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Aggregation of TAR-DNA-binding protein 43 (TDP-43) and of its fragments TDP-25 and TDP-35 occurs in amyotrophic lateral sclerosis (ALS). TDP-25 and TDP-35 act as seeds for TDP-43 aggregation, altering its function and exerting toxicity. Thus, inhibition of TDP-25 and TDP-35 aggregation and promotion of their degradation may protect against cellular damage. Upregulation of HSPB8 is one possible approach for this purpose, since this chaperone promotes the clearance of an ALS associated fragments of TDP-43 and is upregulated in the surviving motor neurones of transgenic ALS mice and human patients. We report that overexpression of HSPB8 in immortalized motor neurones decreased the accumulation of TDP-25 and TDP-35 and that protection against mislocalized/truncated TDP-43 was observed for HSPB8 in Drosophila melanogaster. Overexpression of HSP67Bc, the functional ortholog of human HSPB8, suppressed the eye degeneration caused by the cytoplasmic accumulation of a TDP-43 variant with a mutation in the nuclear localization signal (TDP-43-NLS). TDP-43-NLS accumulation in retinal cells was counteracted by HSP67Bc overexpression. According with this finding, downregulation of HSP67Bc increased eye degeneration, an effect that is consistent with the accumulation of high molecular weight TDP-43 species and ubiquitinated proteins. Moreover, we report a novel Drosophila model expressing TDP-35, and show that while TDP-43 and TDP-25 expression in the fly eyes causes a mild degeneration, TDP-35 expression leads to severe neurodegeneration as revealed by pupae lethality; the latter effect could be rescued by HSP67Bc overexpression. Collectively, our data demonstrate that HSPB8 upregulation mitigates TDP-43 fragment mediated toxicity, in mammalian neuronal cells and flies.

The self-assembly of peptides and peptide mimetics into supramolecular polymers has been established in recent years as a route to biocompatible nanomaterials with novel mechanical, optical, and electronic properties. The morphologies of the resulting polymers are usually dictated by the strengths as well as lifetimes of the noncovalent bonds that lead to the formation of the structures. Together with an often incomplete understanding of the assembly mechanisms, these factors limit the control over the formation of polymers with tailored structures. Here, we have developed a microfluidic flow reactor to measure growth rates directly and accurately on the axial and radial faces of crystalline peptide supramolecular polymers. We show that the structures grow through two-dimensional nucleation mechanisms, with rates that depend exponentially on the concentration of soluble peptide. Using these mechanistic insights into the growth behavior of the axial and radial faces, we have been able to tune the aspect ratio of populations of dipeptide assemblies. These results demonstrate a general strategy to control kinetically self-assembly beyond thermodynamic products governed by the intrinsic properties of the building blocks in order to attain the required morphology and function.

The ability of biological molecules to replicate themselves, achieved with the aid of a complex cellular machinery, is the foundation of life. However, a range of aberrant processes involve the self-replication of pathological protein structures without any additional factors. A dramatic example is the autocatalytic replication of pathological protein aggregates, including amyloid fibrils and prions, involved in neurodegenerative disorders. Here, we use computer simulations to identify the necessary requirements for the self-replication of fibrillar assemblies of proteins. We establish that a key physical determinant for this process is the affinity of proteins for the surfaces of fibrils. We find that self-replication can only take place in a very narrow regime of inter-protein interactions, implying a high level of sensitivity to system parameters and experimental conditions. We then compare our theoretical predictions with kinetic and biosensor measurements of fibrils formed from the Aβ peptide associated with Alzheimer’s disease. Our results show a quantitative connection between the kinetics of self-replication and the surface coverage of fibrils by monomeric proteins. These findings reveal the fundamental physical requirements for the formation of supra-molecular structures able to replicate themselves, and shed light on mechanisms in play in the proliferation of protein aggregates in nature.

Item does not contain fulltext Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.

