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 (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3632608?pdf=render |
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| author | Vasanthy Vigneswara Simon Cass Declan Wayne Edward L Bolt David E Ray Wayne G Carter |
| author_facet | Vasanthy Vigneswara Simon Cass Declan Wayne Edward L Bolt David E Ray Wayne G Carter |
| author_sort | Vasanthy Vigneswara |
| collection | DOAJ |
| 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-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-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 (3)H-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 | 2024-12-11T08:22:12Z |
| format | Article |
| id | doaj.art-bd4901aa710d4234a1cc6185ab57c939 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-11T08:22:12Z |
| publishDate | 2013-01-01 |
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| spelling | doaj.art-bd4901aa710d4234a1cc6185ab57c9392022-12-22T01:14:38ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-0184e6144210.1371/journal.pone.0061442Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms.Vasanthy VigneswaraSimon CassDeclan WayneEdward L BoltDavid E RayWayne G CarterAbnormal 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-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-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 (3)H-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.http://europepmc.org/articles/PMC3632608?pdf=render |
| spellingShingle | Vasanthy Vigneswara Simon Cass Declan Wayne Edward L Bolt David E Ray Wayne G Carter Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms. PLoS ONE |
| 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 | http://europepmc.org/articles/PMC3632608?pdf=render |
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