Interaction of the chaperones alpha B-crystallin and CHIP with fibrillar alpha-synuclein: Effects on internalization by cells and identification of interacting interfaces.

The spread of fibrillar alpha-synuclein from affected to naïve neuronal cells is thought to contribute to the progression of synucleinopathies. The binding of fibrillar alpha-synuclein to the plasma membrane is key in this process. We and others previously showed that coating fibrillar alpha-synuclein by the molecular chaperone Hsc70 affects fibrils properties. Here we assessed the effect of the two molecular chaperones alpha B-crystallin and CHIP on alpha-synuclein fibrils uptake by Neuro-2a cells. We demonstrate that both chaperones diminish fibrils take up by cells. We identify through a cross-linking and mass spectrometry strategy the interaction interfaces between alpha-synuclein fibrils and alpha B-crystallin or CHIP. Our results open the way for designing chaperone-derived polypeptide binders that interfere with the propagation of pathogenic alpha-synuclein assemblies.

Molecular interaction between the chaperone Hsc70 and the N-terminal flank of huntingtin exon 1 modulates aggregation.

The aggregation of polyglutamine (polyQ)-containing proteins is at the origin of nine neurodegenerative diseases. Molecular chaperones prevent the aggregation of polyQ-containing proteins. The exact mechanism by which they interact with polyQ-containing, aggregation-prone proteins and interfere with their assembly is unknown. Here we dissect the mechanism of interaction between a huntingtin exon 1 fragment of increasing polyQ lengths (HttEx1Qn), the aggregation of which is tightly associated with Huntington's disease, and molecular chaperone Hsc70. We show that Hsc70, together with its Hsp40 co-chaperones, inhibits HttEx1Qn aggregation and modifies the structural, seeding, and infectious properties of the resulting fibrils in a polyQ-independent manner. We demonstrate that Hsc70 binds the 17-residue-long N-terminal flank of HttEx1Qn, and we map Hsc70-HttEx1Qn surface interfaces at the residue level. Finally, we show that this interaction competes with homotypic interactions between the N termini of different HttEx1Qn molecules that trigger the aggregation process. Our results lay the foundations of future therapeutic strategies targeting huntingtin aggregation in Huntington disease.

ProFeatMap: a highly customizable tool for 2D feature representation of protein sets.

Motivation: Studies of sets of proteins are a central point in biology. In particular, the application of omics in the last decades has generated lists of several hundreds or thousands of proteins or genes. However, these lists are often not inspected globally, possibly due to the lack of tools capable of simultaneously visualizing the feature architectures of a large number of proteins. Results: Here, we present ProFeatMap, an intuitive Python-based website. For a given set of proteins, it allows to display features such as domains, repeats, disorder or post-translational modifications and their organization along the sequences, into a highly customizable 2D map. Starting from a user-defined protein list of UniProt accession codes, ProFeatMap extracts the most important annotated features available for each protein from one of the well-established databases such as Uniprot or InterPro, allocates shapes and colors, potentially depending on quantitative or qualitative data and sorts the protein list based on homologous feature content. The resulting publication-quality map allows even large protein families to be explored, and to classify them based on shared features. It can help to gain insights, for example, feature redundancy or feature pattern, that were previously overlooked. ProFeatMap is freely available on the web at: https://profeatmap.pythonanywhere.com/. Availability and implementation: Source code is freely accessible at https://github.com/profeatmap/ProFeatMap under the GPL license. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Quantitative fragmentomics allow affinity mapping of interactomes.

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

Kinetic analysis of amyloid formation in the presence of heparan sulfate: faster unfolding and change of pathway.

A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and beta-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis.

Polypeptides derived from α-Synuclein binding partners to prevent α-Synuclein fibrils interaction with and take-up by cells.

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.

Aggregation propensity of the human proteome.

Formation of amyloid-like fibrils is involved in numerous human protein deposition diseases, but is also an intrinsic property of polypeptide chains in general. Progress achieved recently now allows the aggregation propensity of proteins to be analyzed over large scales. In this work we used a previously developed predictive algorithm to analyze the propensity of the 34,180 protein sequences of the human proteome to form amyloid-like fibrils. We show that long proteins have, on average, less intense aggregation peaks than short ones. Human proteins involved in protein deposition diseases do not differ extensively from the rest of the proteome, further demonstrating the generality of protein aggregation. We were also able to reproduce some of the results obtained with other algorithms, demonstrating that they do not depend on the type of computational tool employed. For example, proteins with different subcellular localizations were found to have different aggregation propensities, in relation to the various efficiencies of quality control mechanisms. Membrane proteins, intrinsically disordered proteins, and folded proteins were confirmed to have very different aggregation propensities, as a consequence of their different structures and cellular microenvironments. In addition, gatekeeper residues at strategic positions of the sequences were found to protect human proteins from aggregation. The results of these comparative analyses highlight the existence of intimate links between the propensity of proteins to form aggregates with beta-structure and their biology. In particular, they emphasize the existence of a negative selection pressure that finely modulates protein sequences in order to adapt their aggregation propensity to their biological context.