The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson's disease (PD). Using solution-state nuclear magnetic resonance (NMR) spectroscopy and coarse-grained molecular dynamics (MD) simulations, we find that, although the nascent chain (NC) has a highly disordered conformation, its N-terminal region shows resonance broadening consistent with interactions involving specific regions of the ribosome surface. We also investigated the effects of the ribosome-associated molecular chaperone trigger factor (TF) on αSyn structure and dynamics using resonance broadening to define a footprint of the TF-RNC interactions. We have used these data to construct structural models that suggest specific ways by which emerging NCs can interact with the biosynthesis and quality control machinery.

Conventional approaches for simulating steady-state distributions of dilute particles under diffusive and advective transport involve solving the diffusion and advection equations in at least two dimensions. Here, we present an alternative computational strategy by combining a particle-based rather than a field-based approach with the initialisation of particles in proportion to their flux. This method allows accurate prediction of the steady state and is applicable even at intermediate and high Péclet numbers ( P e > 1 ) $ (Pe \gt 1)$ where traditional particle-based Monte-Carlo methods starting from randomly initialised particle distributions fail. We demonstrate that generating a flux of particles according to a predetermined density and velocity distribution at a single fixed time and initial location allows for accurate simulation of mass transport under flow. Specifically, upon initialisation in proportion to their flux, these particles are propagated individually and detected by summing up their Monte-Carlo trajectories in predefined detection regions. We demonstrate quantitative agreement of the predicted concentration profiles with the results of experiments performed with fluorescent particles in microfluidic channels under continuous flow. This approach is computationally advantageous and readily allows non-trivial initial distributions to be considered. In particular, this method is highly suitable for simulating advective and diffusive transport in microfluidic devices, for instance in the context of diffusive sizing.

It is well-established that widespread transcriptional changes accompany the onset and progression of Alzheimer's disease. Because of the multifactorial nature of this neurodegenerative disorder and its complex relationship with aging, however, it remains unclear whether such changes are the result of nonspecific dysregulation and multisystem failure or instead are part of a coordinated response to cellular dysfunction. To address this problem in a systematic manner, we performed a meta-analysis of about 1,600 microarrays from human central nervous system tissues to identify transcriptional changes upon aging and as a result of Alzheimer's disease. Our strategy to discover a transcriptional signature of Alzheimer's disease revealed a set of down-regulated genes that encode proteins metastable to aggregation. Using this approach, we identified a small number of biochemical pathways, notably oxidative phosphorylation, enriched in proteins vulnerable to aggregation in control brains and encoded by genes down-regulated in Alzheimer's disease. These results suggest that the down-regulation of a metastable subproteome may help mitigate aberrant protein aggregation when protein homeostasis becomes compromised in Alzheimer's disease.

New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

α-Synuclein is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease and other related neurodegenerative disorders. Recently, two single-domain camelid antibodies (nanobodies) were shown to bind α-synuclein with high affinity. Herein, we investigated how these two nanobodies (NbSyn2 and NbSyn87), which are directed to two distinct epitopes within the C-terminal domain of α-synuclein, affect the conformational properties of this protein. Our results suggest that nanobody NbSyn2, which binds to the five C-terminal residues of α-synuclein (residues 136-140), does not disrupt the transient long-range interactions that generate a degree of compaction within the native structural ensemble of α-synuclein. In contrast, the data that we report indicate that NbSyn87, which targets a central region within the C-terminal domain (residues 118-128), has more substantial effects on the fluctuating secondary and tertiary structure of the protein. These results are consistent with the different effects that the two nanobodies have on the aggregation behavior of α-synuclein in vitro. Our findings thus provide new insights into the type of effects that nanobodies can have on the conformational ensemble of α-synuclein.

