Mechanism for deamidation of asparaginyl residues in peptides.

<p>(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).</p

Proposed role of Bcl-x_L deamidation and methylation in apoptosis.

<p>Deamidation-isomerization of two critical Asn residues of Bcl-x_L abolishes its antiapoptotic function. Methylation of the same residues can restore the functional integrity of Bcl-x_L through repair of the isopeptide bonds. Regulation of apoptosis toward antiapoptosis is gained through fine balancing of Bcl-x_L deamidation and methylation.</p

Quantitation of the extent of protein deamidation in cells lysates after oxidative stress.

<p>Cells transfected with <i>PCMT</i> wild type (sense), antisense <i>PCMT</i> (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.</p

Identification of PCMT substrates.

<p>Panel A: <u>Experimental strategy for isolation and characterization of PCMT substrates</u>. Step 1: human recombinant PCMT, purified to homogeneity, was immobilized onto sulfoSBED by <i>N</i>-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: <u>2D gel electrophoresis imaging of comparative proteomics</u>. HUVEC were infected with antisense <i>PCMT</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003258#pone-0003258-t001" target="_blank">Table 1</a>. 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.</p

Effect of PCMT expression levels and mutants on apoptosis induced by oxidative stress on PAE cells.

<p>Column A: <u>Apoptotic DNA ladder in cells overexpressing PCMT constructs and subject to oxidative treatment</u>. Apoptotic DNA ladder patterns, of transfected endothelial cells stimulated with different concentrations of H₂O₂, were detected after transfection with plasmid void (control) or carrying <i>PCMT wild type</i> (sense); antisense <i>PCMT</i>; <i>PCMT</i> Asp₈₃ →Phe₈₃ Mut and PCMT Asp₈₃ →Val₈₃ Mut. Column B: <u>Caspase-3 activation and PARP cleavage in cells overexpressing PCMT constructs and subject to oxidative treatment</u>. 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 <i>wild type</i> (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: <u>Flow cytometry analysis of cells overexpressing PCMT constructs and subject to oxidative treatment</u>. Cells stimulated with 0.3 mM of H₂O₂ after transfection with void plasmid (control), <i>PCMT wild type</i> (sense), Antisense <i>PCMT</i>, PCMT Asp₈₃ →Phe₈₃ Mut. PCMT Asp₈₃ →Val₈₃ Mut. PI: propidium iodide.</p