John J. Monaco

A molecular model of MHC class-I-restricted antigen processing

Cells of higher vertebrates have evolved mechanisms that allow a sample of their intracellular contents to be available for surveillance by the immune system. This display of intracellular material is in the form of peptides bound to cell surface major histocompatibility complex (MHC) class I molecules. In this review, John Monaco presents a model of the mechanisms by which this takes place, based on the recent identification of a number of new genes in the MHC.

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor β subunits

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit‐specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of α, precursor β and processed β subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of α and precursor β but no processed β subunits. These complexes possess short half‐lives, are enzymatically inactive and their molecular weight is ∼300 kDa. Radioactivity chases from these complexes into mature, long‐lived ∼700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing α subunits and the other, precursor β subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor β subunits PSMB2 (C7‐I), PSMB3 (C10‐II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the β subunits and juxtaposition of the two β rings) is accompanied by conformational changes in the (outer) α rings, and may be inefficient. Finally, interferon‐γ had no significant effect on the half‐lives or total amounts of precursor or mature proteasomes.

Compartmentalization of MHC class II gene expression in transgenic mice

A set of transgenic mouse lines carrying Ek alpha genes with promoter region deletions was created in an attempt to compartmentalize MHC class II gene expression. Fine immunohistological analyses established that one transgenic line is essentially devoid of E complex in the thymic cortex, another displays almost no E in the thymic medulla or on peripheral macrophages, and two lines display no E on greater than 98% of B cells. We have assayed these mice for immune function: E-dependent tolerance, antigen presentation, T cell priming, and antibody response. Certain of the findings are difficult to reconcile with currently popular hypotheses, e.g., tolerance induction to E molecules in the virtual absence of E complex in the thymic medulla and efficient antibody responses to E-restricted antigens when almost all B cells are E-.

An altered T cell repertoire in MECL-1-deficient mice

Immunoproteasome subunits low-molecular mass polypeptide (LMP)2 and LMP7 affect Ag presentation by MHC class I molecules. In the present study, we investigated the function of the third immunosubunit LMP10/multicatalytic endopeptidase complex-like (MECL)-1 (β2i) in MECL-1 gene-targeted mice. The number of CD8+ splenocytes in MECL-1−/− mice was 20% lower than in wild-type mice. Infection with lymphocytic choriomeningitis virus (LCMV) elicited a markedly reduced cytotoxic T cell (CTL) response to the LCMV epitopes GP276–286/Db and NP205–212/Kb in MECL-1−/− mice. The weak CTL response to GP276–286/Db was not due to an impaired generation of this epitope but was attributed to a decreased precursor frequency of GP276–286/Db-specific T cells. The expansion of TCR-Vβ10+ T cells, which contain GP276–286/Db-specific cells, was reduced in LCMV-infected MECL-1−/− mice. Taken together, our data reveal an in vivo function of MECL-1 in codetermining the T cell repertoire for an antiviral CTL response.

Pathways for the processing and presentation of antigens to T cells.

Two pathways exist within vertebrate cells to generate peptides for recognition by T cells. The “en dogenous” pathway provides peptides to MHC class I molecules for presentation to CD8+ T cells. These pep tides are derived from proteins synthesized or residing in the cytoplasm or nucleus, and involves proteasomes and the ubiquitin pathway of protein degradation, as well as a specific peptide transporter (TAP) that allows these pep tides access to the lumen of the endoplasmic reticulum. The exogenous pathway provides peptides to MHC class II molecules for presentation to CD4+ T cells. These pep tides are derived from extracellular antigens taken up by endocytosis and degraded in the endosomal/lysosomal pathway. Peptide loading of MHC class II molecules re quires the presence of a molecule (H‐2M in mouse, HLA‐DM in humans) that is structurally related to MHC class II molecules, but the mechanistic basis of this require ment is unknown. The class II region of the MHC con tains a cluster of genes encoding proteins involved in an tigen processing, including genes for two proteasome subunits (LMP2 and LMP7), the peptide transporter hetcrodimer (TAPI and TAP2), and the H‐2M/HLA‐DM molecule (Ma and Mb, or DMA and DMB). J. Lcukoc. Biol. 57: 543–547; 1995.

Immune Defects in 28-kDa Proteasome Activator γ-Deficient Mice

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-γ-inducible PA28α/β complex in Ag processing. Although the noninducible and predominantly nuclear PA28γ complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28γ-deficient mice and investigated their immune function. PA28γ−/− mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28γ−/− mice, like PA28α−/−/β−/− mice, are deficient in the processing of only specific Ags.