Alzheimer&rsquo, s disease is associated with the aggregation of the amyloid-&beta, peptide (A&beta, ), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which A&beta, aggregates induce neuronal dysfunction has highlighted the importance of the A&beta, oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure&ndash, toxicity relationship of A&beta, oligomers. For this purpose, we use Zn2+-stabilized oligomers of the 40-residue form of A&beta, (A&beta, 40) as models of brain A&beta, oligomers and two single-domain antibodies (DesAb18-24 and DesAb34-40), designed to bind to epitopes at residues 18&ndash, 24 and 34&ndash, 40 of A&beta, 40, respectively. We found that the DesAbs induce a change in structure of the Zn2+-stabilized A&beta, 40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure&ndash, toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of A&beta, oligomers.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated α-synuclein (α-syn). These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression. α-Synuclein exists in multiple structural forms, including disordered, nonamyloid oligomers, ordered amyloid oligomers, and fibrils. It is critical to understand which conformers contribute to specific PD phenotypes. Here, we utilized a mouse model to explore the pathological effects of stable β-amyloid-sheet oligomers compared with those of fibrillar α-synuclein. We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays. We then injected these different α-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes. We found that β-sheet oligomers produce a small but significant loss of dopamine neurons in the substantia nigra pars compacta (SNc). Injection of small β-sheet fibril fragments, however, produced the most robust phenotypes, including reduction of striatal dopamine terminals, SNc loss of dopamine neurons, and motor-behavior defects. We conclude that although the β-sheet oligomers cause some toxicity, the potent effects of the short fibrillar fragments can be attributed to their ability to recruit monomeric α-synuclein and spread

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

Identifying the A- and P-site locations on ribosome-protected mRNA fragments from Ribo-Seq experiments is a fundamental step in the quantitative analysis of transcriptome-wide translation properties at the codon level. Many analyses of Ribo-Seq data have utilized heuristic approaches applied to a narrow range of fragment sizes to identify the A-site. In this study, we use Integer Programming to identify the A-site by maximizing an objective function that reflects the fact that the ribosome’s A-site on ribosome-protected fragments must reside between the second and stop codons of an mRNA. This identifies the A-site location as a function of the fragment’s size and its 5′ end reading frame in Ribo-Seq data generated from S. cerevisiae and mouse embryonic stem cells. The correctness of the identified A-site locations is demonstrated by showing that this method, as compared to others, yields the largest ribosome density at established stalling sites. By providing greater accuracy and utilization of a wider range of fragment sizes, our approach increases the signal-to-noise ratio of underlying biological signals associated with translation elongation at the codon length scale.

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the health and fitness of the animals under study, one typically relies on motility readouts, such as the measurement of the number of body bends or the speed of movement. These measurements usually involve manual counting, making it challenging to obtain good statistical significance, as time and labor constraints often limit the number of animals in each experiment to 25 or less. Since high statistical power is necessary to obtain reproducible results and limit false positive and negative results when weak phenotypic effects are investigated, efforts have recently been made to develop automated protocols focused on increasing the sensitivity of motility detection and multi-parametric behavioral profiling. In order to extend the limit of detection to the level needed to capture the small phenotypic changes that are often crucial in genetic studies and drug discovery, we describe here a technological development that enables the study of up to 5,000 individual animals simultaneously, increasing the statistical power of the measurements by about 1,000-fold compared to manual assays and about 100-fold compared to other available automated methods.

The misfolding and aggregation of proteins into linear fibrils is widespread in human biology, for example, in connection with amyloid formation and the pathology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The oligomeric species that are formed in the early stages of protein aggregation are of great interest, having been linked with the cellular toxicity associated with these conditions. However, these species are not characterized in any detail experimentally, and their properties are not well understood. Many of these species have been found to have approximately spherical morphology and to be held together by hydrophobic interactions. We present here an analytical statistical mechanical model of globular oligomer formation from simple idealized amphiphilic protein monomers and show that this correlates well with Monte Carlo simulations of oligomer formation. We identify the controlling parameters of the model, which are closely related to simple quantities that may be fitted directly from experiment. We predict that globular oligomers are unlikely to form at equilibrium in many polypeptide systems but instead form transiently in the early stages of amyloid formation. We contrast the globular model of oligomer formation to a well-established model of linear oligomer formation, highlighting how the differing ensemble properties of linear and globular oligomers offer a potential strategy for characterizing oligomers from experimental measurements.

Oligomeric species populated during the aggregation of the Aβ42 peptide have been identified as potent cytotoxins linked to Alzheimer's disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach that combines theory, experiment and simulation, we reveal, in molecular detail, the mechanisms of Aβ42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we found that most Aβ42 oligomers dissociate into their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar structures. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to those of aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases.

