Assays of proteasome activity in relation to aging.

Proteasomes play a major role in intracellular protein turnover. They exist in cells in several different molecular forms including 20S proteasomes, 26S proteasomes and PA28-20S proteasome complexes. In this study we have compared the properties of these purified proteasome complexes to try to design assays that will distinguish between the different complexes (26S proteasome, 20S proteasome, PA28-20S proteasome) in cell extracts. Although the different purified complexes were found to have differences in stability, and in their sensitivity to low concentrations of SDS and salt, the results suggest that it is not straightforward to assay selectively for each type of complex in cell extracts. The relative contribution of different proteasome complexes varies in different cell types and there may be other proteases present which hydrolyse the chosen substrate. Proteasome assays carried out under defined conditions allow comparisons of activity in cell extracts as a function of age, but separation by gel filtration on a Superose 6 column was found to be a useful method for determining the level of different proteasome related complexes.

Trypanocidal effect of alpha',beta'-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity.

Previous studies have shown that the proteasome of Trypanosoma brucei is a candidate for novel chemotherapy of African sleeping sickness. In this study, two potent and highly selective alpha',beta'-epoxyketones peptide proteasome inhibitors, epoxomicin and YU101, have been tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of T. brucei. Both inhibitors displayed promising anti-trypanosomal activities with ED(50) and ED(90) values in the low to mid nanomolar range. Based on MIC values, epoxomicin exhibited a selectivity index approaching those of commercially available drugs. Enzymatic analyses of proteasomal peptidase activities revealed that, compared with mammalian cells, trypanosomes are particular sensitive to inhibition of the trypsin-like activity of the proteasome. In conclusion, the data suggests that proteasome inhibitors targeting the trypsin-like activity are the rational choice for future anti-trypanosomal drug development.

Evaluation of the anti-trypanosomal activity of tyropeptin A.

The natural compound tyropeptin A, a new peptidyl aldehyde proteasome inhibitor, was tested for its trypanocidal activity in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. The concentrations of tyropeptin A required to reduce the growth rate by 50 % and to kill all cells were 10 and 100 times lower for bloodstream-form trypanosomes than for human leukaemia HL-60 cells, respectively. Enzymatic analysis showed that the trypsin-like activity of the trypanosome proteasome and the chymotrypsin-like activity of the mammalian proteasome are particularly sensitive to inhibition by tyropeptin A. The results suggest that natural compounds targeting the trypsin-like activity of the proteasome may serve as leads for rational drug development of novel anti-trypanosomal agents.

Overexpression of proteasome beta5 assembled subunit increases the amount of proteasome and confers ameliorated response to oxidative stress and higher survival rates.

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta5 subunit. Stable overexpression of the beta5 subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) alpha-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

Trafficking of exogenous peptides into proteasome-dependent major histocompatibility complex class I pathway following enterotoxin B subunit-mediated delivery.

The B-subunit component of Escherichia coli heat-labile enterotoxin (EtxB), which binds to cell surface GM1 ganglioside receptors, was recently shown to be a highly effective vehicle for delivery of conjugated peptides into the major histocompatibility complex (MHC) class I pathway. In this study we have investigated the pathway of epitope delivery. The peptides used contained the epitope either located at the C terminus or with a C-terminal extension. Pretreatment of cells with cholesterol-disrupting agents blocked transport of EtxB conjugates to the Golgi/endoplasmic reticulum, but did not affect EtxB-mediated MHC class I presentation. Under these conditions, EtxB conjugates entered EEA1-positive early endosomes where peptides were cleaved and translocated into the cytosol. Endosome acidification was required for epitope presentation. Purified 20 S immunoproteasomes were able to generate the epitope from peptides in vitro, but 26 S proteasomes were not. Only presentation from the C-terminal extended peptide was proteasome-dependent in cells, and this was found to be significantly slower than presentation from peptides with the epitope at the C terminus. These results implicate the proteasome in the generation of the correct C terminus of the epitope and are consistent with proteasome-independent N-terminal trimming. Epitope presentation was blocked in a TAP-deficient cell line, providing further evidence that conjugated peptides enter the cytosol as well as demonstrating a requirement for the peptide transporter. Our findings demonstrate the utility of EtxB-mediated peptide delivery for rapid and efficient loading of MHC class I epitopes in several different cell types. Conjugated peptides are released from early endosomes into the cytosol where they gain access to proteasomes and TAP in the "classical" pathway of class I presentation.