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...

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.

Epithelial cells lining the murine genital tract act as sentinels for microbial infection, play a major role in the initiation of the early inflammatory response, and can secrete factors that modulate the adaptive immune response when infected with Chlamydia. C. muridarum-infected murine oviduct epithelial cells secrete the inflammatory cytokines IL-6 and GM-CSF in a TLR2-dependent manner. Further, C. muridarum infection induces IFN-β synthesis in the oviduct epithelial cells in a TRIF-dependent manner. Because murine oviduct epithelial cells express TLR3 but not TLRs 4, 7, 8, or 9, we hypothesized that TLR3 or an unknown TRIF-dependent pattern recognition receptor was the critical receptor for IFN-β production. To investigate the role of TLR3 in the Chlamydia-induced IFN-β response in oviduct epithelial cells, we used small interfering RNA, dominant-negative TLR3 mutants, and TLR3-deficient oviduct epithelial cells to show that the IFN-β secreted during C. muridarum infection requires a functional TLR3. Interestingly, we demonstrate that the TLR3 signaling pathway is not required for IFN-β synthesis in C. muridarum-infected macrophages, suggesting that there are alternate and redundant pathways to Chlamydia-induced IFN-β synthesis that seem to be dependent upon the cell type infected. Finally, because there is no obvious dsRNA molecule associated with Chlamydia infection, the requirement for TLR3 in Chlamydia-induced IFN-β synthesis in infected oviduct epithelial cells implicates a novel ligand that binds to and signals through TLR3.