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinson’s and Huntington’s diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine. In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

Small oligomers formed during the aggregation of certain peptides and proteins are highly cytotoxic in numerous neurodegenerative disorders. Because of their transient nature and conformational heterogeneity, however, the structural and biological features of these oligomers are still poorly understood. Here, we describe a method of generating stable oligomers formed by the Alzheimer's Aβ

Oligomeric and protofibrillar aggregates that are populated along the pathway of amyloid fibril formation appear generally to be more toxic than the mature fibrillar state. In particular, α-synuclein, the protein associated with Parkinson's disease, forms kinetically trapped protofibrils in the presence of lipid vesicles. Here, we show that lipid-induced α-synuclein protofibrils can convert rapidly to mature fibrils at higher temperatures. Furthermore, we find that β-synuclein, generally considered less aggregation prone than α-synuclein, forms protofibrils at higher temperatures. These findings highlight the importance of energy barriers in controlling the de novo formation and conversion of amyloid fibrils.

The aggregates of the Aβ peptide associated with Alzheimer’s disease are able to both grow in size as well as generate, through secondary nucleation, new small oligomeric species, that are major cytotoxins associated with neuronal death. Despite the importance of these amyloid fibril-dependent processes, their structural and molecular underpinnings have remained challenging to elucidate. Here, we consider two molecular chaperones: the Brichos domain, which suppresses specifically secondary nucleation processes, and clusterin which our results show is capable of inhibiting, specifically, the elongation of Aβ fibrils at remarkably low substoichiometric ratios. Microfluidic diffusional sizing measurements demonstrate that this inhibition originates from interactions of clusterin with fibril ends with high affinity. Kinetic experiments in the presence of both molecular chaperones reveal that their inhibitory effects are additive and noncooperative, thereby indicating that the reactive sites associated with the formation of new aggregates and the growth of existing aggregates are distinct., Science Advances, 5 (4), ISSN:2375-2548

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the stalk protein bL12, and this information was used to refine a 3D structure of its C-terminal domain (CTD). Orientational constraints on the CTD alignment arising from the semiflexible linker sequence were further probed by a paramagnetic alignment strategy, and provided direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome–nascent chain complexes.

This study is supported by the Michael J. Fox Foundation (10200); The Royal Society with a University Research Fellowship (UF120277) (S.F.L.); a Marie‐Curie Individual Fellowship (S.D.); the Boehringer Ingelheim Fonds (P.F.), the Studienstiftung des Deutschen Volkes (P.F.); the UK Biotechnology and Biological Sciences Research Council (C.M.D.); the Wellcome Trust (C.M.D.); the Cambridge Centre for Misfolding Diseases (P.F. and C.M.D.), and the Royal Society and the European Research Council with an ERC Advanced Grant (669237) (D.K.). Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all-optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin-T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α-synuclein. By studying the diffraction-limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age-matched healthy controls. Publisher PDF

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid‐specific aptamer, we demonstrate that this technique is able to detect and super‐resolve a range of aggregated species, including those formed by α‐synuclein and amyloid‐β. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Understanding how normally soluble peptides and proteins aggregate to form amyloid fibrils is central to many areas of modern biomolecular science, ranging from the development of functional biomaterials to the design of rational therapeutic strategies against increasingly prevalent medical conditions such as Alzheimer's and Parkinson's diseases. As such, there is a great need to develop models to mechanistically describe how amyloid fibrils are formed from precursor peptides and proteins. Here we review and discuss how ideas and concepts from chemical reaction kinetics can help to achieve this objective. In particular, we show how a combination of theory, experiments, and computer simulations, based on chemical kinetics, provides a general formalism for uncovering, at the molecular level, the mechanistic steps that underlie the phenomenon of amyloid fibril formation.

