Proteomic Identification of Novel Substrates of a Protein Isoaspartyl Methyltransferase Repair Enzyme

Abstract

We report the use of a proteomic strategy to identify hitherto unknown substrates for mammalian protein L-isoaspartate O-methyltransferase. This methyltransferase initiates the repair of isoaspartyl residues in aged or stress-damaged proteins in vivo. Tissues from mice lacking the methyltransferase (Pcmt1 ؊/؊ ) accumulate more isoaspartyl residues than their wild-type littermates, with the most "damaged" residues arising in the brain. To identify the proteins containing these residues, brain homogenates from Pcmt1 ؊/؊ mice were methylated by exogenous repair enzyme and the radiolabeled methyl donor S-adenosyl-[methyl-3 H]methionine. Methylated proteins in the homogenates were resolved by both one-dimensional and twodimensional electrophoresis, and methyltransferase substrates were identified by their increased radiolabeling when isolated from Pcmt1 ؊/؊ animals compared with Pcmt1 ؉/؉ littermates.Mass spectrometric analyses of these isolated brain proteins reveal for the first time that microtubule-associated protein-2, calreticulin, clathrin light chains a and b, ubiquitin carboxylterminal hydrolase L1, phosphatidylethanolamine-binding protein, stathmin, ␤-synuclein, and ␣-synuclein, are all substrates for the L-isoaspartate methyltransferase in vivo. Our methodology for methyltransferase substrate identification was further supplemented by demonstrating that one of these methyltransferase targets, microtubule-associated protein-2, could be radiolabeled within Pcmt1 ؊/؊ brain extracts using radioactive methyl donor and exogenous methyltransferase enzyme and then specifically immunoprecipitated with microtubule-associated protein-2 antibodies to recover co-localized protein with radioactivity. We comment on the functional significance of accumulation of relatively high levels of isoaspartate within these methyltransferase targets in the context of the histological and phenotypical changes associated with the methyltransferase knock-out mice.

Common Proteomic Changes in the Hippocampus in Schizophrenia and Bipolar Disorder and Particular Evidence for Involvement of Cornu Ammonis Regions 2 and 3

“…It is highly expressed in the brain and participates in the degradation and/or repair of damaged proteins, including the core CME protein clathrin. 123 In keeping with this latter finding, previous work has shown a downregulation of PCMT1 mRNA in the hippocampus in bipolar disorder. 9 These findings implicate CME in schizophrenia and bipolar disorder.…”

Protein Repairl-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor inArabidopsis

“…However, it is possible that during imbibition, PIMT targets specific proteins that must be repaired for successful germination. Proteomic approaches will help address this question (Vigneswara et al, 2006;Zhu et al, 2006;Dinkins et al, 2008). Since water limitations in soil and salinity stress are frequently encountered today in agronomic conditions (Pasternak and De Malach, 1994;Flagella et al, 2002;Quadir and Oster, 2004), our results stress the paramount importance of this enzyme for plant propagation in nature and for determining crop yields.…”

Methylation Status and Neurodegenerative Markers in Parkinson Disease

“…PIMT is expressed in platelets (37 ). In an animal model of methyl group deficiency, lower SAM was associated with enhanced retention of isoaspartyl-rich ␣-synuclein, which tended to aggregate and cause aberrant synaptic transmission in the deficient animals (36 ). PIMTknockdown cells showed hyperphosphorylation of components of the mitogen-activated protein (MAP) kinase cascade compared to control cells (38 ).…”

“…An increased mRNA for ␣-synuclein has been found in chronic alcoholism (34 ), where disorders of Hcy and methylation status are very common (35 ). Second, SAH can inhibit isoaspartyl methyltransferase (PIMT), an SAM-dependent enzyme responsible for initiating the repair of isoaspartyl residues in aged or stressdamaged proteins including ␣-synuclein (36 ). PIMT is expressed in platelets (37 ).…”