Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms
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-methyltra...
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Public Library of Science
2013
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| Online Access: | https://eprints.nottingham.ac.uk/2971/ |
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| author | Vigneswara, Vasanthy Cass, Simon Wayne, Declan Bolt, Edward L. Ray, David E. Carter, Wayne |
| author_facet | Vigneswara, Vasanthy Cass, Simon Wayne, Declan Bolt, Edward L. Ray, David E. Carter, Wayne |
| author_sort | Vigneswara, Vasanthy |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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. |
| first_indexed | 2025-11-14T18:20:17Z |
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| id | nottingham-2971 |
| institution | University of Nottingham Malaysia Campus |
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| last_indexed | 2025-11-14T18:20:17Z |
| publishDate | 2013 |
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| spelling | nottingham-29712020-05-04T16:36:22Z https://eprints.nottingham.ac.uk/2971/ 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 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. Public Library of Science 2013-04-22 Article PeerReviewed Vigneswara, Vasanthy, Cass, Simon, Wayne, Declan, Bolt, Edward L., Ray, David E. and Carter, Wayne (2013) Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms. PLoS ONE, 8 (4). e61442/1-e61442/12. ISSN 1932-6203 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061442 doi:10.1371/journal.pone.0061442 doi:10.1371/journal.pone.0061442 |
| spellingShingle | Vigneswara, Vasanthy Cass, Simon Wayne, Declan Bolt, Edward L. Ray, David E. Carter, Wayne Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| title | Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| title_full | Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| title_fullStr | Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| title_full_unstemmed | Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| title_short | Molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| title_sort | molecular ageing of alpha- and beta-synucleins: protein damage and repair mechanisms |
| url | https://eprints.nottingham.ac.uk/2971/ https://eprints.nottingham.ac.uk/2971/ https://eprints.nottingham.ac.uk/2971/ |