Native silk fibroin (NSF) is a unique biomaterial with extraordinary mechanical and biochemical properties. These key characteristics are directly associated with the physical transformation of unstructured, soluble NSF into highly organized nano‐ and microscale fibrils rich in β‐sheet content. Here, it is shown that this NSF fibrillation process is accompanied by the development of intrinsic fluorescence in the visible range, upon near‐UV excitation, a phenomenon that has not been investigated in detail to date. Here, the optical and fluorescence characteristics of NSF fibrils are probed and a route for potential applications in the field of self‐assembled optically active biomaterials and systems is explored. In particular, it is demonstrated that NSF can be structured into autofluorescent microcapsules with a controllable level of β‐sheet content and fluorescence properties. Furthermore, a facile and efficient fabrication route that permits arbitrary patterns of NSF microcapsules to be deposited on substrates under ambient conditions is shown. The resulting fluorescent NSF patterns display a high level of photostability. These results demonstrate the potential of using native silk as a new class of biocompatible photonic material., Macromolecular Bioscience, 18 (4), ISSN:1616-5187, ISSN:1616-5195

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H 2 O 2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H 2 O 2 . We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H 2 O 2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

The self-assembly of polypeptides into amyloid structures is associated with a range of increasingly prevalent neurodegenerative diseases as well as with a select set of functional processes in biology. The phenomenon of self-assembly results in species with dramatically different sizes, from small oligomers to large fibrils; however, the kinetic relationship between these species is challenging to characterize. In the case of prion aggregates, these structures can self-replicate and act as infectious agents. Here we use single molecule spectroscopy to obtain quantitative information on the oligomer populations formed during aggregation of the yeast prion protein Ure2. Global analysis of the aggregation kinetics reveals the molecular mechanism underlying oligomer formation and depletion. Quantitative characterization indicates that the majority of Ure2 oligomers are relatively short-lived, and their rate of dissociation is much higher than their rate of conversion into growing fibrils. We identify an initial metastable oligomer, which can subsequently convert into a structurally distinct oligomer, which in turn converts into growing fibrils. We also show that fragmentation is responsible for the autocatalytic self-replication of Ure2 fibrils, but that preformed fibrils do not promote oligomer formation, indicating that secondary nucleation of the type observed for peptides and proteins associated with neurodegenerative disease does not occur at a significant rate for Ure2. These results establish a framework for elucidating the temporal and causal relationship between oligomers and larger fibrillar species in amyloid forming systems, and provide insights into why functional amyloid systems are not toxic to their host organisms.

Item does not contain fulltext The human molecular chaperone DNAJB6, an oligomeric protein with a conserved S/T-rich region, is an efficient suppressor of amyloid fibril formation by highly aggregation-prone peptides such as the Abeta and polyQ peptides associated with Alzheimer's and Huntington's disease, respectively. We previously showed that DNAJB6 can inhibit the processes through which amyloid fibrils are formed via strong interactions with aggregated forms of Abeta42 that become sequestered. Here we report that the concentration-dependent capability of DNAJB6 to suppress fibril formation in thioflavin T fluorescence assays decreases progressively with an increasing number of S/T substitutions, with an almost complete loss of suppression when 18 S/T residues are substituted. The kinetics of primary nucleation in particular are affected. No detectable changes in the structure are caused by the substitutions. Also, the level of binding of DNAJB6 to Abeta42 decreases with the S/T substitutions, as determined by surface plasmon resonance and microscale thermophoresis. The aggregation process monitored using nuclear magnetic resonance spectroscopy showed that DNAJB6, in contrast to a mutational variant with 18 S/T residues substituted, can keep monomeric Abeta42 soluble for an extended time. The inhibition of the primary nucleation is likely to depend on hydroxyl groups in side chains of the S/T residues, and hydrogen bonding with Abeta42 is one plausible molecular mechanism, although other possibilities cannot be excluded. The loss of the ability to suppress fibril formation upon S/T to A substitution was previously observed also for polyQ peptides, suggesting that the S/T residues in the DNAJB6-like chaperones have a general ability to inhibit amyloid fibril formation by different aggregation-prone peptides.

