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essentially the same. However, it was seriously doubted at the time, especially
by Dean and colleagues (240-242, 306), that the used terminally differentiated
cells, as erythrocytes and as lens cells, are really a suitable model and reflect
the behavior of other cell types. Matthews and colleagues (307) expressed
doubts on the role of the proteasome.
However, a series of publications in the mid-1990s showed that by using
metabolically radiolabeled proteins, various cell lines, fully functional and
dividing, are able to respond to oxidative stress with an increase in protein
turnover (268, 308). It is worth noting that several kinds of oxidative stresses
were applied, and for all these stresses, it could be shown that mild oxidation
with moderate oxidant concentrations or fluxes increased the degradation of
endogenous radiolabeled proteins, whereas a further increase of the oxidative
thread decreased proteolysis rates, without killing the cells or destroying pro-
teasomal capacity.
However, the major role of the proteasomal system in the degradation of
oxidized proteins in these living cells still had to be demonstrated. Several
indirect experimental approaches, such as comparison of inhibitor profiles of
isolated proteasomes and cell extracts toward oxidized model proteins, and
size fractionation studies of cell extracts, strongly suggested a major involve-
ment of the proteasome in the removal of oxidized proteins in diverse cell
types as well. Some of the clearest answers came from immunoprecipitation
studies of the proteasome (268, 308). However, the most convincing evidence
for the involvement of the proteasome in the degradation of oxidized proteins
was provided with experiments employing antisense suppression of the C2
proteasomal subunit (268, 275, 276) and with the application of various pro-
teasomal inhibitors (250, 309). These antisense-treated cells are essentially
depleted of the proteasome and are no longer able to increase protein turn-
over after oxidative stress to degrade oxidatively modified proteins (268, 275,
276). However, the most rigorous test of the involvement of proteasome in the
degradation of oxidatively modified proteins in living cells could be tested in
k.o. mutants. However, this approach seems to be impossible since the protea-
some is essential for cell cycle and cell division. A crucial step in the usage of
antisense oligodeoxynucleotides to decrease the proteasomal activity in cells
(268, 308) was the overcoming of the particular long half-life of the enzyme.
Therefore, it was necessary to treat cells with antisense oligodeoxynucleotides
each day, for several days. While cells initially still divided, due to the loss of
ability to synthesize new proteasome, the amount of proteasome dropped
significantly and the cells stopped to divide. Using this approach, we were able
to drop the proteasome content substantially, as indicated by Western blots.
However, the basal proteolysis rates in cells did not change following the
antisense treatment. Only the oxidative stress-induced increase in proteoly-
sis was prevented, indicating that the remaining (approximately 10-15%)
proteasomal activity in the antisense-treated cells was able to maintain the
proteasomal function during normal cellular existence, but was not sufficient
during stress exposure. This labor-extensive approach was later followed, upon
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