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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

Vasanthy Vigneswara

Simon Cass

Declan Wayne

Edward L. Bolt

David E. Ray



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), 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 http://eprints.nottingham.ac.uk/id/eprint/2971
Publisher URL http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061442
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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Copyright Statement
Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0





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