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. 2008 Sep 22;3(9):e3258.
doi: 10.1371/journal.pone.0003258.

Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation

Amelia Cimmino  1 , Rosanna Capasso, Fabbri Muller, Irene Sambri, Lucia Masella, Marianna Raimo, Maria Luigia De Bonis, Stefania D'Angelo, Vincenzo Zappia, Patrizia Galletti, Diego Ingrosso
Affiliations

Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation

Amelia Cimmino et al. PLoS One. .

Retraction in

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mechanism for deamidation of asparaginyl residues in peptides.
(Step 1): the nitrogen of the Asn+1 residue (a Gly in the example) attacks the β-carbonyl carbon of the Asn, thus forming the succinimidyl derivative of the peptide (ASU) with the ammonia elimination. The ASU ring can open spontaneously on either side of the nitrogen atom. In one case the α-aspartyl peptide is formed (Step 2). In the other case the β-isoaspartyl peptide does occur (Step 3).
Figure 2
Figure 2. Characterization of PCMT plasmid constructs.
Panel A) Plasmids were transfected into endothelial cells and 48 h later methyltransferase activity was assayed using a methanol diffusion assay in the presence of saturating concentrations of the methyl donor AdoMet and ovalbumin as a methyl accepting protein. Results are given as the mean of three experiments. Error bars indicate standard deviation; (*) refer to statistically significant differences (p<0.05), as evaluated by t-test. Panel B) Cells transfected were then processed for immunoblotting with PCMT antibody. The immunoblot was reprobed for actin as a loading control. The sample “PCMT” is authentic human recombinant enzyme as a positive control. PAEc = PAE transfected with void plasmid.
Figure 3
Figure 3. Effect of PCMT expression levels and mutants on apoptosis induced by oxidative stress on PAE cells.
Column A: Apoptotic DNA ladder in cells overexpressing PCMT constructs and subject to oxidative treatment. Apoptotic DNA ladder patterns, of transfected endothelial cells stimulated with different concentrations of H2O2, were detected after transfection with plasmid void (control) or carrying PCMT wild type (sense); antisense PCMT; PCMT Asp83 →Phe83 Mut and PCMT Asp83 →Val83 Mut. Column B: Caspase-3 activation and PARP cleavage in cells overexpressing PCMT constructs and subject to oxidative treatment. Immunoblot developed with a polyclonal antibody against caspase-3 and PARP (a final effector of various apoptotic pathways) on transfected endothelial cells stimulated with different concentration of H2O2 after transfection with plasmid void (control) or carring PCMT wild type (sense), antisense PCMT, PCMT Asp83 →Phe83 Mut and PCMT Asp83 →Val83 Mut. CP is a positive control obtained by treating a parallel cell sample with cisplatin. Column C: Flow cytometry analysis of cells overexpressing PCMT constructs and subject to oxidative treatment. Cells stimulated with 0.3 mM of H2O2 after transfection with void plasmid (control), PCMT wild type (sense), Antisense PCMT, PCMT Asp83 →Phe83 Mut. PCMT Asp83 →Val83 Mut. PI: propidium iodide.
Figure 4
Figure 4. Quantitation of the extent of protein deamidation in cells lysates after oxidative stress.
Cells transfected with PCMT wild type (sense), antisense PCMT (anti), PCMT mutants [Asp83 →Phe83 (Phe) and Asp83→Val83 (Val)] and endothelial cells transfected with void plasmid (PAE) were stressed with 0.3 mM of H2O2. Deamidated proteins were quantitated in each lysate preparation using recombinant PCMT. Standard deviation error bars are included for each analysis.
Figure 5
Figure 5. Identification of PCMT substrates.
Panel A: Experimental strategy for isolation and characterization of PCMT substrates. Step 1: human recombinant PCMT, purified to homogeneity, was immobilized onto sulfoSBED by N-hidroxysuccinimide chemistry; Step 2: cell extracts as a source of substrates were added and Step 3: proteins interacting with PCMT were immobilized upon UV photoactivation; Step 4: PCMT was released and biotin “transferred” onto the methyltrasferase substrate (Label Transfer Method); Step 5: purification was accomplished by exploiting streptavidin-biotin interactions. Panel B: 2D gel electrophoresis imaging of comparative proteomics. HUVEC were infected with antisense PCMT carrying retrovirus and then stressed with 0.3 mM of H2O2. Cells lysates were reacted with Sulfo-SBED previously cross-linked with recombinant PCMT (Panel B). Arrows indicate the protein spots which have been characterized as reported in Table 1. Background noise due to aspecific binding was subtracted by comparison with the 2D image obtained from a parallel sample reacted with non-cross-linked Sulfo-SBED.
Figure 6
Figure 6. Proposed role of Bcl-xL deamidation and methylation in apoptosis.
Deamidation-isomerization of two critical Asn residues of Bcl-xL abolishes its antiapoptotic function. Methylation of the same residues can restore the functional integrity of Bcl-xL through repair of the isopeptide bonds. Regulation of apoptosis toward antiapoptosis is gained through fine balancing of Bcl-xL deamidation and methylation.
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