Therapeutics designed to target α-synuclein (α-syn) aggregation may be critical in halting the progression of pathology in Parkinson’s disease (PD) patients. Nanobodies are single-domain antibody fragments that bind with antibody specificity, but allow readier genetic engineering and delivery. When expressed intracellularly as intrabodies, anti-α-syn nanobodies fused to a proteasome-targeting proline, aspartate or glutamate, serine, and threonine (PEST) motif can modulate monomeric concentrations of target proteins. Here we aimed to validate and compare the in vivo therapeutic potential of gene therapy delivery of two proteasome-directed nanobodies selectively targeting α-syn in a synuclein overexpression-based PD model: VH14*PEST (non-amyloid component region) and NbSyn87*PEST (C-terminal region). Stereotaxic injections of adeno-associated viral 5-α-syn (AAV5-α-syn) into the substantia nigra (SN) were performed in Sprague–Dawley rats that were sorted into three cohorts based on pre-operative behavioral testing. Rats were treated with unilateral SN injections of vectors for VH14*PEST, NbSyn87*PEST, or injected with saline 3 weeks post lesion. Post-mortem assessments of the SN showed that both nanobodies markedly reduced the level of phosphorylated Serine-129 α-syn labeling relative to saline-treated animals. VH14*PEST showed considerable maintenance of striatal dopaminergic tone in comparison to saline-treated and NbSyn87*PEST-treated animals as measured by tyrosine hydroxylase immunoreactivity (optical density), DAT immunoreactivity (optical density), and dopamine concentration (high-performance liquid chromatography). Microglial accumulation and inflammatory response, assessed by stereological counts of Iba-1-labeled cells, was modestly increased in NbSyn87*PEST-injected rats but not in VH14*PEST-treated or saline-treated animals. Modest behavioral rescue was also observed, although there was pronounced variability among individual animals. These data validate in vivo therapeutic efficacy of vector-delivered intracellular nanobodies targeting α-syn misfolding and aggregation in synucleinopathies such as PD., Nanomedicine: A disease-modifying approach Two nanobodies – fragments of antibodies – that target the highly conserved protein alpha-synuclein for degradation reduce neurotoxicity in a rat model of Parkinson’s Disease (PD). Misfolded aggregated alpha-synuclein is the major component of the characteristic protein deposits found in PD, Lewy bodies. Previous studies have shown that alpha-synuclein-binding nanobodies containing a peptide sequence that acts as a signal for protein degradation interfere with alpha-synuclein aggregation in neurons. Jeffrey Kordower at Rush University Medical Center in Chicago, USA, and colleagues have used viruses to deliver these nanobodies into the substantia nigra of rats overexpressing alpha-synuclein in this brain region. The nanobodies not only reduced alpha-synuclein aggregation, they also improved motor function. These findings highlight a promising approach for halting the progression of PD and other neurodegenerative diseases characterized by the accumulation of abnormal alpha-synuclein aggregates.

Scanning probe microscopy provides a unique window into the morphology, mechanics, and structure of proteins and their complexes on the nanoscale. Such measurements require, however, deposition of samples onto substrates. This process can affect conformations and assembly states of the molecular species under investigation and can bias the molecular populations observed in heterogeneous samples through differential adsorption. Here, we show that these limitations can be overcome with a single-step microfluidic spray deposition platform. This method transfers biological solutions to substrates as microdroplets with subpicoliter volume, drying in milliseconds, a timescale that is shorter than typical diffusion times of proteins on liquid–solid interfaces, thus avoiding surface mass transport and change to the assembly state. Finally, the single-step deposition ensures the attachment of the full molecular content of the sample to the substrate, allowing quantitative measurements of different molecular populations within heterogeneous systems, including protein aggregates.

We have studied two misfolded oligomeric forms of the protein HypF-N, which show similar morphologies but very different toxicities. We measured over 80 intermolecular distance-dependent parameters for each oligomer type using FRET, in conjunction with solution- and solid-state NMR and other biophysical techniques. The results indicate that the formation of a highly organised hydrogen bonded core in the toxic oligomers results in the exposure of a larger number of hydrophobic residues than in the nontoxic species, causing the former to form aberrant interactions with cellular components.

