Considerations in the identification of endogenous substrates for protein L-isoaspartyl methyltransferase: the case of synuclein.
Protein L-isoaspartyl methyltransferase (PIMT) repairs abnormal isoaspartyl peptide bonds in age-damaged proteins. It has been reported that synuclein, a protein implicated in neurodegenerative diseases, is a major target of PIMT in mouse brain. To extend this finding and explore its possible relevance to neurodegenerative diseases, we attempted to determine the stoichiometry of isoaspartate accumulation in synuclein in vivo and in vitro. Brain proteins from PIMT knockout mice were separated by 2D electrophoresis followed by on-blot [(3)H]-methylation to label isoaspartyl proteins, and by immunoblotting to confirm the coincident presence of synuclein. On-blot (3)H-methylation revealed numerous isoaspartyl proteins, but no signal in the position of synuclein. This finding was corroborated by immunoprecipitation of synuclein followed by on-blot (3)H-methylation. To assess the propensity of synuclein to form isoaspartyl sites in vitro, samples of recombinant mouse and human α-synucleins were aged for two weeks by incubation at pH 7.5 and 37 °C. The stoichiometries of isoaspartate accumulation were extremely low at 0.02 and 0.07 mol of isoaspartate per mol of protein respectively. Using a simple mathematical model based on the first order kinetics of isoaspartyl protein methyl ester hydrolysis, we ascribe the discrepancy between our results and the previous report to methodological limitations of the latter stemming from an inherent, and somewhat counterintuitive, relationship between the propensity of proteins to form isoaspartyl sites and the instability of the (3)H-methyl esters used to tag them. The results presented here indicate that synuclein is not a major target of PIMT in vivo, and emphasize the need to minimize methyl ester hydrolysis when using methylation to assess the abundance of isoaspartyl sites in proteins.
Department of Molecular Biology & Biochemistry, University of California Irvine, Irvine, California, United States of America.
Gareth J
GJ
Morrison
Ranjani
R
Ganesan
Zhenxia
Z
Qin
Dana W
DW
Aswad
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002384:Catalysis; D019175:DNA Methylation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000378:metabolism; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D051843:Synucleins / Q000378:metabolism / Q000502:physiology*
D051843:Synucleins; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase