Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

BACKGROUND:Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS:Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. CONCLUSION/SIGNIFICANCE:Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein

D-aspartate content of erythrocyte membrane proteins is decreased in uremia: Implications for the repair of damaged proteins

The authors of this article have demonstrated that erythrocytes from patients affected by either chronic renal failure or ESRD, conditions associated with erythrocyte membrane disorders, show reduced levels of methyl esterified membrane proteins because of elevated S-adenosylhomocysteine concentration. The enzyme protein L-isospartyl (D-aspartyl) O-methyltransferase, responsible for the bulk of this methyl esterification, is implicated in the repair of proteins containing isomerized and racemized aspartyl residues, which arise from L-asparaginyl and L-aspartyl residues. The presence of these altered residues, spontaneously generated during protein aging, can adversely affect protein function. The amount of D- and L-aspartyl residues (and their isomerized derivatives) in erythrocyte membranes from hemodialysis patients was determined. The total level of D-aspartyl derivatives (D-Asx) actually was found to be lower than in controls. In contrast, neither the abundance of several other amino acids, nor of total non-Asx D-amino acids, differs between patients and controls. Mathematical simulation of relevant reactions supports the hypothesis that these effects reflect the lessening of the normal D-isoaspartyl residue accumulation that occurs as a side reaction in the methyltransferase-induced repair process. This evidence is the first that D-Asx content is influenced in vivo by L-isoaspartyl (D-aspartyl) O-methyltransferase activity and can be significantly altered in a disease where this activity is inhibited, thus representing a red flag in a disrupted circuit

Protein damage and repair controlling seed vigor and longevity

International audienceThe 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

D-amino acids in aging erythrocytes.

Mature human erythrocytes are highly differentiated cells which have lost the ability to biosynthesize proteins de novo. During cell aging in circulation, erythrocyte proteins undergo spontaneous postbiosynthetic modifications, regarded as "protein fatigue" damage, which include formation of isomerized and/or racemized aspartyl residues. These damaged proteins cannot be replaced by new molecules; nevertheless, data support the notion that they can be repaired to a significant extent, through an enzymatic transmethylation reaction. This repair reaction has therefore been used as a means to monitor the increase of altered aspartyl residues in erythrocyte membrane proteins during cell aging. The relationship between protein repair and aspartyl racemization in red blood cell stress and disease is discussed

Deuteration protects asparagine residues against racemization

Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age