As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remainunknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature. Here, we used ensemble and single-molecule fluorescence measurements to dissect how Hsp70 counteracts the selfassembly process of K18 tau with the pathological deletion ΔK280. We found that Hsp70 blocks the early stages of tau aggregationby suppressing the formation of tau nuclei. Additionally, Hsp70 sequesters oligomers and mature tau fibrils with nanomolar affinity into a protective complex, efficiently neutralizing their ability to damage membranes and seed further tau aggregation. Our results provide novel insights into the molecular mechanisms by which the chaperone Hsp70 counteracts the formation, propagation and toxicity of tau aggregates.

The aggregation process of peptides and proteins is of great relevance as it is associated with a wide range of highly debilitating disorders, including Alzheimer's and Parkinson's diseases. The natural product (-)-epigallocatechin-3-gallate (EGCG) can redirect this process away from amyloid fibrils and towards non-toxic oligomers. In this study we used nuclear magnetic resonance (NMR) spectroscopy to characterize the binding of EGCG to a set of natively structured and unstructured proteins. The results show that the binding process is dramatically dependent on the conformational properties of the protein involved, as EGCG interacts with different binding modes depending on the folding state of the protein. We used replica exchange molecular dynamics simulations to reproduce the trends observed in the NMR experiments, and analyzed the resulting samplings to identify the dominant direct interactions between EGCG and ordered and disordered proteins.

Small oligomers formed during the aggregation of certain peptides and proteins are highly cytotoxic in numerous neurodegenerative disorders. Because of their transient nature and conformational heterogeneity, however, the structural and biological features of these oligomers are still poorly understood. Here, we describe a method of generating stable oligomers formed by the Alzheimer's Aβ40 peptide by carrying out an aggregation reaction in the presence of zinc ions. The resulting oligomers are amenable to detailed biophysical and biological characterization, which reveals a homogeneous population with small size, high cross-β sheet structure content, and extended hydrophobic surface patches. We also show that these oligomers decrease the viability of neuroblastoma cells and impair the motility of C. elegans. The availability of these oligomers offers novel opportunities for studying the mechanisms of Aβ 40 toxicity in vitro and in cellular and animal models of Alzheimer's disease.

Alzheimer’s disease represents one of the greatest challenges to the social fabric and health care systems of the world since the great plagues of the Middle Ages. At the present time, there are some 40 million sufferers from this disease worldwide, but it is predicted that this number will rise to nearly 150 million by 2050. The predominant reason for this rapidly increasing prevalence is the increase in longevity that has resulted from the tremendous advances in public health and hygiene and in medical and surgical interventions over the last century. This article discusses recent progress in our understanding of the molecular origins of Alzheimer’s disease and emerging ideas for new and rational therapeutic strategies by which to combat its onset and progression.

Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinson's disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of ?S are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of ?S oligomers and can help to rationalize some aspects of ?S amyloid fibril formation. (Figure Presented).

The generation of mechanical forces are central to a wide range of vital biological processes, including the function of the cytoskeleton. Although the forces emerging from the polymerization of native proteins have been studied in detail, the potential for force generation by aberrant protein polymerization has not yet been explored. Here, we show that the growth of amyloid fibrils, archetypical aberrant protein polymers, is capable of unleashing mechanical forces on the piconewton scale for individual filaments. We apply microfluidic techniques to measure the forces released by amyloid growth for two systems: insulin and lysozyme. The level of force measured for amyloid growth in both systems is comparable to that observed for actin and tubulin, systems that have evolved to generate force during their native functions and, unlike amyloid growth, rely on the input of external energy in the form of nucleotide hydrolysis for maximum force generation. Furthermore, we find that the power density released from growing amyloid fibrils is comparable to that of high-performance synthetic polymer actuators. These findings highlight the potential of amyloid structures as active materials and shed light on the criteria for regulation and reversibility that guide molecular evolution of functional polymers.

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions.

