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

• Autor(a) principal: Pacheco, Fabio Juliano [UNIFESP]
• Data de Publicação: 2005
• Outros Autores: Servin, J., Dang, D., Kim, J., Molinaro, C., Daniels, T., Brown-Bryan, T. A., Egami, Mizue Imoto [UNIFESP], Casiano, C. A.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: http://dx.doi.org/10.1002/art.21147; http://repositorio.unifesp.br/handle/11600/28349

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.