title: Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells.
abstract: In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.
authors.affiliation: Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.
authors.forename: K
authors.initials: K
authors.lastname: Yu
authors.forename: C A
authors.initials: CA
authors.lastname: Kennedy
authors.forename: M M
authors.initials: MM
authors.lastname: O'Neill
authors.forename: R W
authors.initials: RW
authors.lastname: Barton
authors.forename: R J
authors.initials: RJ
authors.lastname: Tatake
mesh_terms: D017209:Apoptosis; D001665:Binding Sites; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019169:Jurkat Cells; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D019014:fas Receptor / Q000378:metabolism
chemical_list: D010455:Peptides; D012333:RNA, Messenger; D019014:fas Receptor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases