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.

Vertebrate proteasomes are structurally heterogeneous, consisting of both "constitutive" (or "standard") proteasomes and "immunoproteasomes." Constitutive proteasomes contain three ubiquitously expressed catalytic subunits, Delta (␤1), Z (␤2), and X (␤5), whereas immunoproteasomes contain three interferon-␥-inducible catalytic subunits, LMP2 (␤1i), MECL (␤2i), and LMP7 (␤5i). We recently have demonstrated that proteasome assembly is biased to promote immunoproteasome homogeneity when both types of catalytic subunits are expressed in the same cell. This cooperative assembly is due in part to differences between the LMP7 (␤5i) and X (␤5) propeptides. In the current study we demonstrate that differences between the MECL (␤2i) and Z (␤2) propeptides also influence cooperative assembly. Specifically, replacing the MECL propeptide with that of Z enables MECL incorporation into otherwise constitutive (Delta ؉ /X ؉ ) proteasomes and facilitates X incorporation into otherwise immunoproteasomes (MECL ؉ / LMP2 ؉ ). We also show, using MECL ؊/؊ mice, that LMP2 incorporation does not require MECL, in contrast with previous suggestions that their incorporation is mutually codependent. These results enable us to refine our model for cooperative proteasome assembly by determining which combinations of inducible and constitutive subunits are favored over others, and we propose a mechanism for how propeptides mediate cooperative assembly.Eukaryotic proteasomes are an integral component of ubiquitin-mediated protein degradation, which plays a major role in the turnover of cytoplasmic and nuclear proteins (1-5). By virtue of their role in protein metabolism, proteasomes are involved in a number of cellular processes, including cell cycle control, cellular stress responses, intracellular signaling, and major histocompatibility complex class I antigen processing (6). Immunoproteasomes are a specialized subset of vertebrate proteasomes that contain three interferon-␥-inducible catalytic subunits, LMP2 (␤1i), MECL (␤2i), and LMP7 (␤5i) (7,8).Immunoproteasomes are thought to possess enhanced capability for generating major histocompatibility complex class Ibinding peptides with basic or hydrophobic C termini as compared with constitutive proteasomes, which contain three constitutively synthesized catalytic subunits, Delta (␤1), Z (␤2), and X (␤5) (9 -14).The 20 S catalytic proteasome core is comprised of 28 subunits arranged in four stacked seven-member rings (15-17). Each outer ring contains seven different non-catalytic ␣-type subunits, ␣1-␣7, and each inner ring contains seven different ␤-type subunits, ␤1-␤7 (18), at least three of which are catalytic (␤1 or ␤1i, ␤2 or ␤2i, and ␤5 or ␤5i). The N-terminal proteolytic active sites are on the inner surface of the ␤ rings, whereas the C termini of ␤ subunits are on the outer surface of proteasomes (19 -21), enabling us to use C-terminal "epitope tags" to immunoprecipitate and track specific subunits because these tags do not appear to interfere with proteasome structure or catalytic act...

The mechanisms by which prolonged estrogen exposures, such as estrogen therapy and pregnancy, reduce thymus weight, cellularity, and CD4 and CD8 phenotype expression, have not been well defined. In this study, the roles played by the membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), and the intracellular estrogen receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta), in 17beta-estradiol (E2)-induced thymic atrophy were distinguished by construction and the side-by-side comparison of GPR30-deficient mice with ERalpha and ERbeta gene-deficient mice. Our study shows that whereas ERalpha mediated exclusively the early developmental blockage of thymocytes, GPR30 was indispensable for thymocyte apoptosis that preferentially occurs in T cell receptor beta chain(-/low) double-positive thymocytes. Additionally, G1, a specific GPR30 agonist, induces thymic atrophy and thymocyte apoptosis, but not developmental blockage. Finally, E2 treatment attenuates the activation of nuclear factor-kappa B in CD25(-)CD4(-)CD8(-) double-negative thymocytes through an ERalpha-dependent yet ERbeta- and GPR30-independent pathway. Differential inhibition of nuclear factor-kappaB by ERalpha and GPR30 might underlie their disparate physiological effects on thymocytes. Our study distinguishes, for the first time, the respective contributions of nuclear and membrane E2 receptors in negative regulation of thymic development.

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.