Involvement of lysosomal cathepsins in the cleavage of DNA topoisomerase I during necrotic cell death.
OBJECTIVE
Autoantibodies to DNA topoisomerase I (topo I) are associated with diffuse systemic sclerosis (SSc), appear to be antigen driven, and may be triggered by cryptic epitopes exposed during in vivo topo I fragmentation. These autoantibodies recognize topo I and fragments of this autoantigen generated during apoptosis and necrosis. We undertook this study to determine whether lysosomal cathepsins are involved in topo I fragmentation during necrosis.


METHODS
Topo I cleavage during necrosis was assessed by immunoblotting of lysates from L929 fibroblasts exposed to tumor necrosis factor alpha (TNFalpha) and the broad caspase inhibitor Z-VAD-FMK, and by immunoblotting of lysates from endothelial cells treated with HgCl2. Purified topo I and L929 nuclei were incubated with cathepsins B, D, G, H, and L, and topo I cleavage was detected by immunoblotting. The intracellular localization of cathepsin L activity and topo I in necrotic cells was examined using fluorescence microscopy.


RESULTS
Treatment of L929 cells with TNFalpha and Z-VAD-FMK induced caspase-independent cell death with necrotic morphology. This cell death involved topo I cleavage into fragments of approximately 70 kd and 45 kd. This cleavage profile was reproduced in vitro by cathepsins L and H and was inhibited by the cathepsin L inhibitor Z-FY-CHO. During necrosis, cathepsin L activity diffused from lysosomes into the cytoplasm and nucleus, whereas topo I partially relocalized to the cytoplasm. Z-FY-CHO delayed necrosis and partially blocked topo I cleavage. The topo I cleavage fragments were also detected in necrotic endothelial cells and recognized by SSc sera containing anti-topo I antibodies.


CONCLUSIONS
These results implicate cathepsins, particularly cathepsin L, in the cleavage of topo I during necrosis. This cleavage may generate potentially immunogenic fragments that could trigger anti-topo I immune responses in SSc.
Loma Linda University School of Medicine, Loma Linda, California 92350, USA.
Fabio J
FJ
Pacheco
Jacqueline
J
Servin
David
D
Dang
Jim
J
Kim
Christine
C
Molinaro
Tracy
T
Daniels
Terry A
TA
Brown-Bryan
Mizue
M
Imoto-Egami
Carlos A
CA
Casiano
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D002403:Cathepsins / Q000378:metabolism*; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D007739:L Cells; D008247:Lysosomes / Q000201:enzymology*; D008627:Mercuric Chloride / Q000494:pharmacology; D051379:Mice; D009336:Necrosis / Q000201:enzymology*; D012595:Scleroderma, Systemic / Q000276:immunology / Q000503:physiopathology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D008627:Mercuric Chloride; D002403:Cathepsins; D004264:DNA Topoisomerases, Type I