Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex.
Diverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, 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. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that 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.
Cardinale
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Department of Neuroscience, University of Rome Tor Vergata, Via di Tor Vergata 135, 00133 Rome, Italy.
Filesi
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Biocca
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D000936:Antigen-Antibody Complex / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism / Q000528:radiation effects / Q000648:ultrastructure; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014452:Ubiquitins / Q000378:metabolism
D000936:Antigen-Antibody Complex; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D016283:Proto-Oncogene Proteins p21(ras)