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The proteasome is a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells that controls numerous cellular processes through regulated protein degradation. Proteasome inhibitors have significantly improved the survival of multiple myeloma patients. However, clinically approved proteasome inhibitors have failed to show efficacy against solid tumors, neither alone nor in combination with other therapies. Targeting the immunoproteasome with selective inhibitors has been therapeutically effective in preclinical models for several autoimmune diseases and colon cancer. Moreover, immunoproteasome inhibitors prevented the chronic rejection of allogeneic organ transplants. In recent years, it has become apparent that inhibition of one single active center of the proteasome is insufficient to achieve therapeutic benefits. In this review we summarize the latest insights how targeting multiple catalytically active proteasome subunits can interfere with disease progression in autoimmunity, growth of solid tumors, and allograft rejection.

MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome (IP), a special type of proteasome mainly expressed in hematopoietic cells. Targeting the IP in autoimmune diseases proved to be therapeutically effective in preclinical mouse models. In endotoxin-stimulated human PBMCs, IP inhibition reduces the secretion of several proinflammatory cytokines, with the suppression of IL-23 being the most prominent. In this study, we investigated why the production of IL-23, a key mediator of inflammation in autoimmunity, is blocked when the IP is inhibited in LPS-stimulated human PBMCs. CD14 + monocytes could be identified as the main producers of IL-23 in LPS-stimulated PBMCs. We found that IP inhibition with the irreversible LMP7/LMP2 inhibitor ONX 0914 induced apoptosis in CD14 + monocytes, whereas CD4 + , CD3 + , CD19 + , and CD56 + cells remained unaffected. A high expression of IPs renders monocytes susceptible to IP inhibition, leading to an accumulation of polyubiquitylated proteins and the induction of the unfolded protein response. Similar to IP inhibition, inducers of the unfolded protein response selectively kill CD14 + monocytes in human PBMCs. The blockage of the translation in CD14 + monocytes protects these cells from ONX 0914-induced cell death, indicating that the IP is required to maintain protein turnover in monocytes. Taken together, our data reveal why IP inhibition is particularly effective in the suppression of IL-23-driven autoimmunity., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The immunoproteasome is expressed in cells of hematopoietic origin and is induced during inflammation by IFN-γ. Targeting the immunoproteasome with selective inhibitors has been shown to be therapeutically effective in pre-clinical models for autoimmune diseases, colitis-associated cancer formation, and transplantation. Immunoproteasome inhibition prevents activation and proliferation of lymphocytes, lowers MHC class I cell surface expression, reduces the expression of cytokines of activated immune cells, and curtails  T helper 1 and 17 cell differentiation. This might explain the in vivo efficacy of immunoproteasome inhibition in different pre-clinical disease models for autoimmunity, cancer, and transplantation. In this review, we summarize the effect of immunoproteasome inhibition in different animal models for transplantation.

Chronic antibody-mediated rejection is the leading cause of allograft dysfunction and loss after kidney transplantation, and current immunosuppressive regimens fail to target the plasma cells that produce alloantibodies. We previously showed that treatment with the immunoproteasome inhibitor ONX 0914 prevented the expansion of plasma cells and prevented chronic allograft nephropathy and organ failure after kidney transplantation in rats, but the mechanism has remained elusive. In the current study, we confirmed a long-term reduction in alloantibody production and improvements in allograft histology in rats treated with ONX 0914 or with the broad-spectrum proteasome inhibitor bortezomib. Plasma cells from allotransplanted rats expressed immunoproteasomes at high levels. Immunoproteasome inhibition with ONX 0914 led to ubiquitin-conjugate accumulation, activation of the unfolded protein response, and induction of apoptosis in plasma cells. In addition, ONX 0914 suppressed the expression of adhesion molecules (VLA-4 and LFA-1), plasma cell survival factors (APRIL and IL-6), and IFN-γ-inducible chemokines in bone marrow, while the APRIL receptor BCMA, the IL-6 receptor, and the chemokine receptors CXCR4 and CXCR3 were down-regulated on plasma cells. Taken together, immunoproteasome inhibition blocked alloantibody production by inducing apoptosis of plasma cells through activating the unfolded protein response and suppressing plasma cell survival factors in the bone marrow., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases., (© 2018 The Authors.)

