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Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases.

Abstract
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.
AuthorsS Gobeil, C C Boucher, D Nadeau, G G Poirier
JournalCell death and differentiation (Cell Death Differ) Vol. 8 Issue 6 Pg. 588-94 (Jun 2001) ISSN: 1350-9047 [Print] England
PMID11536009 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Cell Extracts
  • Peptide Fragments
  • Poly(ADP-ribose) Polymerases
  • Cathepsins
  • Endopeptidases
  • Peptide Hydrolases
  • Serine Endopeptidases
  • CTSG protein, human
  • Cathepsin G
  • Caspases
  • Cathepsin B
  • DEVDase
Topics
  • Amino Acid Sequence
  • Animals
  • Apoptosis
  • Blotting, Western
  • Caspases (metabolism)
  • Cathepsin B (metabolism)
  • Cathepsin G
  • Cathepsins (metabolism)
  • Cattle
  • Cell Extracts
  • Endopeptidases (metabolism)
  • Enzyme Activation
  • Humans
  • Jurkat Cells
  • Lysosomes (enzymology, metabolism)
  • Molecular Weight
  • Necrosis
  • Peptide Fragments (chemistry, metabolism)
  • Peptide Hydrolases (metabolism)
  • Poly(ADP-ribose) Polymerases (chemistry, metabolism)
  • Protein Structure, Tertiary
  • Serine Endopeptidases
  • Time Factors

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