scholarly journals Structural dissection of the first events following membrane binding of the islet amyloid polypeptide

2021 ◽  
Author(s):  
Lucie Khemtemourian ◽  
Hebah Fatafta ◽  
Benoit Davion ◽  
Sophie Lecomte ◽  
Sabine Castano ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Intrinsically Disordered Proteins ◽  
Lipid Membrane ◽  
Islet Amyloid Polypeptide ◽  
Membrane Binding ◽  
Disordered Proteins ◽  
Helical Conformation ◽  
Islet Amyloid ◽  
Membrane Interaction ◽  
Intrinsically Disordered

Amyloid forming proteins are involved in many pathologies and often belong to the class of intrinsically disordered proteins. One of these proteins is the islet amyloid polypeptide (IAPP), which is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The molecular mechanism of IAPP-induced cell death is not yet understood, however it is known that the cell membrane plays a dual role, being a catalyst of IAPP aggregation and the target of IAPP toxicity. Using FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Both our experiments and simulations reveal distinct IAPP-membrane interaction modes for the various IAPP variants. Membrane binding causes IAPP to fold into an amphipathic helix, which in the case of H18K- and H18R-IAPP can easily insert below the lipid headgroups. For all IAPP variants but H18E-IAPP, the membrane-bound α-helical structure is an intermediate on the way to IAPP amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel β-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel β-sheets as well as amorphous aggregates. In summary, our results emphasize the importance of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good target for destabilizing amyloid fibrils of IAPP and inhibit its toxic actions by possible therapeutic molecules.

scholarly journals Hsp70 inhibits aggregation of Islet amyloid polypeptide by binding to the heterogeneous prenucleation oligomers

2020 ◽  
Author(s):  
Neeraja Chilukoti ◽  
Bankanidhi Sahoo ◽  
S Deepa ◽  
Sreelakshmi Cherakara ◽  
Mithun Maddheshiya ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Islet Amyloid Polypeptide ◽  
Correlation Spectroscopy ◽  
Size Exclusion ◽  
Disordered Proteins ◽  
Strong Interactions ◽  
Islet Amyloid ◽  
Intrinsically Disordered ◽  
Exclusion Chromatography ◽  
In The Beginning

AbstractMolecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, mechanism of the interactions of Hsp70 with the amyloidogenic intrinsically disordered proteins (IDPs) is not clear. Here, we use Hsp70 from different organisms to show that it inhibits aggregation of Islet amyloid polypeptide (IAPP) at substoichiometric concentrations even in absence of ATP. The effect is found to be the strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating role of Hsp70 in preventing primary nucleation possibly via interactions with the prefibrillar oligomers of IAPP. Fluorescence Correlation Spectroscopy (FCS) measurements of the solutions containing fluorescently labelled Hsp70 and IAPP exhibit fluorescence bursts suggesting formation of heterogeneous complexes of oligomeric IAPP binding to multiple molecules of Hsp70. Size exclusion chromatography and field flow fractionation are then used to fractionate the smaller complexes. Multiangle light scattering and FCS measurements suggest that these complexes comprise of monomers of Hsp70 and small oligomers of IAPP. However, concentration of the complexes is measured to be a few nanomolar amidst several μmolar of free Hsp70 and IAPP. Hence, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a common feature of the interactions between the chaperones and the amyloidogenic IDPs. While strong interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers avert interference with the functions of the IDPs.

scholarly journals Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

2020 ◽  
Vol 117 (21) ◽  
pp. 11421-11431 ◽  
Author(s):  
Benjamin S. Schuster ◽  
Gregory L. Dignon ◽  
Wai Shing Tang ◽  
Fleurie M. Kelley ◽  
Aishwarya Kanchi Ranganath ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Lipid Membrane ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Sequence Features ◽  
Intrinsically Disordered ◽  
Arginine Residues

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

scholarly journals NSP 11 of SARS-CoV-2 is an Intrinsically Disordered Protein

2020 ◽  
Author(s):  
Kundlik Gadhave ◽  
Prateek Kumar ◽  
Ankur Kumar ◽  
Taniya Bhardwaj ◽  
Neha Garg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Alpha Helix ◽  
Intrinsically Disordered Protein ◽  
Disordered Proteins ◽  
Helical Conformation ◽  
Structural Protein ◽  
Intrinsically Disordered ◽  
Protein Size

AbstractThe intrinsically disordered proteins/regions (IDPs/IDPRs) are known to be responsible for multiple cellular processes and are associated with many chronic diseases. In viruses, the existence of disordered proteome is also proven and are related with its conformational dynamics inside the host. The SARS-CoV-2 virus has a large proteome, in which, structure and functions of many proteins are not known as of yet. Previously, we have investigated the dark proteome of SARS-CoV-2. However, the disorder status of non-structural protein 11 (nsp11) was not possible because of very small in size, just 13 amino acid long, and for most of the IDP predictors, the protein size should be at least 30 amino acid long. Also, the structural dynamics and function status of nsp11 was not known. Hence, we have performed extensive experimentation on nsp11. Our results, based on the Circular dichroism spectroscopy gives characteristic disordered spectrum for IDPs. Further, we investigated the conformational behaviour of nsp11 in the presence of membrane mimetic environment, alpha helix inducer, and natural osmolyte. In the presence of negatively charged and neutral liposomes, nsp11 remains disordered. However, with SDS micelle, it adopted an α-helical conformation, suggesting the helical propensity of nsp11. At the end, we again confirmed the IDP behaviour of nsp11 using molecular dynamics simulations.

