tailieunhanh - Báo cáo khoa học: Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies

Anti-Ras intracellular antibodies inhibit cell proliferation in vivo by sequestering the antigen and diverting it from its physiological location [Lener,M.,Horn, ., Messina, S., Nielsen, ., Rybak, ., Hoogenboom, ., Cattaneo, A., Biocca, S. (2000)Eur. J. , 1196–1205]. Here we demonstrate that strongly aggregating single-chain antibody fragments (scFv), binding to Ras, induce apoptosis, and this effect is strictly related to the antibody-mediated aggregation of p21Ras. Proteasomes are quickly recruited to the newly formed aggregates, and their activity is strongly inhibited | Eur. J. Biochem. 270 3389-3397 2003 FEBS 2003 doi Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies Alessio Cardinale Ilaria Filesi Sonia Mattei and Silvia Biocca Department of Neuroscience University of Rome Tor Vergata Rome Italy Anti-Ras intracellular antibodies inhibit cell proliferation in vivo by sequestering the antigen and diverting it from its physiologicallocation Lener M. Horn I. R. Cardinale A. Messina S. Nielsen . Rybak . Hoogenboom . Cattaneo A. Biocca S. 2000 Eur. J. Biodwm. 267 1196-1205 . Here we demonstrate that strongly aggregating single-chain antibody fragments scFv binding to Ras induce apoptosis and this effect is strictly related to the antibody-mediated aggregation of p21Ras. Proteasomes are quickly recruited to the newly formed aggregates and their activity is strongly inhibited. This leads to the formation of aggresome-like structures which become evident in the vast majority of apoptotic cells. A combination of anti-Ras scFv fragments with a nontoxic concentration of the proteasome inhibitor lactacystin markedly increases proteasome dysfunction and apoptosis. The dominant-negative H-ras N17-H-ras which is mostly soluble and does not induce aggresome formation or inhibit proteasome activity only affects cell viability slightly. Together these observations suggest a mechanism linking antibody-mediated Ras aggregation impairment of the ubiquitin-proteasome system and cytotoxicity. Keywords aggresome anti-p21Ras apoptosis proteasome scFv fragment. Ras is a membrane-bound GTP GDP-binding protein which functions as a molecular switch in a large network of signaling pathways 1 . Mutations in the ras gene have been identified in about 30 of all human cancers indicating that this molecule is a preferential target for the development of anticancer strategies. Indeed Ras protein has been inhibited through different approaches such as ribozymes antisense .