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.
Mesh Terms:
Amino Acid Sequence, Animals, Apoptosis, Blotting, Western, Caspases, Cathepsin B, Cathepsin G, Cathepsins, Cattle, Cell Extracts, Endopeptidases, Enzyme Activation, Humans, Jurkat Cells, Lysosomes, Molecular Weight, Necrosis, Peptide Fragments, Peptide Hydrolases, Poly(ADP-ribose) Polymerases, Protein Structure, Tertiary, Serine Endopeptidases, Time Factors
Cell Death Differ.
Date: Jun. 01, 2001
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