Molecular ageing of alpha- and Beta-synucleins: protein damage and repair mechanisms.
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-[(3)H-methyl]methionine as a methyl donor, and liquid scintillation counting of liberated (3)H-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 (3)H-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.
Mesh Terms:
Amino Acid Sequence, Animals, Brain, Chromatography, Gel, Cytoplasm, Humans, Isoaspartic Acid, Isoelectric Point, Methylation, Mice, Mice, Knockout, Molecular Sequence Data, Mutation, Missense, Parkinson Disease, Protein D-Aspartate-L-Isoaspartate Methyltransferase, Protein Processing, Post-Translational, Sequence Homology, Amino Acid, alpha-Synuclein, beta-Synuclein
Amino Acid Sequence, Animals, Brain, Chromatography, Gel, Cytoplasm, Humans, Isoaspartic Acid, Isoelectric Point, Methylation, Mice, Mice, Knockout, Molecular Sequence Data, Mutation, Missense, Parkinson Disease, Protein D-Aspartate-L-Isoaspartate Methyltransferase, Protein Processing, Post-Translational, Sequence Homology, Amino Acid, alpha-Synuclein, beta-Synuclein
PLoS One
Date: May. 01, 2013
PubMed ID: 23630590
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