Amyloidosis is a group of proteinopathies characterized by the systemic or organ-specific deposition of proteins in the form of amyloid fibers. Nearly 40 proteins play a role in these pathologies, and the structures of the associated fibers are beginning to be determined by Cryo-EM. However, the molecular events underlying the process, such as fiber nucleation and elongation, are poorly understood, which impairs developing efficient therapies. In most cases, only a few dozen amino acids of the pathological protein are found in the final structure of the fibers, while amyloid peptides comprising five to 10 amino acids are involved in the fiber nucleation process. The identification and biochemical characterization of these peptides are therefore of major scientific and clinical importance. We demonstrated that in silico approaches are limited due to the peptides' small size and long-distance intra- and intermolecular interactions that occur during nucleation. To address this problem, we developed a novel biochemical method for characterizing and classifying batches of related peptides. Initial work to optimize our approach is based on the reference peptide PHF6 (β1) from Microtubule-Associated Protein Tau (MAPT) as compared to 22 related peptides. Depending on their biochemical properties and using the Garnier-Delamarche plot we propose, we classified these peptides into three groups: aggregative, amyloid, and soluble (neither aggregative nor amyloid). We emphasize that our biochemical classification method is applicable to any family of peptides and could be scaled up for high-throughput analyses.

Amyloidosis are proteinopathies characterized by systemic or organ-specific deposition of proteins in the form of amyloid fibers. Nearly forty proteins have been identified to play a role in these pathologies and the structures of the associated fibers are beginning to be determined by Cryo-EM. However, the molecular events underlying the process, such as fiber nucleation and elongation, are poorly understood, which impairs developing efficient therapies. In most cases, only a few dozen amino acids of the pathological protein are found in the final structure of the fibers, while amyloid peptides comprising 5 to 10 amino acids are involved in fibers nucleation process. The identification and biochemical characterization of these peptides is therefore of major scientific and clinical importance. In silico approaches are limited due to the peptides’ small size and long-distance intra- and intermolecular interactions that occur during nucleation. To address this problem, we developed a novel biochemical method for characterizing and classifying batches of related peptides. Initial work to optimize our approach is based on the reference peptide PHF6 (ß1) from Microtubule Associated Protein Tau (MAPT) as compared to 22 related peptides. We classified these peptides into groups displaying different biochemical properties, and thereby identified new amyloid peptides and peptides with therapeutic potential. We underline that our method is applicable to any family of peptides and could be scaled up for high-throughput analyses.### Competing Interest StatementThe authors have declared no competing interest.

Proposed models of the function of Hsp90 are characterised by high flexibility of the dimeric state and conformational changes regulated by both nucleotide binding and hydrolysis, and by co-chaperone interactions. In addition to its dimeric state, Hsp90 self-associates upon particular stimuli. The Hsp90 dimer is the building block up to the hexamer that we named "cosy nest", and the dodecamer results from the association of two hexamers. Oligomers exhibit chaperone activity, but their exact mechanism of action has not yet been determined. One of the best ways to elucidate how oligomers might operate is to study their interactions with co-chaperone proteins known to regulate the Hsp90 chaperone cycle, such as p23 and Aha1. In this review, we summarise recent results and conclude that Hsp90 oligomers are key players in the chaperone cycle. Crucible-shaped quaternary structures likely provide an ideal environment for client protein accommodation and folding, as is the case for other Hsp families. Confirmation of the involvement of Hsp90 oligomers in the chaperone cycle and a better understanding of their functionality will allow us to address some of the more enigmatic aspects of Hsp90 activity. Utilising this knowledge, future work will highlight how Hsp90 oligomers and co-chaperones cooperate to build the structures required to fold or refold numerous different client proteins.

The "Bioénergétique et Ingénierie des Protéines (BIP)" laboratory, CNRS (France), organized its first French workshop on molecular chaperone proteins and protein folding in November 2017. The goal of this workshop was to gather scientists working in France on chaperone proteins and protein folding. This initiative was a great success with excellent talks and fruitful discussions. The highlights were on the description of unexpected functions and post-translational regulation of known molecular chaperones (such as Hsp90, Hsp33, SecB, GroEL) and on state-of-the-art methods to tackle questions related to this theme, including Cryo-electron microscopy, Nuclear Magnetic Resonance (NMR), Electron Paramagnetic Resonance (EPR), simulation and modeling. We expect to organize a second workshop in two years that will include more scientists working in France in the chaperone field.

The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient's kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for β-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-β-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis.

Aggregation of TDP-43 (transactive response DNA binding protein 43 kDa) is a hallmark of certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, intracellular TDP-43-positive inclusions are often found in other neurodegenerative diseases. Recently it was shown that zinc ions can provoke the aggregation of endogenous TDP-43 in cells, allowing to assume a direct interaction of TDP-43 with zinc ions. In this work, we investigated zinc binding to the 102–269 TDP-43 fragment, which comprise the two RNA recognition motifs. Using isothermal titration calorimetry, mass spectrometry, and differential scanning fluorimetry, we showed that zinc binds to this TDP-43 domain with a dissociation constant in the micromolar range and modifies its tertiary structure leading to a decrease of its thermostability. Moreover, the study by dynamic light scattering and negative stain electron microscopy demonstrated that zinc ions induce auto-association process of this TDP-43 fragment into rope-like structures. These structures are thioflavin-T-positive allowing to hypothesize the direct implication of zinc ions in pathological aggregation of TDP-43.

