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% H 2 O 2 , 10% EtOH or 100 mM HgCl 2 . 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. Cell Death and Differentiation (2001) 8, 588 ± 594.

Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death

“…The specific cathepsin L inhibitor Z-FY-CHO inhibited cathepsin L-mediated cleavage of purified and nuclear topo I (Figures 3C and D). The concentration ranges of cathepsins and the incubation conditions used in these experiments were based on reported studies (26,27). It should be noted that untreated topo I, either in purified form or in the isolated nuclei, underwent limited degradation into a 70-kd product during protein or nuclear preparation (Figure 3), consistent with previous reports (3)(4)(5).…”

“…A previous report implicated cathepsins B and G in the necrotic cleavage of PARP, a lupus-associated autoantigen (26). Our results indicated that cathepsins B, G, H, and L can induce topo I fragmentation in vitro.…”

Zonal necrosis prevented by transduction of the artificial anti-death FNK protein

“…The 50 kDa fragment was designated as a major necrotic fragment. [38][39][40] From these results, we confirmed that the death of HepG2 cells induced by CCl 4 is predominantly due to necrosis.…”

“…Detection of a 50 kDa fragment derived from PARP-1 in CCl 4 -treated cells is strong evidence for necrosis, because the apoptotic PARP-1 fragment of 85 kDa induced with TNFa/CHX is distinct from the 50 kDa fragment 38,39 (Figure 1d). These results clearly indicated that a majority of HepG2 cells exposed to CCl 4 died in a necrotic manner, with a good agreement with previous results.…”

Apoptosis caused by cathepsins does not require Bid signaling in an in vivo model of progressive myoclonus epilepsy (EPM1)

“…23 One possibility is that cathepsins could nonspecifically degrade important cellular proteins, thereby causing the cell to initiate apoptosis. 24 Alternatively, cathepsins could cleave and activate caspases or their downstream death effector substrates, 25 thereby causing apoptosis. In support of the former mechanism, it has been shown that cathepsin B can activate the inflammatory caspases 1 and 11, 17,26 and that cathepsin L may activate caspase-3.…”