol 6, Iss , Pp 100069- (2022)

مصطلحات موضوعية: Magic Angle Spinning (MAS), Solid-state NMR, Amyloid fibrils, Structure, Cryo electron microscopy (EM), Biology (General), QH301-705.5

الوصف: AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the β-sheets identified in the NMR experiments are similar to the β-sheets found in the cryo-EM study, with the exception of amino acids 33–42. These residues cannot be assigned by solid-state NMR, while they adopt a stable β-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33–42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion.

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2590152422000101Test; https://doaj.org/toc/2590-1524Test

الوصول الحر: https://doaj.org/article/e7a120bbadc8491d9c9f5041614681f5Test

المؤلفون: Christoph Göbl, Vanessa K. Morris, Loes van Dam, Marieke Visscher, Paulien E. Polderman, Christoph Hartlmüller, Hesther de Ruiter, Manuel Hora, Laura Liesinger, Ruth Birner-Gruenberger, Harmjan R. Vos, Bernd Reif, Tobias Madl, Tobias B. Dansen

المصدر: Redox Biology, Vol 28, Iss , Pp - (2020)

مصطلحات موضوعية: Medicine (General), R5-920, Biology (General), QH301-705.5

الوصف: The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils. Keywords: Amyloids, Protein aggregation, Redox signaling, Cysteine oxidation, Structural biology

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2213231719307918Test; https://doaj.org/toc/2213-2317Test

الوصول الحر: https://doaj.org/article/455304bdf2544f08bd2e21616c0f6c16Test

المؤلفون: William Close, Matthias Neumann, Andreas Schmidt, Manuel Hora, Karthikeyan Annamalai, Matthias Schmidt, Bernd Reif, Volker Schmidt, Nikolaus Grigorieff, Marcus Fändrich

المصدر: Nature Communications, Vol 9, Iss 1, Pp 1-7 (2018)

مصطلحات موضوعية: Science

الوصف: Amyloid fibril structures can display polymorphism. Here the authors reveal the cryo-EM structures of several different fibril morphologies of a peptide derived from an amyloidogenic immunoglobulin light chain and present a mathematical analysis of physical factors that influence fibril polymorphism.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2041-1723Test

الوصول الحر: https://doaj.org/article/0e804c5e89db443289443ff9451e6c39Test

المؤلفون: Kai Xue, Riddhiman Sarkar, Carina Motz, Sam Asami, Diana C. Rodriguez Camargo, Venita Decker, Sebastian Wegner, Zdenek Tosner, Bernd Reif

المصدر: Scientific Reports, Vol 7, Iss 1, Pp 1-7 (2017)

مصطلحات موضوعية: Medicine, Science

الوصف: Abstract MAS solid-state NMR is capable of determining structures of protonated solid proteins using proton-detected experiments. These experiments are performed at MAS rotation frequency of around 110 kHz, employing 0.5 mg of material. Here, we compare 1H, 13C correlation spectra obtained from protonated and deuterated microcrystalline proteins at MAS rotation frequency of 111 kHz, and show that the spectral quality obtained from deuterated samples is superior to those acquired using protonated samples in terms of resolution and sensitivity. In comparison to protonated samples, spectra obtained from deuterated samples yield a gain in resolution on the order of 3 and 2 in the proton and carbon dimensions, respectively. Additionally, the spectrum from the deuterated sample yields approximately 2–3 times more sensitivity compared to the spectrum of a protonated sample. This gain could be further increased by a factor of 2 by making use of stereospecific precursors for biosynthesis. Although the overall resolution and sensitivity of 1H, 13C correlation spectra obtained using protonated solid samples with rotation frequencies on the order of 110 kHz is high, the spectral quality is still poor when compared to the deuterated samples. We believe that experiments involving large protein complexes in which sensitivity is limiting will benefit from the application of deuteration schemes.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2045-2322Test

الوصول الحر: https://doaj.org/article/d086e727126b43d6b7851cd83e778236Test

المؤلفون: Diana C Rodriguez Camargo, Kyle J Korshavn, Alexander Jussupow, Kolio Raltchev, David Goricanec, Markus Fleisch, Riddhiman Sarkar, Kai Xue, Michaela Aichler, Gabriele Mettenleiter, Axel Karl Walch, Carlo Camilloni, Franz Hagn, Bernd Reif, Ayyalusamy Ramamoorthy

المصدر: eLife, Vol 6 (2017)

مصطلحات موضوعية: membrane, Amyloid peptide, Structure, NMR, Medicine, Science, Biology (General), QH301-705.5

الوصف: Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

وصف الملف: electronic resource

العلاقة: https://elifesciences.org/articles/31226Test; https://doaj.org/toc/2050-084XTest

الوصول الحر: https://doaj.org/article/83e9a3672fef478bbca4e26152d644a1Test

المؤلفون: Manuel Hora, Riddhiman Sarkar, Vanessa Morris, Kai Xue, Elke Prade, Emma Harding, Johannes Buchner, Bernd Reif

المصدر: PLoS ONE, Vol 12, Iss 7, p e0181799 (2017)

مصطلحات موضوعية: Medicine, Science

الوصف: Little structural information is available so far on amyloid fibrils consisting of immunoglobulin light chains. It is not understood which features of the primary sequence of the protein result in fibril formation. We report here MAS solid-state NMR studies to identify the structured core of κ-type variable domain light chain fibrils. The core contains residues of the CDR2 and the β-strands D, E, F and G of the native immunoglobulin fold. The assigned core region of the fibril is distinct in comparison to the core identified in a previous solid-state NMR study on AL-09 by Piehl at. al, suggesting that VL fibrils can adopt different topologies. In addition, we investigated a soluble oligomeric intermediate state, previously termed the alternatively folded state (AFS), using NMR and FTIR spectroscopy. The NMR oligomer spectra display a high degree of similarity when compared to the fibril spectra, indicating a high structural similarity of the two aggregation states. Based on comparison to the native state NMR chemical shifts, we suggest that fibril formation via domain-swapping seems unlikely. Moreover, we used our results to test the quality of different amyloid prediction algorithms.

وصف الملف: electronic resource

العلاقة: http://europepmc.org/articles/PMC5528828?pdf=renderTest; https://doaj.org/toc/1932-6203Test

الوصول الحر: https://doaj.org/article/8385323faf984c73a88bfdb03891255dTest