A. Cardinale et al., Aggresome formation by anti-Ras intracellular scFv fragments - The fate ofthe antigen-antibody complex, EUR J BIOCH, 268(2), 2001, pp. 268-277
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.