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution.

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of "sequence scrambled" variants of the first 29 residues of horse heart apomyoglobin (apoMb(1-29)), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k(agg)) and a remarkable shortening of the lag phase (t(lag)) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k(agg) and t(lag) values of the apoMb(1-29) variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms.

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.

α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane.

Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain.

Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength.

The fluorescence of tryptophan is used as a signal to monitor the unfolding of proteins, in particular the intensity of fluorescence and the wavelength of its maximum lambda(max). The law of the signal is linear with respect to the concentrations of the reactants for the intensity but not for lambda(max). Consequently, the stability of a protein and its variation upon mutation cannot be deduced directly from measurements made with lambda(max). Here, we established a rigorous law of the signal for lambda(max). We then compared the stability DeltaG(H(2)O) and coefficient of cooperativity m for a two-state equilibrium of unfolding, monitored with lambda(max), when the rigorous and empirical linear laws of the signal are applied. The corrective terms involve the curvature of the emission spectra at their lambda(max) and can be determined experimentally. The rigorous and empirical values of the cooperativity coefficient m are equal within the experimental error for this parameter. In contrast, the rigorous and empirical values of the stability DeltaG(H(2)O) generally differ. However, they are equal within the experimental error if the curvatures of the spectra for the native and unfolded states are identical. We validated this analysis experimentally using domain 3 of the envelope glycoprotein of the dengue virus and the single-chain variable fragment (scFv) of antibody mAbD1.3, directed against lysozyme.

DNAJB6 is a peptide-binding chaperone which can suppress amyloid fibrillation of polyglutamine peptides at substoichiometric molar ratios.

Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation in cells (Hageman et al. in Mol Cell 37(3):355-369, 2010). Furthermore, also aggregation of polyQ peptides expressed in cells was recently found to be efficiently suppressed by co-expression of DNAJB6 (Gillis et al. in J Biol Chem 288:17225-17237, 2013). These suppression effects can be due to an indirect effect of DNAJB6 on other cellular components or to a direct interaction between DNAJB6 and polyQ peptides that may depend on other cellular components. Here, we have purified the DNAJB6 protein to investigate the suppression mechanism. The purified DNAJB6 protein formed large heterogeneous oligomers, in contrast to the more canonical family member DNAJB1 which is dimeric. Purified DNAJB6 protein, at substoichiometric molar ratios, efficiently suppressed fibrillation of polyQ peptides with 45°Q in a thioflavin T fibrillation. No suppression was obtained with DNAJB1, but with the closest homologue to DNAJB6, DNAJB8. The suppression effect was independent of HSPA1 and ATP. These data, based on purified proteins and controlled fibrillation in vitro, strongly suggest that the fibrillation suppression is due to a direct protein-protein interaction between the polyQ peptides and DNAJB6 and that the DNAJB6 has unique fibrillation suppression properties lacking in DNAJB1. Together, the data obtained in cells and in vitro support the view that DNAJB6 is a peptide-binding chaperone that can interact with polyQ peptides that are incompletely degraded by and released from the proteasome.

Polyglutamine repeats are associated to specific sequence biases that are conserved among eukaryotes.

Nine human neurodegenerative diseases, including Huntington's disease and several spinocerebellar ataxia, are associated to the aggregation of proteins comprising an extended tract of consecutive glutamine residues (polyQs) once it exceeds a certain length threshold. This event is believed to be the consequence of the expansion of polyCAG codons during the replication process. This is in apparent contradiction with the fact that many polyQs-containing proteins remain soluble and are encoded by invariant genes in a number of eukaryotes. The latter suggests that polyQs expansion and/or aggregation might be counter-selected through a genetic and/or protein context. To identify this context, we designed a software that scrutinize entire proteomes in search for imperfect polyQs. The nature of residues flanking the polyQs and that of residues other than Gln within polyQs (insertions) were assessed. We discovered strong amino acid residue biases robustly associated to polyQs in the 15 eukaryotic proteomes we examined, with an over-representation of Pro, Leu and His and an under-representation of Asp, Cys and Gly amino acid residues. These biases are conserved amongst unrelated proteins and are independent of specific functional classes. Our findings suggest that specific residues have been co-selected with polyQs during evolution. We discuss the possible selective pressures responsible of the observed biases.

