Biology subjects

Vendruscolo, M.

Publications and source records attributed to Vendruscolo, M..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

While aggregation-prone proteins are known to accelerate ageing and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A and SERF2 have recently been identified as cellular modifiers of such cytotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with specific patterns of negatively charged and hydrophobic, aromatic amino acids. The absence of such patterns, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In a protein aggregation model in the nematode C. elegans, protein aggregation was suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that charge interactions are required for MOAG-4 and SERF2 to promote aggregation. Such charged interactions might accelerate the primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our finding that negatively charged segments are overrepresented in amyloid-forming proteins suggests that inhibition of charge interactions deserves exploration as a strategy to target age-related protein toxicity. Significance StatementHow aging causes relatively common diseases such as Alzheimers and Parkinsons is still a mystery. Since toxic structural changes in proteins are likely to be responsible, we investigated biological mechanisms that could drive such changes. We made use of a modifying factor called SERF2, which accelerates structural changes and aggregation of several disease-related proteins. Through a peptide-binding screen, we found that SERF2 acts on negatively charged protein regions. The abundance of such regions in the disease-related proteins explains why SERF has its effect. Removing positive charge in SERF was sufficient to suppress protein aggregation in models for disease. We propose that blocking charge-interactions with SERF or other modifiers could serve as a general approach to treat age-related protein toxicity.

molecular biology↗

bioRxiv2020

Sequence determinants for the aggregation and cytotoxicity of proteins within condensates generated by liquid-liquid phase separation

The transition between the native and amyloid states of proteins can proceed via a deposition pathway via oligomeric intermediates or via a condensation pathway via liquid droplet intermediates generated through liquid-liquid phase separation. Here we investigate the sequence determinants of aggregation from within the droplet state based on generic interactions. We describe a model in which these sequence determinants can be captured by three features, the droplet-promoting propensity, the aggregation-promoting propensity and the binding mode entropy. By using this approach, we propose a formula to identify aggregation-promoting mutations in droplet-forming proteins. This analysis provides insights into the amino acid code for the conversion of proteins between liquid-like and solid-like condensates.

cell biology↗

bioRxiv2020

A Structural Ensemble of a Tau-Microtubule Complex Reveals Regulatory Tau Phosphorylation and Acetylation Mechanisms

Tau is a microtubule-associated protein that regulates the stability of microtubules. The affinity of tau for microtubules is modulated by post-translational modifications, and the dysregulation of these events has been associated with the aberrant aggregation of tau in Alzheimer's disease and related tauopathies. Here, we use the metainference cryo-electron microscopy approach to determine an ensemble of structures representing the structure and dynamics of a tau-microtubule complex comprising an extended microtubule-binding region of tau (residues 202-395). We thus identify the ground state of the complex and a series of excited states of lower populations. An analysis of the interactions in these states of structures reveals positions in the tau sequence that are important to determine the overall stability of the tau-microtubule complex. This analysis leads to the identification of positions where phosphorylation and acetylation events have destabilising effects, which we validate by using site-specific post-translationally modified tau variants obtained by chemical mutagenesis. Taken together, these results illustrate how the simultaneous determination of ground and excited states of macromolecular complexes reveals functional and regulatory mechanisms.

biophysics↗

bioRxiv2020

The Hsc70 Disaggregation Machinery RemovesMonomer Units Directly from α-Synuclein FibrilEnds

Molecular chaperones contribute to the maintenance of cellular protein homeostasis through a wide range of mechanisms, including the assistance of de novo protein folding, the rescue of misfolded proteins, and the prevention of amyloid formation. Chaperones of the Hsp70 family have a striking capability of disaggregating otherwise irreversible aggregate structures such as amyloid fibrils that accumulate during the development of neurodegenerative diseases. However, the mechanisms of this key emerging functionality remain largely unknown. Here, we bring together microfluidic measurements with kinetic analysis and show that that the Hsp70 protein heat chock complement Hsc70 together with its two co-chaperones DnaJB1 and the nucleotide exchange factor Apg2 is able to completely reverse the aggregation process of alpha-synuclein, associated with Parkinsons disease, back to its soluble monomeric state. Moreover, we show that this reaction proceeds with first order kinetics in a process where monomer units are taken off directly from the fibril ends. Our results demonstrate that all components of the chaperone triad are essential for fibril disaggregation. Lastly, we quantify the interactions between the three chaperones as well as between the chaperones and the fibrils in solution, yielding both binding stoichiometries and dissociation constants. Crucially, we find that the stoichiometry of Hsc70 binding to fibrils suggests Hsc70 clustering at the fibril ends. Taken together, our results show that the mechanism of action of the Hsc70-DnaJB1-Apg2 chaperone system in disaggregating -synuclein fibrils involves the removal of monomer units without any intermediate fragmentation steps. These findings are fundamental to our understanding of the suppression of amyloid proliferation early in life and the natural clearance mechanisms of fibrillar deposits in Parkinsons disease, and inform on the possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies and related neurodegenerative diseases.

