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 ␤-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 ␤ 5 subunit. Stable overexpression of the ␤ 5 subunit in WI38/T and HL60 cells resulted in elevated levels of other ␤-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) ␣-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in ␤ 5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that ␤ 5 -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 ␤ 5 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.

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The B-subunit component of Escherichia coli heatlabile 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.Cytotoxic CD8 ϩ T lymphocytes recognize and clear host cells infected with intracellular bacteria or viruses, or tumors that display non-self or tumor-derived peptides on their surface in the context of major histocompatibility complex (MHC) 1 class I molecules (1). The majority of peptides presented are generated from endogenously synthesized proteins that are degraded by the proteasome (reviewed in Refs. 2 and 3). These are subsequently transported into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen presentation (TAP). In the ER, peptides bind to newly synthesized MHC class I molecules, which are then transported to the cell surface for recognition by CD8 ϩ T-cells. This leads to T-cell activation, and eventually to cytotoxic CD8 ϩ T lymphocyte-mediated lysis of peptide-presenting cells. Activation of cytotoxic CD8 ϩ T lymphocytes is obviously an important strategy for vaccination. However, exogenous soluble antigens generally fail to gain access to the cytosolic compartment of cells, and thus novel strategies of targeting this pathway are needed.One strategy that is receiving attention is the use of protein delivery vehicles that possess ...

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.