Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases.
The poly(ADP-ribose) polymerase (PARP-1), a 113 kDa nuclear enzyme, is cleaved in fragments of 89 and 24 kDa during apoptosis. This cleavage has become a useful hallmark of apoptosis and has been shown to be done by DEVD-ase caspases, a family of proteases activated during apoptosis. Interestingly, PARP-1 is also processed during necrosis but a major fragment of 50 kDa is observed. This event is not inhibited by zVAD-fmk, a broad spectrum caspase inhibitor, suggesting that these proteases are not implicated in the necrotic cleavage of PARP-1. Since lysosomes release their content into the cytosol during necrosis, the proteases liberated could produce the cleavage of PARP-1. We therefore isolated lysosomal rich-fractions from Jurkat T cells. Our results reveal that the in vitro lysosomal proteolytic cleavage of affinity purified bovine PARP-1 is composed of fragments corresponding, in apparent molecular weight and function, to those found in Jurkat T cells treated with necrotic inducers like 0.1% H2O2, 10% EtOH or 100 microM HgCl2. Moreover, we used purified lysosomal proteases (cathepsins B, D and G) in an in vitro cleavage assay and found that cathepsins B and G cleaved PARP-1 in fragments also found with the lysosomal rich-fractions. These findings suggest that the necrotic cleavage of PARP-1 is caused in part or in totality by lysosomal proteases released during necrosis.
Gobeil
S
S
Health and Environment Unit, Laval University Medical Research Center, CHUQ, and Faculty of Medicine, Laval University, Québec, Canada.
Boucher
C C
CC
Nadeau
D
D
Poirier
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D002457:Cell Extracts; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D008247:Lysosomes / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D009336:Necrosis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases; D013997:Time Factors
D002457:Cell Extracts; D010446:Peptide Fragments; D011065:Poly(ADP-ribose) Polymerases; D002403:Cathepsins; D010450:Endopeptidases; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D020169:Caspases; D002401:Cathepsin B; C108711:DEVDase