biophysics↗

bioRxiv2020

Widespread occurrence of the droplet state of proteins in the human proteome

A wide range of proteins have been reported to condensate into a dense liquid phase, forming a reversible droplet state. Failure in the control of the droplet state can lead to the formation of the more stable amyloid state, which is often disease-related. These observations prompt the question of how many proteins can undergo liquid-liquid phase separation. Here, in order to address this problem, we discuss the biophysical principles underlying the droplet state of proteins by analyzing current evidence for droplet-driver and droplet-client proteins. Based on the concept that the droplet state is stabilized by the large conformational entropy associated with non-specific side-chain interactions, we develop the FuzDrop method to predict droplet-promoting regions and proteins, which can spontaneously phase separate. We use this approach to carry out a proteome-level study to rank proteins according to their propensity to form the droplet state, spontaneously or via partner interactions. Our results lead to the conclusion that the droplet state could be, at least transiently, accessible to most proteins under conditions found in the cellular environment. SignificanceLiquid-liquid phase separation of proteins results in biomolecular condensates, which contribute to the organisation of cellular matter into membraneless organelles. It is still unclear, however, whether these condensates represent a common state of proteins. Here, based on biophysical principles driving phase separation, we report a proteome-wide ranking of proteins according to their propensity to condensate into a droplet state. We analyze two mechanisms for droplet formation - driver proteins can spontaneously phase separate, while client proteins require additional components. We conclude that the droplet state, as the native and amyloid states, is a fundamental state of proteins, with most proteins expected to be capable of undergoing liquid-liquid phase separation via either of these two mechanisms.

bioinformatics↗

bioRxiv2020

Kinetic analysis reveals the rates and mechanisms of protein aggregation in a multicellular organism

The accumulation of insoluble protein aggregates containing amyloid fibrils has been observed in many different human protein misfolding diseases1,2, and their pathological features have been recapitulated in diverse model systems3. In vitro kinetic studies have provided a quantitative understanding of how the fundamental molecular level processes of nucleation and growth lead to amyloid formation4. However, it is not yet clear to what extent these basic biophysical processes translate to amyloid formation in vivo, given the complexity of the cellular and organismal environment. Here we show that the aggregation of a fluorescently tagged polyglutamine (polyQ) protein into {micro}m-sized inclusions in the muscle tissue of living C. elegans can be quantitatively described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h-1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results are consistent with observations for the aggregation of polyQ proteins in vitro5 and in cell culture6, and highlight how nucleation events control the overall progression of aggregation in the organism through the spatial confinement into individual cells. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.

biophysics↗

bioRxiv2020

Infrared Nanospectroscopy Reveals the Molecular Interaction Fingerprint of an Aggregation Inhibitor with Single Aβ42 Oligomers

Very significant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimers and Parkinsons diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule level morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the interaction between single A{beta}42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, originally an anticancer drug capable recently shown to be able to inhibit A{beta}42 aggregation in animal models of Alzheimers disease. Our results demonstrate that the carbonyl group of this compound interacts with A{beta}42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as powerful tool in structure-based drug discovery for protein misfolding diseases.

biophysics↗

bioRxiv2020

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Misfolded -synuclein is a major component of Lewy bodies, which are a hallmark of Parkinsons disease. A large body of evidence shows that -synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinsons disease, that -synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of -synuclein condensates in the presence of lipids and other cellular components.

molecular biology↗

bioRxiv2020

Cholesterol rich, highly phase separated, naked mole-rat brain lipids are exquisitely sensitive to amyloid induced membrane damage

