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

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

Autor Pacheco, Fabio Juliano Autor UNIFESP Google Scholar
Servin, J. Google Scholar
Dang, D. Google Scholar
Kim, J. Google Scholar
Molinaro, C. Google Scholar
Daniels, T. Google Scholar
Brown-Bryan, T. A. Google Scholar
Egami, Mizue Imoto Autor UNIFESP Google Scholar
Casiano, C. A. Google Scholar
Instituição Loma Linda Univ
Universidade Federal de São Paulo (UNIFESP)
Resumo Objective. Autoantibodies to DNA topoisomerase I (topo 1) 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 a (TNF alpha) 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 TNFa 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 Land 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.Conclusion. 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.
Idioma Inglês
Data 2005-07-01
Publicado em Arthritis and Rheumatism. Hoboken: Wiley-liss, v. 52, n. 7, p. 2133-2145, 2005.
ISSN 0004-3591 (Sherpa/Romeo, fator de impacto)
Editor Wiley-Blackwell
Extensão 2133-2145
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000230608100026

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