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

ABSTRACT: The protein alpha-synuclein (?S) 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 ?S 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 ?S. The resulting explicit kinetic model of ?S 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.

SUBMITTER: Iljina M

PROVIDER: S-EPMC4780632 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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

Iljina Marija M Garcia Gonzalo A GA Horrocks Mathew H MH Tosatto Laura L Choi Minee L ML Ganzinger Kristina A KA Abramov Andrey Y AY Gandhi Sonia S Wood Nicholas W NW Cremades Nunilo N Dobson Christopher M CM Knowles Tuomas P J TP Klenerman David D

Proceedings of the National Academy of Sciences of the United States of America 20160216 9

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-m ...[more]

PMID: 26884195

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Project description:Aggregation of neuronal protein ?-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of ?-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to ?-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such ?-synuclein sequence extension on ?-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of ?-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of ?-synuclein. To address these questions, we prepared mutants of ?-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type ?-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of ?-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of ?-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

Dataset Information

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

Publications

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

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