Background: The 90 kDa heat shock protein (Hsp90) participates in regulating the homeostasis of cellular proteins and was considered one of the key chaperones involved in the control and regulation of amyloid deposits. Hsp90 interacts with the amyloid protein tau through tau aggregation-prone regions, including the VQIVYK hexapeptide motif. This hexapeptide, which self-aggregates, forming amyloid fibrils, is widely used to model amyloid formation mechanisms. Despite evidence showing that Hsp90 interacts directly with Ac-VQIVYK-NH2, its role in the hexapeptide fibrillation process and its binding to peptide structures have not yet been determined.Methods: Various biochemical and biophysical techniques, including ultracentrifugation, spectrophotometry, spectrofluorimetry, and electron microscopy, were employed to assess the effects of Hsp90 on Ac-VQIVYK-NH2 assembly and disassembly processes.Results: At sub-stoichiometric concentrations, Hsp90 bound directly to Ac-VQIVYK-NH2 amyloid structures in vitro, with each Hsp90 dimer interacting with an amyloid structure made of around 50 hexapeptide subunits. Hsp90 inhibited Ac-VQIVYK-NH2 assembly by increasing the critical concentrations of Ac-VQIVYK-NH2 required for assembly. Hsp90 also inhibited the disassembly of Ac-VQIVYK-NH2 amyloid fibrils and promoted their rescue.Conclusions: A model explaining the dual effect of Hsp90 on the Ac-VQIVYK-NH2 amyloid fibrillation process has been proposed.General significance: These in vitro results provide new insights into the possible roles of molecular chaperones in modulating amyloid structures by limiting the spread of toxic species. (C) 2016 Elsevier B.V. All rights reserved.

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer–oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a “protein wedge” regarding the Hsp90 dimer closure and the Hsp90 oligomerization process.

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer able to self-associate in the presence of divalent cations or under heat shock. This study investigated the relationship between Hsp90 oligomers and the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase). The interactions of Aha1 with Hsp90 dimers and oligomers were evaluated by ultracentrifugation, size-exclusion chromatography coupled to multiangle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Hsp90 dimer was able to bind up to four Aha1 molecules, and Hsp90 oligomers are also able to interact with Aha1. The binding of Aha1 did not interfere with the Hsp90 oligomerization process. Except for Hsp90 dimer, the stoichiometry of the interaction remained constant, at 2 Aha1 molecules per Hsp90 dimer, regardless of the degree of Hsp90 oligomerization. Moreover, Aha1 predominantly bound to Hsp90 oligomers. Thus, the ability of Hsp90 oligomers to bind the Aha1 ATPase activator reinforces their role within the Hsp90 chaperone machineries.

Since noncovalent protein macrocomplexes are implicated in many cellular functions, their characterization is essential to understand how they drive several biological processes. Over the past 20 years, because of its high sensitivity, mass spectrometry has been described as a powerful tool for both the protein identification in macrocomplexes and the understanding of the macrocomplexes organization. Nonetheless, stabilizing these protein macrocomplexes, by introducing covalent bonds, is a prerequisite before their analysis by the denaturing mass spectrometry technique. In this study, using the Hsp90/Aha1 macrocomplex as a model (where Hsp denotes a heat shock protein), we optimized a double cross-linking protocol with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). This protocol takes place in a two-step process: initially, a cross-linking is performed according to a previously optimized protocol, and then a second cross-linking is performed by increasing the EDC concentration, counterbalanced by a high dilution of sample and, thus, protein macrocomplexes. Using matrix-assisted laser desorption ionization (MALDI) mass spectrometry, we verified the efficiency of our optimized protocol by submitting (or not submitting) samples to the K200 MALDI MS analysis kit containing N-succinimidyl iodo-acetate, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester), suberic acid bis(N-hydroxysuccinimide ester), disuccinimidyl tartrate, and dithiobis(succinimidyl) propionate, developed by the CovalX Company. Results obtained show that our optimized cross-linking protocol allows a complete stabilization of protein macrocomplexes and appears to be very accurate. Indeed, contrary to other cross-linkers, the "zero-length" feature of the EDC reagent prevents overdetermination of the mass of complexes, because EDC does not remain as part of the linkage.