Naked mole-rats are extraordinarily long-lived rodents that do not develop age-related neurodegenerative diseases. Remarkably, they do not accumulate amyloid plaques, even though their brains contain high concentrations of amyloid beta peptide, even from a young age Therefore, these animals offer an opportunity to investigate mechanisms of resistance against the neurotoxicity of amyloid beta aggregation. Working in this direction, here we examine the composition, phase behaviour, and amyloid beta interactions of naked mole-rat brain lipids. Relative to mouse, naked mole-rat brain lipids are rich in cholesterol and contain sphingomyelin in lower amounts and of shorter chain lengths. Proteins associated with metabolism of ceramides, sphingomyelin and ceramide receptor activity were also found to be decreased in naked mole-rat brain lysates. Correspondingly, we find that naked mole-rat brain lipid membranes exhibit a high degree of phase separation, with the liquid ordered phase occupying up to 80% of the supported lipid bilayer. These observations are consistent with the membrane pacemaker hypothesis of ageing, according to which long-living species have lipid membranes particularly resistant to oxidative damage. However, we found that exposure to amyloid beta disrupts the naked mole-rat brain lipid membranes while those formed from mouse brain lipids exhibit small, well-defined footprints, whereby the amyloid beta penetrates deeply into the lipid membranes. These results suggest that in naked mole-rats the lipid composition of cell membranes may offer neuroprotection through resistance to oxidative processes rather than through mechanical effects.

biophysics↗

bioRxiv2020

A kinetic ensemble of the Alzheimer's Aβ peptide

The discovery that disordered proteins are widespread in the human proteome has prompted the quest for methods to characterize the conformational properties that determine their functional and dysfunctional behaviour. It has become customary to describe these proteins in terms of structural ensembles and free energy landscapes, which offer conformational and thermodynamic insight. However, a current major challenge is to generalize this description to kinetic ensembles, thereby also providing information on transition rates between states. Approaches based on the theory of stochastic processes can be particularly suitable for this purpose. Here, we develop a Markov state model and illustrate its application by determining a kinetic ensemble of the 42-residue form of the amyloid-{beta} peptide (A{beta}42), whose aggregation is associated with Alzheimers disease. Using the Google Compute Engine, we generated 315 s all-atom, explicit solvent molecular dynamics trajectories, validated with experimental data from nuclear magnetic resonance spectroscopy. Using a probabilistic-based definition of conformational states in a neural network approach, we found that A{beta}42 is characterized by inter-state transitions no longer than the microsecond timescale, exhibiting only fully unfolded or short-lived, partially-folded states. We contextualize our findings by performing additional simulations of the oxidized form of A{beta}42. Our results illustrate how the use of kinetic ensembles offers an effective means to provide information about the structure, thermodynamics, and kinetics of disordered proteins towards an understanding of these ubiquitous biomolecules.

biophysics↗

bioRxiv2020

Thermodynamic and kinetic design principles for protein aggregation inhibitors

Understanding the mechanism of action of compounds capable of inhibiting protein aggregation is critical to the development of potential ther-apeutics against protein misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of protein aggregation inhibition which reveals the fundamental thermodynamic and kinetic signatures characterising effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimisation of protein aggregation inhibitors.

biophysics↗

bioRxiv2020

Dynamics of oligomer populations formed during the aggregation of Alzheimer's Aβ42 peptide

Oligomeric aggregates populated during the aggregation of the A{beta}42 peptide have been identified as potent cytotoxins linked to Alzheimers disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach combining theory, experiment, and simulation, we reveal in molecular detail the mechanisms of A{beta}42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we find that most A{beta}42 oligomers dissociate to their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar species. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

biophysics↗

bioRxiv2019

Kinetic fingerprint of antibody therapies predicts outcomes of Alzheimer clinical trials

The amyloid cascade hypothesis, according to which the self-assembly of amyloid-{beta} peptide (A{beta}) is a causative process in Alzheimers disease, has driven many therapeutic efforts for the past 20 years. Failures of clinical trials investigating A{beta}-targeted therapies have been interpreted as evidence against this hypothesis, irrespective of the characteristics and mechanisms of action of the therapeutic agents, which are highly challenging to assess. We bring together kinetic analysis with quantitative binding measurements to address the mechanisms of action of four clinical stage anti-A{beta} antibodies, aducanumab, gantenerumab, bapineuzumab and solanezumab. We reveal and quantify the striking differences of these antibodies on the aggregation kinetics and on the production of oligomeric aggregates, and link these effects to the affinity and stoichiometry of each antibody for monomeric and fibrillar forms of A{beta}. Our results uncover that, uniquely amongst these four antibodies, aducanumab dramatically reduces the flux of oligomeric forms of A{beta}.

biophysics↗

bioRxiv2019

Escalating protein supersaturation underlies inclusion formation in muscle proteinopathies

