Protein isoaspartyl methyltransferase

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartatedamaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and 3 H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of~75e80 kDa,~95e100 kDa, and~155e160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the~160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine Smethyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (~2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartatedamaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and 3 H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of~75e80 kDa,~95e100 kDa, and~155e160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the~160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine Smethyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (~2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

-Le vieillissement et la perte de viabilité sont universels auxêtres vivants. Chez les plantesà fleurs, la graine mature disséminée dans le sol matérialise la phase du cycle de vie exposée au vieillissement. Dans nos climats tempérés les graines, dont le succès germinatif conditionne la propagation de la descendance, ont acquis une aptitudeà la survie qui en fait un modèle d'étude de choix. Outre les propriétés physicochimiques de la semence qui lui confèrent une résistance particulière au stress oxydatif, le métabolisme des protéines semble jouer un rôle déterminant dans le contrôle de la longévité. De récentesétudes génétiques ont permis de mettre enévidence le rôle important de l'enzyme de réparation, la Protéine L-isoaspartyl méthyltransférase, dans la survie des graines ainsi que dans la vigueur germinative. Ces mécanismes de détoxification cellulaire par la réparation des protéines sont universels et l'étude de leur mode d'action dans la graine va contribuerà une meilleure compréhension des mécanismes de protection contre le vieillissement.

The: form:.nion of a bno rmal isoaspanyl residuc:s ckr,,'ed from aspartyl or asparaginyl residw:s is a major source of spontanc:ous prote: in misfolding in cc: Ils. The: re: pair enzyme prote in L·isoospartyl methyltr.lJlS. fc:rasc: ( PI.MT) counteracts such damage: by catalyzing the con,·c:rsion of abnormal isoasp.ut)•l residues to the ir nonnal aspart)•l forms. Thus, this enzyme: conributes to the survival of m3Jly organisms, including plants. Analysis of the accumulation o f iso.tspartyl·c· :mtai.ni.ng protc:ins and its modulation by the: Pll\.IT rc:pair path\\'3)\ using germination tests, immunodc:tc:ccion, eru:ymacic assays, and HPLC anaJysis, givc:s new insi.gbts in undc:rstandi.ng controlling mc:chanisms of sc:c:d longc:vity and \i.gor.

The formation of abnormal isoaspartyl residues derived from aspartyl or asparaginyl residues is a major source of spontaneous protein misfolding in cells. The repair enzyme protein L: -isoaspartyl methyltransferase (PIMT) counteracts such damage by catalyzing the conversion of abnormal isoaspartyl residues to their normal aspartyl forms. Thus, this enzyme contributes to the survival of many organisms, including plants. Analysis of the accumulation of isoaspartyl-containing proteins and its modulation by the PIMT repair pathway, using germination tests, immunodetection, enzymatic assays, and HPLC analysis, gives new insights in understanding controlling mechanisms of seed longevity and vigor.