Activity and expression of the 20S proteasome are increased in skeletal muscle during sepsis

Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, ubiquitinated proteins are recognized, unfolded, and degraded by the multicatalytic 26S protease complex. The 20S proteasome is the catalytic core of the 26S protease complex. The role of the 20S proteasome in the regulation of sepsis-induced muscle proteolysis is not known. We tested the hypothesis that sepsis increases 20S proteasome activity and the expression of mRNA for various subunits of this complex. Proteolytic activity of isolated 20S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from septic rats. The proteolytic activity was inhibited by specific proteasome blockers. Northern blot analysis revealed an approximately twofold increase in the relative abundance of mRNA for the 20S α-subunits RC3 and RC9 and the β-subunit RC7. However, Western blot analysis did not show any difference in RC9 protein content between sham-operated and septic rats. The increased activity and expression of the 20S proteasome in muscles from septic rats lend further support for a role of the ubiquitin-proteasome-pathway in the regulation of sepsis-induced muscle proteolysis.

T Cells Lacking Immunoproteasome Subunits MECL-1 and LMP7 Hyperproliferate in Response to Polyclonal Mitogens

Immunoproteasomes comprise a specialized subset of proteasomes that is defined by the presence of three catalytic immunosubunits: LMP2, MECL-1 (LMP10), and LMP7. Proteasomes in general serve many cellular functions through protein degradation, whereas the specific function of immunoproteasomes has been thought to be largely, if not exclusively, optimization of MHC class I Ag processing. In this report, we demonstrate that T cells from double knockout mice lacking two of the immunosubunits, MECL-1 and LMP7, hyperproliferate in vitro in response to various polyclonal mitogens. We observe hyperproliferation of both CD4+ and CD8+ T cell subsets and demonstrate accelerated cell cycling. We do not observe hyperproliferation of T cells lacking only one of these subunits, and thus hyperproliferation is independent of either reduced MHC class I expression in LMP7−/− mice or reduced CD8+ T cell numbers in MECL-1−/− mice. We observe both of these latter two phenotypes in MECL-1/LMP7−/− mice, which indicates that they also are independent of each other. Finally, we provide evidence of in vivo T cell dysfunction by demonstrating increased numbers of central memory phenotype CD8+ T cells in MECL-1/LMP7−/− mice. In summary, this novel phenotype of hyperproliferation of T cells lacking both MECL-1 and LMP7 implicates a specific role for immunoproteasomes in T cell proliferation that is not obviously connected to MHC class I Ag processing.

Sepsis-induced increase in muscle proteolysis is blocked by specific proteasome inhibitors

Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. The 20S proteasome is the catalytic core of the ubiquitin-dependent proteolytic pathway. We tested the effects in vitro of the proteasome inhibitors N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) and lactacystin on protein breakdown in incubated muscles from septic rats. LLnL resulted in a dose- and time-dependent inhibition of protein breakdown in muscles from septic rats. Lactacystin blocked both total and myofibrillar muscle protein breakdown. In addition to inhibiting protein breakdown, LLnL reduced muscle protein synthesis and increased ubiquitin mRNA levels, probably reflecting inhibited proteasome-associated ribonuclease activity. Inhibited muscle protein breakdown caused by LLnL or lactacystin supports the concept that the ubiquitin-proteasome pathway plays a central role in sepsis-induced muscle proteolysis. The results suggest that muscle catabolism during sepsis may be inhibited by targeting specific molecular mechanisms of muscle proteolysis.

The LMP antigens: A stable MHC-controlled multisubunit protein complex☆

In addition to encoding the well-known class I (H-2), II (Ia), and III (complement components C2, C4, and factor B) antigens, the murine MHC controls the expression of a large, intracellular protein complex of unknown function. This complex is composed of a large number of noncovalently linked low molecular weight polypeptide subunits (hence the name, LMP) which are biochemically, serologically, and genetically distinct from class I, II, and III antigens. Only two of these subunits display electrophoretic polymorphism within the standard inbred mouse strains, and both of these polymorphisms map within the H-2 complex, between the H-2K and I-A subregions. The remainder of the LMP complex subunits have not been mapped, and may be encoded elsewhere in the genome. A biochemically similar complex has been detected in human cells, although linkage to HLA remains to be established. In this article we will review the biochemistry, serology, and genetics of the LMP antigens, and will speculate on their biological function.