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

Abstract

Abnormal ?-synuclein aggregates are hallmarks of a number of neurodegenerative diseases. Alpha synuclein and ?-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 ?-synuclein, the ?-synuclein familial mutants A30P and A53T that give rise to Parkinsonian phenotypes, and ?-synuclein, at physiological pH and temperature for a time course of up to 20 days. Resolution of native ?-synuclein and ?-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 ?-synuclein proteins accumulated isoaspartate at ~1% of molecules/day, ~20 times faster than for ?-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 ?-synucleins accumulated stable oligomers that were resistant to denaturing conditions, and which also contained isoaspartate. Co-incubation of approximately equimolar ?-synuclein with ?-synuclein resulted in a significant reduction of isoaspartate formed in all ?-synucleins after 20 days of ageing. Co-incubated ?- and ?-synucleins, or ?, or ? synucleins alone, were resolved by non-denaturing size exclusion chromatography and all formed oligomers of ~57.5 kDa; consistent with tetramerization. Direct association of ?-synuclein with ?-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 ?- and ?-synucleins during ageing, and highlight the susceptibility of ?-synuclein to protein damage, and the potential protective role of ?-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