scholarly journals Structural dynamics and transient lipid binding of synaptobrevin-2 tune SNARE assembly and membrane fusion

2019 ◽  
Vol 116 (18) ◽  
pp. 8699-8708 ◽  
Author(s):  
Nils-Alexander Lakomek ◽  
Halenur Yavuz ◽  
Reinhard Jahn ◽  
Ángel Pérez-Lara
Keyword(s):  
Membrane Fusion ◽  
Intrinsically Disordered Proteins ◽  
Membrane Binding ◽  
Lipid Binding ◽  
Snare Complex ◽  
Snare Proteins ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Repulsive Forces

Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial. Here, by a combination of NMR and fluorescence spectroscopy methods, we find that vesicular synaptobrevin-2 (syb-2) in its monomeric prefusion conformation shows high flexibility, characteristic for an IDP, but also a high dynamic range and increasing rigidity from the N to C terminus. The gradual increase in rigidity correlates with an increase in lipid binding affinity from the N to C terminus. It could also explain the increased rate for C-terminal SNARE zippering, known to be faster than N-terminal SNARE zippering. Also, the syb-2 SNARE motif and, in particular, the linker domain show transient and weak membrane binding, characterized by a high off-rate and low (millimolar) affinity. The transient membrane binding of syb-2 may compensate for the repulsive forces between the two membranes and/or the SNARE motifs and the membranes, helping to destabilize the hydrophilic-hydrophobic boundary in the bilayer. Therefore, we propose that optimum flexibility and membrane binding of syb-2 regulate SNARE assembly and minimize repulsive forces during membrane fusion.

scholarly journals Living in Promiscuity: The Multiple Partners of Alpha-Synuclein at the Synapse in Physiology and Pathology

2019 ◽  
Vol 20 (1) ◽  
pp. 141 ◽  
Author(s):  
Francesca Longhena ◽  
Gaia Faustini ◽  
Maria Grazia Spillantini ◽  
Arianna Bellucci
Keyword(s):  
Lipid Membranes ◽  
Intrinsically Disordered Proteins ◽  
Lipid Membrane ◽  
Lewy Bodies ◽  
Alpha Synuclein ◽  
Disordered Proteins ◽  
Monoamine Transporters ◽  
Post Translational Modification ◽  
Intrinsically Disordered ◽  
Multiple Partners

Alpha-synuclein (α-syn) is a small protein that, in neurons, localizes predominantly to presynaptic terminals. Due to elevated conformational plasticity, which can be affected by environmental factors, in addition to undergoing disorder-to-order transition upon interaction with different interactants, α-syn is counted among the intrinsically disordered proteins (IDPs) family. As with many other IDPs, α-syn is considered a hub protein. This function is particularly relevant at synaptic sites, where α-syn is abundant and interacts with many partners, such as monoamine transporters, cytoskeletal components, lipid membranes, chaperones and synaptic vesicles (SV)-associated proteins. These protein–protein and protein–lipid membrane interactions are crucial for synaptic functional homeostasis, and alterations in α-syn can cause disruption of this complex network, and thus a failure of the synaptic machinery. Alterations of the synaptic environment or post-translational modification of α-syn can induce its misfolding, resulting in the formation of oligomers or fibrillary aggregates. These α-syn species are thought to play a pathological role in neurodegenerative disorders with α-syn deposits such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are referred to as synucleinopathies. Here, we aim at revising the complex and promiscuous role of α-syn at synaptic terminals in order to decipher whether α-syn molecular interactants may influence its conformational state, contributing to its aggregation, or whether they are just affected by it.

scholarly journals Coupling Bulk Phase Separation of Disordered Proteins to Membrane Domain Formation in Molecular Simulations on a Bespoke Compute Fabric

Membranes ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 17
Author(s):  
Julian C. Shillcock ◽  
David B. Thomas ◽  
Jonathan R. Beaumont ◽  
Graeme M. Bragg ◽  
Mark L. Vousden ◽  
...  
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Lipid Membrane ◽  
Molecular Simulations ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Computational Techniques ◽  
Domain Formation ◽  
Intrinsically Disordered ◽  
Ideal Mixing

