title: Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation.
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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine and Surgery, Second University of Naples, Naples, Italy.
authors.forename: Amelia
authors.initials: A
authors.lastname: Cimmino
authors.forename: Rosanna
authors.initials: R
authors.lastname: Capasso
authors.forename: Fabbri
authors.initials: F
authors.lastname: Muller
authors.forename: Irene
authors.initials: I
authors.lastname: Sambri
authors.forename: Lucia
authors.initials: L
authors.lastname: Masella
authors.forename: Marianna
authors.initials: M
authors.lastname: Raimo
authors.forename: Maria Luigia
authors.initials: ML
authors.lastname: De Bonis
authors.forename: Stefania
authors.initials: S
authors.lastname: D'Angelo
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Zappia
authors.forename: Patrizia
authors.initials: P
authors.lastname: Galletti
authors.forename: Diego
authors.initials: D
authors.lastname: Ingrosso
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D017209:Apoptosis; D019175:DNA Methylation; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D026581:Isoaspartic Acid / Q000737:chemistry*; D008954:Models, Biological; D018384:Oxidative Stress; D011496:Protein Methyltransferases / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013552:Swine; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D026581:Isoaspartic Acid; D051020:bcl-X Protein; D006861:Hydrogen Peroxide; D011496:Protein Methyltransferases