Vasanthy Vigneswara
Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms
Vigneswara, Vasanthy; Cass, Simon; Wayne, Declan; Bolt, Edward L.; Ray, David E.; Carter, Wayne
Authors
Simon Cass
Declan Wayne
Edward L. Bolt
David E. Ray
Dr WAYNE CARTER WAYNE.CARTER@NOTTINGHAM.AC.UK
ASSOCIATE PROFESSOR
Abstract
Abnormal α-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and β-synucleins are susceptible to post-translational modification as isoaspartate protein damage, which is regulated in vivo by the action of the repair enzyme protein L-isoaspartyl O-methyltransferase (PIMT). We aged in vitro native α-synuclein, the α-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and β-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native α-synuclein and β-synuclein by two dimensional techniques showed the accumulation of a number of post-translationally modified forms of both proteins. The levels of isoaspartate formed over the 20 day time course were quantified by exogenous methylation with PIMT using S-Adenosyl-L-[3H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated 3H-methanol. All α-synuclein proteins accumulated isoaspartate at ~1% of molecules/day, ~20 times faster than for β-synuclein. This disparity between rates of isoaspartate was confirmed by exogenous methylation of synucleins by PIMT, protein resolution by one-dimensional denaturing gel electrophoresis, and visualisation of 3H-methyl esters by autoradiography. Protein silver staining and autoradiography also revealed that α-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar β-synuclein with α-synuclein resulted in a significant reduction of isoaspartate formed in all α-synucleins after 20 days of ageing. Co-incubated α- and β-synucleins, or α, or β synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ~57.5 kDa; consistent with tetramerization. Direct association of α-synuclein with β-synuclein in column fractions or from in vitro ageing co-incubations was demonstrated by their co-immunoprecipitation. These results provide an insight into the molecular differences between α- and β-synucleins during ageing, and highlight the susceptibility of α-synuclein to protein damage, and the potential protective role of β-synuclein.
Citation
Vigneswara, V., Cass, S., Wayne, D., Bolt, E. L., Ray, D. E., & Carter, W. (2013). Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms. PLoS ONE, 8(4), Article e61442. https://doi.org/10.1371/journal.pone.0061442
Journal Article Type | Article |
---|---|
Acceptance Date | Apr 14, 2013 |
Publication Date | Apr 22, 2013 |
Deposit Date | Apr 22, 2014 |
Publicly Available Date | Apr 22, 2014 |
Journal | PLoS ONE |
Electronic ISSN | 1932-6203 |
Publisher | Public Library of Science |
Peer Reviewed | Peer Reviewed |
Volume | 8 |
Issue | 4 |
Article Number | e61442 |
DOI | https://doi.org/10.1371/journal.pone.0061442 |
Public URL | https://nottingham-repository.worktribe.com/output/714347 |
Publisher URL | http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061442 |
Contract Date | Apr 22, 2014 |
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Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0
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