The glycoprotein (GP) molecular fraction structure of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using high-performance size exclusion chromatography-multi angle laser light scattering (HPSEC-MALLS), small angle X-ray scattering (SAXS), synchrotron radiation circular dichroism (SRCD) and transmission electron microscopy (TEM) observations. In solution, GP would be a mixture of spheroidal monomers and more anisotropic oligomers as suggested by the two exponent values found in the Rg vs. Mw relationship and TEM observations. The GP conformation probed by SAXS was ascribed to a thin object with a triaxial ellipsoid morphology, certainly attributed to GP oligomers. A 9 nm diameter particle was also identified by SAXS in agreement with the dimensions identified by TEM on single isolated ring-like structures. The GP oligomerization process, as probed by TEM, would be the result of ring-like subunits self-association. This self-association would lead to more linear or, sometimes, cyclised assembly. At the molecular level, GP fraction was found to have secondary structures mainly made of β-sheets and turns (64%) but also, to a lesser extent, made of polyproline II (PPII) and α-helices (19%). These features were characteristic of hydroxyprolin-rich glycoproteins with arabinosylated and arabinogalactan polysaccharide side chains grafted to the polypeptide backbone. The GP molecular fraction structure from Acacia gum would be an assembly of ring-like glycoproteins modules. These ring-like structures were certainly due to hydroxyproline (Hyp)-arabinogalactan (AG) subunits.

The structure of the arabinogalactan-protein (AGP) fraction of the gum exudate of Acacia senegal (gum Arabic) isolated from hydrophobic interaction chromatography was investigated using HPSEC-MALLS, small angle neutron scattering and TEM observations. Literature reported that the AGP structure of gum Arabic adopts a very compact conformation in solution due to the attachment of short arabinoside side chains and much larger blocks of carbohydrate to the polypeptidic backbone. The present study revealed that AGP in solution had a weight average molecular weight Mw of 1.86×10(6) g mol(-1) and a radius of gyration Rg of 30 nm. In addition, two exponent values were identified in the Rg, [η], Rh and ρ vs. Mw relationships highlighting two types of conformations depending on the molecular weight range considered: a low molar mass population with long-chain branching and a compact conformation and a high molar mass population with short-chain branching and an elongated conformation. AGP would behave in solution as a branched or hyper-branched polymer with conformations ranging from globular to elongated shape depending on the size of the carbohydrate branches. Small angle scattering form factor revealed an elongated average conformation corresponding to a triaxial ellipsoid while inverse Fourier transform of the scattering form factor gave a maximum dimension for AGP of 64 nm. Transmission electron microscopy highlighted the existence of two types of flat objects with thicknesses below 3-5 nm, single particles with a more or less anisotropic spheroidal shape and aggregated structures with a more elongated shape. A remarkable feature of all particle morphologies was the presence of an outer structure combined to an inner more or less porous network of interspersed chains or interacting structural blocks, as previously found for the arabinogalactan (AG) main molecular fraction of Acacia gum. However, clear differences were observed in the density and morphology of the inner porous network, probably highlighting differences in the degree of branching. The existence of assembled AG as part of the AGP family was confirmed using TEM micrographs at high resolution. Fused AGP dimers, trimers, tetramers and multimers were also identified. These molecular assemblies questioned about the nature of interactions involved.

1 EB1 promotes microtubule dynamics and regulates tubulin sheet closure in vitro. End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles 1, 2. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells 3-6 and purified systems 7-13 , the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates microtubule spontaneous nucleation and growth rate, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets that rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing ends, together with the elimination of stressed microtubule lattices, may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues. Microtubules are unstable polymers that switch stochastically between growing and shrinking states, a particular behaviour known as dynamic instability 14. Essential for this process is the tubulin-bound GTP hydrolysis that occurs during microtubule assembly, since it creates an unstable lattice that can rapidly depolymerise 14. Microtubule growth involves the formation of outwardly curved tubulin sheets that gradually close into tubes, and which are thought to protect the lattice from catastrophe events 15, 16. Catastrophes may arise from loss of this capping structure through occasional closure of the sheets onto energetically unfavourable configurations 17. Conversely, rescues may involve a metastable intermediate state between disassembly and assembly 18. Catastrophes and rescues are finely regulated in cells and can be either stimulated or inhibited depending on the cell activity 14. Several proteins interact with unpolymerised tubulin and/or microtubules to modulate their dynamic properties 19. Amongst them, EB1 is a highly conserved +TIP that was first described as a partner of the tumour suppressor APC 1. EB1 accumulates at microtubule plus ends through a treadmilling process characterized by an association with the newly growing end coupled with a rapid dissociation from the lattice older part 2. The recognition of a specific structure at microtubule plus end (e.g. the growing tubulin sheet) may be the targeting mechanism involved, rather …

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg2+-induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a “nest-like” shape that forms a “cozy chaperoning chamber” where the client protein folding/protection could occur.

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.

BACKGROUND INFORMATION:Hsp90 (90 kDa heat-shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide-binding-dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue).RESULTS AND DISCUSSION:Here, we present the first nucleotide-free structures of the entire eukaryotic Hsp90 (apo-Hsp90) obtained by small-angle X-ray scattering and single-particle cryo-EM (cryo-electron microscopy). We show that, in solution, apo-Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo-EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo-forms require large movements of the NTD (N-terminal domain) and MD (middle domain) around two flexible hinge regions.CONCLUSIONS:The present study shows, for the first time, the structure of the entire eukaryotic apo-Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.