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

Abstract

BackgroundNatural 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 α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-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 FindingsEndothelial 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 (H2O2; 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 H2O2 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 H2O2 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/SignificanceApoptosis 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.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line

“…3E). The oxidative stress is considered to be a favorable condition for Bcl-xL deamidation by a mechanism that is not dependent on the increased NHE-1 activity [26]. However, in our study, no significant increase of the ROS levels was induced by any of the drugs.…”

The MicroRNA 15a/16–1 Cluster Down-regulates Protein Repair Isoaspartyl Methyltransferase in Hepatoma Cells

“…This was accomplished by using a purification protocol that employed a resin linked to human recombinant PCMT1, which can only recognize isomerized proteins but not the one carrying normal aspartyl (27). These latter experiments provided the first evidence that Bcl xL undergoes deamidation under cell stress conditions, yielding its isomerized derivative, which is devoid of antiapoptotic function.…”

“…The overall scenario may be even more complex, because of the pleiotropic activity of this microRNA cluster on a number of other antiapoptotic genes (23, 28 -30, 35-37, 44), not just PCMT1 and also because other PCMT1 substrates, such as Hsp70, Hsp90, are involved in apoptosis (27), and both of these aspects may further complicate this model and certainly deserve further investigations. These results, as a whole, support a novel interpretation that PCMT1 should not be simply interpreted as a repair methyltransferase of aged/damaged proteins, but it may act as an effective modulator of apoptotic cell death, whose derangement may play a role in proliferative or degenerative disorders.…”

“…Consistently, reduced levels of Bcl xL deamidation were detected in hepatocellular carcinomas compared with normal liver tissue and this has been linked to the typical apoptosis resistance of such transformed cells (26). In our previous work (27) we demonstrated the antiapoptotic action of PCMT1 and proposed a mechanism of action, based on the ability of this methyltransferase to maintain the structural stability of some crucial antiapoptotic proteins, including Bcl xL , through methylation and repair of their malfunctional L-isoaspartyl-containing derivatives.…”

Current advances in the application of proteomics in apoptosis research

“…JAB1 could compete with Bcl-XL/Bcl-2 to bind to BclGs and thus promote apoptosis. Cimmino et al [60] found that cells overexpressing "wild-type" human isoaspartyl protein carboxyl-O-methyltransferase (PCMT) were resistant to apoptosis, whereas overexpression of antisense PCMT induced high sensitivity to apoptosis, even at low H 2 O 2 concentrations. By incubating the purified human recombinant PCMT with cell lysate, they identified that Bcl-xL was a specific methyl-accepting substrate of PCMT and thus lost its anti-apoptotic activity for the methylation modification.…”