Abundant, aggregation prone or \"supersaturated\" proteins are a feature of neurodegeneration. Whether the principle of supersaturation can similarly explain the widespread aggregation that occurs in non-neuronal protein conformational disorders and underlies pathogenic protein aggregate formation is not established. To test this prediction we analyzed proteomic datasets of biopsies from genetic and acquired protein aggregate myopathy (PAM) patients by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that similar to neurodegeneration, a supersaturated subproteome of aggregate prone proteins is present in skeletal muscle from healthy patients. This subproteome escalates in degree of supersaturation as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion containing fibers, supersaturated proteins within the aggregate subproteome rise in abundance, suggesting they escape normal regulation. We show in the context of a human conformational disorder that the level of supersaturation of a metastable subproteome helps to explain widespread aggregation and correlates with the histopathological state of the tissue.\n\nSignificanceIncreasing evidence implicates the phenomenon of protein supersaturation with the selective vulnerability of specific cells to protein misfolding disorders. Quantitative studies of this phenomenon, however, have only been possible post mortem in the case of neurodegenerative diseases. To overcome this limitation, we study here protein aggregate myopathies (PAMs), for which we were able to carry out systematic single fiber proteomic studies on patient-derived samples. We found not only that proteins associated with PAM inclusions are highly supersaturated in muscle but also that their supersaturation levels increases further in affected fibers. These results provide a clear illustration of how an escalation in supersaturation leads protein inclusions in vulnerable cells.

biophysics↗

bioRxiv2019

Small molecule sequestration of amyloid-β as a drug discovery strategy for Alzheimer’s disease

Disordered proteins are challenging therapeutic targets, and no drug is currently in clinical use that has been shown to modify the properties of their monomeric states. Here, we identify a small molecule capable of binding and sequestering the amyloid-{beta} peptide (A{beta}) in its monomeric, soluble state. Our analysis reveals that this compound interacts with A{beta} and inhibits both the primary and secondary nucleation pathways in its aggregation process. We characterise this interaction using biophysical experiments and integrative structural ensemble determination methods. We thus observe that this small molecule has the remarkable effect of increasing the conformational entropy of monomeric A{beta} while decreasing its hydrophobic surface area. We then show that this small molecule rescues a Caenorhabditis elegans model of A{beta}-associated toxicity in a manner consistent with the mechanism of action identified from the in silico and in vitro studies. These results provide an illustration of the strategy of targeting the monomeric states of disordered proteins with small molecules to alter their behaviour for therapeutic purposes.

biophysics↗

bioRxiv2019

Widespread remodelling of proteome solubility in response to different protein homeostasis stresses

The accumulation of protein deposits in neurodegenerative diseases involves the presence of a metastable subproteome vulnerable to aggregation. To investigate this subproteome and the mechanisms that regulates it, we measured the proteome solubility of the Neuro2a cell line under protein homeostasis stresses induced by Huntington Disease proteotoxicity; Hsp70, Hsp90, proteasome and ER-mediated folding inhibition; and oxidative stress. We found one-quarter of the proteome extensively changed solubility. Remarkably, almost all the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodelling of protein complexes involved in adaptation to perturbation, most notably stress granule proteins, which responded differently to different stresses. These results indicate that the robustness of protein homeostasis relies on the absence of proteins highly vulnerable to aggregation and on large changes in aggregation state of regulatory mechanisms that restore protein solubility upon specific perturbations.

biochemistry↗

bioRxiv2019

ADSoluble aggregates present in cerebrospinal fluid change in size and mechanism of toxicity during Alzheimer's disease progression

Soluble aggregates of amyloid-{beta} (A{beta}) have been associated with neuronal and synaptic loss in Alzheimers disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we studied the soluble A{beta} aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls. We found that these aggregates vary structurally and in their mechanisms of toxicity. More small aggregates of A{beta} that can cause membrane permeabilization already found in MCI; in established AD, the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells. These results suggest that different neurotoxic mechanisms are prevalent at different stages of AD.

neuroscience↗

bioRxiv2019

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly / dis-assembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is the major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration. HighlightsO_LIProgressive loss of stoichiometry affects multiple protein complexes C_LIO_LIRibosomes aggregate in old brains C_LIO_LIPartial reduction of proteasome activity is sufficient to induce loss of stoichiometry C_LIO_LIReduced proteasome levels are a major risk factor for early death in killifish C_LI

systems biology↗