Phospholipid membranes surround the cell and its internal organelles, and their multicomponent nature allows the formation of domains that are important in cellular signalling, the immune system, and bacterial infection. Cytoplasmic compartments are also created by the phase separation of intrinsically disordered proteins into biomolecular condensates. The ubiquity of lipid membranes and protein condensates raises the question of how three-dimensional droplets might interact with two-dimensional domains, and whether this coupling has physiological or pathological importance. Here, we explore the equilibrium morphologies of a dilute phase of a model disordered protein interacting with an ideal-mixing, two-component lipid membrane using coarse-grained molecular simulations. We find that the proteins can wet the membrane with and without domain formation, and form phase separated droplets bound to membrane domains. Results from much larger simulations performed on a novel non-von-Neumann compute architecture called POETS, which greatly accelerates their execution compared to conventional hardware, confirm the observations. Reducing the wall clock time for such simulations requires new architectures and computational techniques. We demonstrate here an inter-disciplinary approach that uses real-world biophysical questions to drive the development of new computing hardware and simulation algorithms.

About TFE: Old and New Findings

2019 ◽  
Vol 20 (5) ◽  
pp. 425-451 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia A. Mercurio ◽  
Marilisa Leone
Keyword(s):  
Secondary Structure ◽  
Intrinsically Disordered Proteins ◽  
Tertiary Structure ◽  
Disordered Proteins ◽  
Helical Conformation ◽  
Specific Amino Acid ◽  
Molecular Systems ◽  
Intrinsically Disordered ◽  
New Findings ◽  
Fluorinated Alcohol

The fluorinated alcohol 2,2,2-Trifluoroethanol (TFE) has been implemented for many decades now in conformational studies of proteins and peptides. In peptides, which are often disordered in aqueous solutions, TFE acts as secondary structure stabilizer and primarily induces an α -helical conformation. The exact mechanism through which TFE plays its stabilizing roles is still debated and direct and indirect routes, relying either on straight interaction between TFE and molecules or indirect pathways based on perturbation of solvation sphere, have been proposed. Another still unanswered question is the capacity of TFE to favor in peptides a bioactive or a native-like conformation rather than simply stimulate the raise of secondary structure elements that reflect only the inherent propensity of a specific amino-acid sequence. In protein studies, TFE destroys unique protein tertiary structure and often leads to the formation of non-native secondary structure elements, but, interestingly, gives some hints about early folding intermediates. In this review, we will summarize proposed mechanisms of TFE actions. We will also describe several examples, in which TFE has been successfully used to reveal structural properties of different molecular systems, including antimicrobial and aggregation-prone peptides, as well as globular folded and intrinsically disordered proteins.

scholarly journals β-Hairpin Peptide Mimics Decrease Human Islet Amyloid Polypeptide (hIAPP) Aggregation

2021 ◽  
Vol 9 ◽  
Author(s):  
Jacopo Lesma ◽  
Faustine Bizet ◽  
Corentin Berardet ◽  
Nicolo Tonali ◽  
Sara Pellegrino ◽  
...  
Keyword(s):  
Rational Design ◽  
Islet Amyloid Polypeptide ◽  
Human Islet ◽  
Disordered Proteins ◽  
Diabetic Patients ◽  
Amyloid Β Peptide ◽  
Aggregation Process ◽  
Islet Amyloid ◽  
Intrinsically Disordered ◽  
Human Islet Amyloid Polypeptide

Amyloid diseases are degenerative pathologies, highly prevalent today because they are closely related to aging, that have in common the erroneous folding of intrinsically disordered proteins (IDPs) which aggregate and lead to cell death. Type 2 Diabetes involves a peptide called human islet amyloid polypeptide (hIAPP), which undergoes a conformational change, triggering the aggregation process leading to amyloid aggregates and fibers rich in β-sheets mainly found in the pancreas of all diabetic patients. Inhibiting the aggregation of amyloid proteins has emerged as a relevant therapeutic approach and we have recently developed the design of acyclic flexible hairpins based on peptidic recognition sequences of the amyloid β peptide (Aβ1–42) as a successful strategy to inhibit its aggregation involved in Alzheimer’s disease. The present work reports the extension of our strategy to hIAPP aggregation inhibitors. The design, synthesis, conformational analyses, and biophysical evaluations of dynamic β-hairpin like structures built on a piperidine-pyrrolidine β-turn inducer are described. By linking to this β-turn inducer three different arms (i) pentapeptide, (ii) tripeptide, and (iii) α/aza/aza/pseudotripeptide, we demonstrate that the careful selection of the peptide-based arms from the sequence of hIAPP allowed to selectively modulate its aggregation, while the peptide character can be decreased. Biophysical assays combining, Thioflavin-T fluorescence, transmission electronic microscopy, capillary electrophoresis, and mass spectrometry showed that the designed compounds inhibit both the oligomerization and the fibrillization of hIAPP. They are also capable to decrease the aggregation process in the presence of membrane models and to strongly delay the membrane-leakage induced by hIAPP. More generally, this work provides the proof of concept that our rational design is a versatile and relevant strategy for developing efficient and selective inhibitors of aggregation of amyloidogenic proteins.

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