Aggresome formation by anti-Ras intracellular scFv fragments - The fate ofthe antigen-antibody complex

Diverting the antigen from its normal intracellular location to other compa rtments in an antibody-mediated way represents a mode of action for intrace llular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Bi occa, S. (1998) FEBS Lett., 439, 197-202; 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, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. W e have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation a nd the intracellular localization of the aggregates. Overexpression of anti -Ras scFv fragments or inhibition of proteasomes activity leads to the form ation of large perinuclear aggresomes formed of ubiquitinated-scFv fragment s in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulat ion of scFv fragments in a single aggresome and induces the dispersion of t hese structures in the periphery of the cell. Cotransfection of the GFP-scF v with a myc-tagged ubiquitin and colocalization with specific anti-proteas ome antibodies indicate the recruitment of exogenous ubiquitin and proteaso mes to the newly formed aggresomes. Taken together these results suggest th at the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function.