Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson's disease genetically related mutants.

Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson's disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.LT has been recipient of a grant PAT Post Doc Outgoing 2009 – 7th Framework Program Marie Curie COFUND actions. NC was funded by a Royal Society Dorothy Hodgkin Research Fellowship and is currently a Ramón y Cajal Research Fellow (Spanish Ministry of Economy and Competitiveness). MHH thanks the Royal Society of Chemistry (Analytical Chemistry Trust Fund) for his studentship. AJD is funded by the Schiff Foundation.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1669

Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-synuclein oligomers.

α-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 αS 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 αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.M.H.H. thanks the Royal Society of Chemistry (Analytical Chemistry Trust Fund) for his studentship. L.T. has been the recipient of a grant PAT Post Doc Outgoing 2009 – 7th Framework Program Marie Curie COFUND actions. A.J.D. is funded by the Schiff Foundation.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acs.analchem.5b0181

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading.

The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.We thank Dr. Nadia Shivji and Beata Blaszczyk for ɑS protein expression, Dr. Peter Jönsson for help with preliminary TIRFM imaging experiments, Chris Taylor for help with preliminary autodilution experiments and Prof. Michel Goedert for critical reading of the manuscript. M.I. is funded by Dr. Tayyeb-Hussain Scholarship. G.A.G. is funded by the Schiff Foundation . S.G. is funded through a Wellcome Trust Intermediate Clinical Fellowship. Funding from the Frances and Augustus Newman Foundation, the European Research Council and the Biothechnology and Biophysical Sciences Research Council is gratefully acknowledged.This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.152412811

Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids.

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.This research study was funded in part by the Wellcome Trust/MRC Joint Call in Neurodegeneration award (WT089698) to the UK Parkinson's Disease Consortium (UKPDC) and the NIHR rare disease translational research collaboration and supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. We are also grateful to the Augustus Newman and Wolfson Foundations for their support. We thank the Royal Society for the University Research Fellowship of Dr. Steven F. Lee (UF120277).This is the final version of the article. It first appeared from ACS via http://dx.doi.org/10.1021/acschemneuro.5b00324

Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.M.I. acknowledges Dr. Tayyeb-Hussain Scholarship. L.T. has been recipient of a grant PAT Post Doc Outgoing 2009 7th Framework Program Marie Curie COFUND actions. C.D.H. and C.E.B. acknowledge funding from Alzheimer’s Research UK. Augustus Newman Foundation is acknowledged

RepeatsDB in 2021: Improved data and extended classification for protein tandem repeat structures

The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures.Fil: Paladin, Lisanna. Università di Padova; ItaliaFil: Bevilacqua, Martina. Università di Padova; ItaliaFil: Errigo, Sara. Università di Padova; ItaliaFil: Piovesan, Damiano. Università di Padova; ItaliaFil: Mičetić, Ivan. Università di Padova; ItaliaFil: Necci, Marco. Università di Padova; ItaliaFil: Monzon, Alexander Miguel. Università di Padova; ItaliaFil: Fabre, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; ArgentinaFil: López, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; ArgentinaFil: Nilsson, Juliet Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas; ArgentinaFil: Ríos, Javier Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Lorenzano Menna, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Cabrera, Maia Diana Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: González Buitrón, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Gonçalves Kulik, Mariane. Johannes Gutenberg Universitat Mainz; AlemaniaFil: Fernández Alberti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Fornasari, Maria Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Parisi, Gustavo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Lagares, Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas; ArgentinaFil: Hirsh, Layla. Pontificia Universidad Católica de Perú; PerúFil: Andrade Navarro, Miguel A.. Johannes Gutenberg Universitat Mainz; AlemaniaFil: Kajava, Andrey V. Centre National de la Recherche Scientifique; FranciaFil: Tosatto, Silvio C E. Università di Padova; Itali

Tools and data services registry: a community effort to document bioinformatics resources

Life sciences are yielding huge data sets that underpin scientific discoveries fundamental to improvement in human health, agriculture and the environment. In support of these discoveries, a plethora of databases and tools are deployed, in technically complex and diverse implementations, across a spectrum of scientific disciplines. The corpus of documentation of these resources is fragmented across the Web, with much redundancy, and has lacked a common standard of information. The outcome is that scientists must often struggle to find, understand, compare and use the best resources for the task at hand. Here we present a community-driven curation effort, supported by ELIXIR—the European infrastructure for biological information—that aspires to a comprehensive and consistent registry of information about bioinformatics resources. The sustainable upkeep of this Tools and Data Services Registry is assured by a curation effort driven by and tailored to local needs, and shared amongst a network of engaged partners. As of November 2015, the registry includes 1785 resources, with depositions from 126 individual registrations including 52 institutional providers and 74 individuals. With community support, the registry can become a standard for dissemination of information about bioinformatics resources: we welcome everyone to join us in this common endeavour. The registry is freely available at https://bio.tools

RepeatsDB in 2021: improved data and extended classification for protein tandem repeat structures

The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Conformational properties of the SDS-bound state of α-synuclein probed by limited proteolysis: Unexpected rigidity of the acidic C-terminal tail

alpha-Synuclein (alpha-syn) is a "natively unfolded" protein constituting the major component of intracellular inclusions in several neurodegenerative disorders. Here, we describe proteolysis experiments conducted on human alpha-syn in the presence of SDS micelles. Our aim was to unravel molecular features of micelle-bound alpha-syn using the limited proteolysis approach. The nonspecific proteases thermolysin and proteinase K, as well as the Glu-specific V8-protease, were used as proteolytic probes. While alpha-syn at neutral pH is easily degraded to a variety of relatively small fragments, in the presence of 10 mM SDS the proteolysis of the protein is rather selective. Complementary fragments 1-111 and 112-140, 1-113 and 114-140, and 1-123 and 124-140 are obtained when thermolysin, proteinase K, and V8 protease, respectively, are used. These results are in line with a conformational model of alpha-syn in which it acquires a folded helical structure in the N-terminal region in its membrane-bound state. At the same time, they indicate that the C-terminal portion of the molecule is rather rigid, as seen in its relative resistance to extensive proteolytic degradation. It is likely that, under the specific experimental conditions of proteolysis in the presence of SDS, the negatively charged C-terminal region can be rigidified by binding a calcium ion, as shown before with intact alpha-syn. In this study, some evidence of calcium binding properties of isolated C-terminal fragments 112-140, 114-140, and 124-140 was obtained by mass spectrometry measurements, since molecular masses for calcium-loaded fragments were obtained. Our results indicate that the C-terminal portion of the membrane-bound alpha-syn is quite rigid and structured, at variance from current models of the membrane-bound protein deduced mostly from NMR. Considering that the aggregation process of alpha-syn is modulated by its C-terminal tail, the results of this study may provide useful insights into the behavior of alpha-syn in a membrane-mimetic environmen