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Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies
Alessio Cardinale1, Ilaria Filesi, Sonia Mattei
1Department of Neuroscience, University of Rome Tor Vergata, Rome, Italy.
Abstract:
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.
Insights
Single-chain antibody fragments targeting Ras induce cell death by causing protein aggregation and inhibiting proteasome function. This mechanism, involving aggresome formation, offers a novel approach to cancer therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Intracellular antibodies targeting Ras inhibit proliferation by sequestering the antigen.
- Ras proteins are key regulators of cell growth and survival.
Purpose of the Study:
- To investigate the mechanism by which anti-Ras single-chain antibody fragments (scFv) induce apoptosis.
- To explore the role of protein aggregation and proteasome inhibition in anti-Ras scFv-mediated cytotoxicity.
Main Methods:
- Utilized anti-Ras scFv fragments to induce aggregation of p21Ras in cells.
- Assessed proteasome recruitment and activity in response to Ras aggregation.
- Investigated the formation of aggresome-like structures.
- Examined the combined effects of anti-Ras scFv and proteasome inhibitors (lactacystin).
- Compared effects with a soluble dominant-negative H-ras mutant (N17-H-ras).
Main Results:
- Strongly aggregating anti-Ras scFv induced apoptosis, strictly linked to p21Ras aggregation.
- Proteasomes were recruited to aggregates and their activity was inhibited, leading to aggresome-like structures in apoptotic cells.
- Combination therapy with lactacystin enhanced proteasome dysfunction and apoptosis.
- Soluble N17-H-ras had minimal effects on cell viability, aggresome formation, or proteasome activity.
Conclusions:
- Antibody-mediated Ras aggregation impairs the ubiquitin-proteasome system, leading to cytotoxicity.
- This mechanism highlights a novel pathway linking Ras signaling, protein aggregation, and apoptosis.
- Targeting Ras aggregation presents a potential therapeutic strategy for cancers driven by Ras signaling.