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 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 Multisite Phosphorylation and Binding Alter Conformational Dynamics of the 4E-BP2 Protein

2022 ◽  
Author(s):  
Spencer Smyth ◽  
Zhenfu Zhang ◽  
Alaji Bah ◽  
Thomas Tsangaris ◽  
Jennifer Dawson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Intrinsically Disordered Proteins ◽  
Regulatory Protein ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Terminal Region ◽  
Disordered Proteins ◽  
Initiation Factor ◽  
Dependent Manner ◽  
Intrinsically Disordered

Intrinsically disordered proteins (IDPs) play critical roles in regulatory protein interactions, but detailed structural/dynamics characterization of their ensembles remain challenging, both in isolation and they form dynamic fuzzy complexes. Such is the case for mRNA cap-dependent translation initiation, which is regulated by the interaction of the predominantly folded eukaryotic initiation factor 4E (eIF4E) with the intrinsically disordered eIF4E binding proteins (4E-BPs) in a phosphorylation-dependent manner. Single-molecule Forster resonance energy transfer showed that the conformational changes of 4E-BP2 induced by binding to eIF4E are non-uniform along the sequence; while a central region containing both motifs that bind to eIF4E expands and becomes stiffer, the C-terminal region is less affected. Fluorescence anisotropy decay revealed a nonuniform segmental flexibility around six different labelling sites along the chain. Dynamic quenching of these fluorescent probes by intrinsic aromatic residues measured via fluorescence correlation spectroscopy report on transient intra- and inter-molecular contacts on nanosecond-microsecond timescales. Upon hyperphosphorylation, which induces folding of ~40 residues in 4E-BP2, the quenching rates decreased at labelling sites closest to the phosphorylation sites and within the folded domain, and increased at the other sites. The chain dynamics around sites in the C-terminal region far away from the two binding motifs were significantly reduced upon binding to eIF4E, suggesting that this region is also involved in the highly dynamic 4E-BP2:eIF4E complex. Our time-resolved fluorescence data paint a sequence-level rigidity map of three states of 4E-BP2 differing in phosphorylation or binding status and distinguish regions that form contacts with eIF4E. This study adds complementary structural and dynamics information to recent studies of 4E-BP2, and it constitutes an important step towards a mechanistic understanding of this important IDP via integrative modelling.

scholarly journals Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS

2016 ◽  
Vol 113 (37) ◽  
pp. E5389-E5398 ◽  
Author(s):  
Mikayel Aznauryan ◽  
Leonildo Delgado ◽  
Andrea Soranno ◽  
Daniel Nettels ◽  
Jie-rong Huang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Local Level ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Disordered Proteins ◽  
Time Range ◽  
Unfolded Proteins ◽  
Intrinsically Disordered ◽  
Unfolded State

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

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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