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, I. R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A., Biocca, S. (2000) Eur. J. Biochem.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.
Cardinale
Alessio
A
Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy.
Filesi
Ilaria
I
Mattei
Sonia
S
Biocca
Silvia
S
D016475:3T3 Cells; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D017209:Apoptosis / Q000276:immunology / Q000502:physiology; D002455:Cell Division / Q000276:immunology / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D051379:Mice; D009097:Multienzyme Complexes / Q000276:immunology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D018631:ras Proteins / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; D007128:Immunoglobulin Fragments; D009097:Multienzyme Complexes; C067713:lactacystin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D018631:ras Proteins; D000111:Acetylcysteine