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The immunoproteasome generates peptides presented on MHC class I molecules to cytotoxic T cells. ONX 0914 (formerly called PR-957) is a selective inhibitor of the immunoproteasome subunit low molecular mass polypeptide (LMP) 7 (β5i) that attenuates disease progression in mouse models of diabetes, colitis, and arthritis. The aim of this study was to investigate the effect of LMP7-specific inhibition on major Th cell differentiation pathways involved in the progression of autoimmune diseases in vitro and in vivo. We used ONX 0914-treated wild-type CD4(+) T cells and also LMP7(-/-) CD4(+) T cells under different Th cell-polarizing conditions, focusing on the effector cytokines and transcription factors involved, and compared them with wild-type CD4(+) T cells. Mouse models of dextran sodium sulfate-induced colitis and a T cell transfer model of colitis were used for in vivo assessment. Deletion or inhibition of LMP7 suppressed generation of Th17 but promoted regulatory T cell (Treg) development. In developing Th17 cells, immunoproteasome inhibition blocked phosphorylation of STAT3, whereas in Tregs, SMAD phosphorylation was enhanced. Additionally, LMP7 inhibition led to reduced STAT1 phosphorylation and Th1 differentiation. These findings were confirmed in vivo as LMP7 inhibition or deficiency resulted in reduced Th1 and Th17 expansion while promoting Treg development in dextran sodium sulfate-induced colitis. Also, in a T cell-dependent transfer model of colitis, LMP7-specific inhibition led to reduced Th1 and Th17 differentiation in vivo. LMP7 governs Th cell lineage determination by affecting the balance of receptor proximal signals during differentiation. These data render LMP7 a promising drug target for the treatment of autoimmune diseases.

Innate immunity to viruses involves receptors such as Retinoic Acid Induced Gene-1 (RIG-I), which senses viral RNA and triggers a signaling pathway involving the outer mitochondrial membrane protein mitochondrial antiviral signaling (MAVS). Recent work has identified that NLRX1, a member of another class of innate immune receptors, sequesters MAVS away from RIG-I and thereby prevents mitochondrial antiviral immunity. In this study, we demonstrate that the proteasome PSMA7 (alpha4) subunit associates with MAVS in vivo and in vitro. Expression of PSMA7 results in a potent inhibition of RIG-1 and MAVS-mediated IFN-beta promoter activity; conversely, depletion of PSMA7 with small interference RNA enhances virus-induced type I IFN production, with consequent reduction of virus replication. Furthermore, a striking reduction in the abundance of endogenous MAVS with overexpressed PSMA7 was found and virus infection leads to transient increase in the endogenous PSMA7 protein level. Cumulatively, these results suggest that PSMA7 is a negative regulator of the MAVS-mediated innate immunity that probably serves to attenuate the establishment of an antiviral state during viral infection, highlighting the biological significance of PSMA7-MAVS association as an important cellular regulatory control.

Downregulation of p53 by MDM2-mediated proteasomal degradation makes cells resistant to apoptosis. The MDM2-p53 interaction is well characterized, but the mechanisms that regulate the interaction are not well understood. Here, we show that PA28gamma, a proteasome activator that inhibits apoptosis and promotes cell cycle progression through unknown mechanisms, exerts an effect as a cofactor in the MDM2-p53 interaction. The polymer form of PA28gamma interacts with both MDM2 and p53 proteins and facilitates their physical interaction. This promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28gamma in human cancer cells abrogates MDM2-mediated p53 degradation, increases the activity of p53, and enhances apoptosis. These findings reveal the mechanism by which PA28gamma affects apoptosis and proliferation. Manipulation of the level of PA28gamma, an approach that would regulate the cellular content of p53, may improve the efficacy of current cancer therapies.