A computational approach for identifying the chemical factors involved in the glycosaminoglycans-mediated acceleration of amyloid fibril formation.

BACKGROUND: Amyloid fibril formation is the hallmark of many human diseases, including Alzheimer's disease, type II diabetes and amyloidosis. Amyloid fibrils deposit in the extracellular space and generally co-localize with the glycosaminoglycans (GAGs) of the basement membrane. GAGs have been shown to accelerate the formation of amyloid fibrils in vitro for a number of protein systems. The high number of data accumulated so far has created the grounds for the construction of a database on the effects of a number of GAGs on different proteins. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have constructed such a database and have used a computational approach that uses a combination of single parameter and multivariate analyses to identify the main chemical factors that determine the GAG-induced acceleration of amyloid formation. We show that the GAG accelerating effect is mainly governed by three parameters that account for three-fourths of the observed experimental variability: the GAG sulfation state, the solute molarity, and the ratio of protein and GAG molar concentrations. We then combined these three parameters into a single equation that predicts, with reasonable accuracy, the acceleration provided by a given GAG in a given condition. CONCLUSIONS/SIGNIFICANCE: In addition to shedding light on the chemical determinants of the protein:GAG interaction and to providing a novel mathematical predictive tool, our findings highlight the possibility that GAGs may not have such an accelerating effect on protein aggregation under the conditions existing in the basement membrane, given the values of salt molarity and protein:GAG molar ratio existing under such conditions.

Amyloid formation by the model protein muscle acylphosphatase is accelerated by heparin and heparan sulphate through a scaffolding-based mechanism.

Amyloid formation is the hallmark of many diseases. The propensity of a protein to aggregate depends on a number of biological factors like the presence of sulphated polysaccharides termed as glycosaminoglycans (GAGs). Here we assessed whether the polymeric nature of GAGs is responsible for their protein aggregation-promoting effect. We studied the effect of different monosaccharide derivatives, featuring the main characteristics of heparin and heparan sulphate (HS) building blocks, on the aggregation kinetics of human muscle acylphosphatase (mAcP), a useful model protein for these studies. We observed that while heparin and HS changed the mAcP aggregation kinetic profile, the monosaccharide derivatives had no effect, whatever their concentration could be and both when they are studied separately or in combination. In contrast, heparin fragments with six or more monosaccharides reproduced the effects of HS and in part those of heparin. We conclude that the effect of heparin and HS on protein aggregation arises from the clustering and regular distribution of their composing units on a polymeric structure. We propose a model in which heparin and HS promote mAcP aggregation through a scaffolding-based mechanism, in which the regularly spaced sulphate moieties of the polymer interact with mAcP molecules increasing their local concentration and facilitating their orientation.

XYLab: an interactive plotting tool for mixed multivariate data observation and interpretation.

UNLABELLED: The correct display of data is often a key point for interpreting the results of experimental procedures. Multivariate data sets suffer from the problem of representation, since a dimensionality above 3 is beyond the capability of plotting programs. Moreover, non numerical variables such as protein annotations are usually fundamental for a full comprehension of biological data. Here we present a novel interactive XY plotter designed to take the full control of large datasets containing mixed-type variables, provided with an intuitive data management, a powerful labelling system and other features aimed at facilitating data interpretation and sub-setting. AVAILABILITY: XYLab program, test dataset and manual is available at www4.unifi.it/scibio/bioinfo/ XYLab.html.

Prevention of amyloid-like aggregation as a driving force of protein evolution.

Uncontrolled protein aggregation is a constant challenge in all compartments of living organisms. The failure of a peptide or protein to remain soluble often results in pathology. So far, more than 40 human diseases have been associated with the formation of extracellular fibrillar aggregates - known as amyloid fibrils - or structurally related intracellular deposits. It is well known that molecular chaperones and elaborate quality control mechanisms exist in the cell to counteract aggregation. However, an increasing number of reports during the past few years indicate that proteins have also evolved structural and sequence-based strategies to prevent aggregation. This review describes these strategies and the selection pressures that exist on protein sequences to combat their uncontrolled aggregation. We will describe the different types of mechanism evolved by proteins that adopt different conformational states including normally folded proteins, intrinsically disordered polypeptide chains, elastomeric systems and multimodular proteins.