Colette F. Gramm

Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules

Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.

Lactacystin and clasto-Lactacystin β-Lactone Modify Multiple Proteasome β-Subunits and Inhibit Intracellular Protein Degradation and Major Histocompatibility Complex Class I Antigen Presentation

The antibiotic lactacystin was reported to covalently modify β-subunit X of the mammalian 20 S proteasome and inhibit several of its peptidase activities. However, we demonstrate that [3H]lactacystin treatment modifies all the proteasome’s catalytic β-subunits. Lactacystin and its more potent derivative β-lactone irreversibly inhibit protein breakdown and the chymotryptic, tryptic, and peptidylglutamyl activities of purified 20 S and 26 S particles, although at different rates. Exposure to these agents for 1 to 2 h reduced the degradation of short- and long-lived proteins in four different mammalian cell lines. Unlike peptide aldehyde inhibitors, lactacystin and the β-lactone do not inhibit lysosomal degradation of an endocytosed protein. These agents block class I antigen presentation of a model protein, ovalbumin (synthesized endogenously or loaded exogenously), but do not affect presentation of the peptide epitope SIINFEKL, which does not require proteolysis for presentation. Generation of most peptides required for formation of stable class I heterodimers is also inhibited. Because these agents inhibited protein breakdown and antigen presentation similarly in interferon-γ-treated cells (where proteasomes contain LMP2 and LMP7 subunits in place of X and Y), all β-subunits must be affected similarly. These findings confirm our prior conclusions that proteasomes catalyze the bulk of protein breakdown in mammalian cells and generate the majority of class I-bound epitopes for immune recognition.

Dissociation of β2-microglobulin leads to the accumulation of a substantial pool of inactive class I MHC heavy chains on the cell surface

A large pool of free class I heavy chains is detected in situ on the plasma membrane of living cells. These chains are present on cells of different MHC genotypes and appear to exist under physiological conditions in vivo. These molecules arise from the dissociation of previously assembled class I heterodimers at the cell surface. The ratio of intact to dissociated heterodimers is strongly affected by the occupancy of the peptide-binding site of the class I molecule. Upon dissociation of the heterodimer, the class I molecule is functionally inactive. These findings may help to explain why class I molecules on the cell surface are unreceptive to binding peptides yet readily associate with peptides in the presence of exogenous beta 2-microglobulin. These results have implications for understanding the distinct functions of class I versus class II molecules and how the immunological identity of cells is preserved.

Antibody responses to trinitrophenyl (TNP)-l-glutamic acid60-l-alanine30-l-tyrosine10 (GAT) in microcultures: Anti-hapten and anti-carrier responses appear to be under separable control

Abstract We have previously reported that the IgM anti-hapten plaque-forming cell (PFC) response to trinitrophenyl (TNP)-conjugated l -glutamic acid 60 - l -alanine 30 - l -tyrosine 10 (GAT) is not under conventional Ir gene control in an in vitro microculture system. To explore this phenomenon, we examined both the anti-hapten and anti-carrier antibody responses to TNP-GAT in the microculture supernatants using a solid-phase radioimmunoassay (RIA). We show that splenocytes from GAT-responder BALB/c mice produce anti-hapten and anti-carrier antibody responses to TNP-GAT in microcultures, while splenocytes from GAT-nonresponder DBA/ 1 mice produce anti-hapten but not anti-carrier responses to TNP-GAT in these culture conditions. We further demonstrate that supernatants from rat splenocyte cultures stimulated by concanavalin A (Con A) similarly drive unprimed, T-lymphocyte-depleted splenocytes to produce anti-hapten but not anti-carrier antibody responses to TNP-GAT in microcultures. This observation suggests that T-cell-B-cell contact may not be required for the generation of anti-hapten responses by splenocytes to TNP-GAT in microcultures and may explain the absence of conventional Ir gene control of such responses in these cultures.