Aging has been associated with a progressive decline of proteostasis, but how this process manifests itself at the level of proteome composition remains largely unexplored. Here we profiled more than 5,000 proteins along the lifespan of the nematode C. elegans. We find that one third of proteins change in abundance at least 2-fold during aging, resulting in a severe proteome imbalance. These changes are reduced in the long-lived daf-2 mutant, but enhanced in the short-lived daf-16 mutant. While ribosomal proteins decline and lose normal stoichiometry, proteasome complexes increase. Proteome imbalance is accompanied by widespread protein aggregation, with abundant proteins that exceed solubility contributing most to aggregate load. Notably, the properties by which proteins are selected for aggregation differ in the daf-2 mutant, and an increased formation of aggregates associated with small heat shock proteins is observed. We suggest that sequestering proteins into chaperone-enriched aggregates is a protective strategy to slow proteostasis decline during nematode aging.

Investigations of the fundamental physical principles underlying Alzheimer’s and Parkinson’s diseases are providing new insights into their origins and are suggesting rational strategies through which to combat their onset and progression.

Nanofibrillar forms of proteins were initially recognized in the context of pathology, but more recently have been discovered in a range of functional roles in nature, including as active catalytic scaffolds and bacterial coatings. Here we show that protein nanofibrils can be used to form the basis of monodisperse microgels and gel shells composed of naturally occurring proteins. We explore the potential of these protein microgels to act as drug carrier agents, and demonstrate the controlled release of four different encapsulated drug-like small molecules, as well as the component proteins themselves. Furthermore, we show that protein nanofibril self-assembly can continue after the initial formation of the microgel particles, and that this process results in active materials with network densities that can be modulated in situ. We demonstrate that these materials are nontoxic to human cells and that they can be used to enhance the efficacy of antibiotics relative to delivery in homogeneous solution. Because of the biocompatibility and biodegradability of natural proteins used in the fabrication of the microgels, as well as their ability to control the release of small molecules and biopolymers, protein nanofibril microgels represent a promising class of functional artificial multiscale materials generated from natural building blocks.

Intracellular deposits of α‐synuclein in the form of Lewy bodies are major hallmarks of Parkinson's disease (PD) and a range of related neurodegenerative disorders. Post‐translational modifications (PTMs) of α‐synuclein are increasingly thought to be major modulators of its structure, function, degradation and toxicity. Among these PTMs, phosphorylation near the C‐terminus at S129 has emerged as a dominant pathogenic modification as it is consistently observed to occur within the brain and cerebrospinal fluid (CSF) of post‐mortem PD patients, and its level appears to correlate with disease progression. Phosphorylation at the neighboring tyrosine residue Y125 has also been shown to protect against α‐synuclein toxicity in a Drosophila model of PD. In the present study we address the potential roles of C‐terminal phosphorylation in modulating the interaction of α‐synuclein with other protein partners, using a single domain antibody fragment (NbSyn87) that binds to the C‐terminal region of α‐synuclein with nanomolar affinity. The results reveal that phosphorylation at S129 has negligible effect on the binding affinity of NbSyn87 to α‐synuclein while phosphorylation at Y125, only four residues away, decreases the binding affinity by a factor of 400. These findings show that, despite the fact that α‐synuclein is intrinsically disordered in solution, selective phosphorylation can modulate significantly its interactions with other molecules and suggest how this particular form of modification could play a key role in regulating the normal and aberrant function of α‐synuclein.

The nematode worm Caenorhabditis elegans (C. elegans) is a versatile and widely used animal model for in vivo studies of a broad range of human diseases, in particular for understanding their genetic origins and for screening drug candidates. Nevertheless, the challenges associated with the administration of native proteins to C. elegans have limited the range of applications of this animal model in protein-based drug discovery programs. Here, we describe a readily usable protocol for the transduction of native proteins in C. elegans, which is based on the encapsulation of the proteins of interest within cationic lipid vesicles, prior to their administration to worms. This procedure limits the degradation of the proteins in the guts of the animals, and promotes their adsorption into body tissues. To illustrate the efficacy of this approach we apply it to deliver an antibody designed to inhibit α-synuclein aggregation, and show that it can lead to the rescue of the disease phenotype in a C. elegans model of Parkinson’s disease. As this transduction protocol is fast and inexpensive, we anticipate that it will be readily applicable to protein-based drug discovery studies that utilize C. elegans as a model organism.

It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease. Electronic supplementary material The online version of this article (10.1186/s40478-017-0481-1) contains supplementary material, which is available to authorized users.