Chronic antibody-mediated rejection is the major cause of fading allograft function and loss after renal transplantation. Currently, pharmacological agents for the suppression of chronic antibody-mediated rejection are lacking. Non-selective proteasome inhibitors suppress antibody-mediated allograft rejection. However, extensive adverse side effects of these inhibitors severely limit their application. In contrast, immunoproteasome inhibition is effective in preclinical models of autoimmune diseases and was applied over weeks without obvious adverse side effects. ONX 0914, an immunoproteasome subunit LMP7 (β5i)-selective inhibitor, impeded the chronic rejection of kidneys transplanted from Fischer to allogeneic Lewis rats. ONX 0914 inhibited immunoproteasome induction both in immune organs and renal allografts. Selective immunoproteasome inhibition reduced the numbers of B and plasma cells, and suppressed donor-specific alloantibody production. The infiltration of T cells, B cells and macrophages as well as interferon-γ, interleukin-17, IgG and complement deposition were reduced in renal allografts of ONX 0914-treated recipients. Chronic nephropathy was ameliorated and renal allograft function preserved, enabling long-term survival of recipients. Thus, our studies define a critical role of the immunoproteasome in chronic kidney allograft rejection and suggest immunoproteasome inhibition as a promising therapeutic approach to suppress chronic antibody-mediated rejection., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Multicatalytic endopeptidase complex-like-1 (β2i), low molecular mass polypeptide (LMP) 2 (β1i) and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in haematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in preclinical models of autoimmune diseases. In this study, we investigated the selectivity and biological activity of LU-005i, a recently described inhibitor of the immunoproteasome., Experimental Approach: The specificity of LU-005i and other immunoproteasome-selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated in endotoxin-stimulated mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). The effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)-induced colitis model was assessed by measuring weight loss and colon length., Key Results: LU-005i is the first human and mouse immunoproteasome-selective inhibitor that targets all three proteolytically active immunoproteasome subunits. LU-005i inhibited cytokine secretion from endotoxin-stimulated mouse splenocytes or human PBMCs. Furthermore, differentiation of naïve T helper cells to T helper 17 cells was impaired in the presence of LU-005i. Additionally, LU-005i ameliorated DSS-induced colitis., Conclusion and Implications: This study with a novel pan-immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer., (© 2017 The British Pharmacological Society.)

The immunoproteasome, a distinct class of proteasomes, which is inducible under inflammatory conditions and constitutively expressed in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Moreover, inhibition of the immunoproteasome subunit LMP7 ameliorates clinical symptoms of autoimmune diseases in vivo and was shown to suppress the development of T helper cell (Th) 1 and Th17 cells and to promote regulatory T-cell (Treg) generation independently of its function in antigen processing. Since Th1 and Th17 cells are detrimental and Treg cells are critical for transplant acceptance, we investigated the influence of the LMP7-selective inhibitor ONX 0914 in a mixed lymphocyte reaction (MLR) in vitro as well as on allograft rejection in a MHC-disparate (C57BL/6 to BALB/c) and a multiple minor histocompatibility antigen (miHA)-disparate (B10.Br to C3H) model of skin transplantation in vivo. Although we observed reduced allo-specific IL-17 production of T cells in vitro, we found that selective inhibition of LMP7 had neither an influence on allograft survival in an MHC-mismatch model nor in a multiple minor mismatch skin transplantation model. We conclude that inhibition of the immunoproteasome is not effective in prolonging skin allograft survival in skin allotransplantation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The immunoproteasome, a special class of the proteasome, is mainly expressed in cells of haematopoietic origin. Additionally, during inflammation, the immunoproteasome is induced by IFN-γ or TNF-α. In recent years it became apparent that the immunoproteasome has important functions other than processing proteins for MHC class I restricted presentation. The immunoproteasome plays a critical role in T cell expansion, cytokine production, and T helper cell differentiation. Inhibition of the immunoproteasome ameliorated disease symptoms in different animal models for autoimmune diseases. Hence, the unique role for LMP7 in controlling pathogenic immune responses provides a therapeutic rationale for targeting LMP7 in autoimmune disorders. In this review we summarise the effect of immunoproteasome inhibition in animal models for rheumatoid arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, systemic lupus erythematosus, and multiple sclerosis.

Specialized variants of the constitutive 20S proteasome in the immune system like the immunoproteasomes and the thymoproteasome contain active site-bearing subunits which differ in their cleavage priorities and substrate binding pockets. The immunoproteasome plays a crucial role in antigen processing and for the differentiation of pro-inflammatory T helper cells which are involved in the pathogenesis of autoimmunity. Selective inhibitors of the immunoproteasome and constitutive proteasome have recently been generated which interfere with the development and progression of autoimmune diseases. Here we describe these inhibitors and their therapeutic potential as predicted from preclinical models., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG₃₅-₅₅ and PLP₁₃₉₋₁₅₁-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4(+) cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.

Immunoproteasomes (IPs) containing the interferon-inducible subunits β1i (LMP2), β2i (MECL-1), and β5i (LMP7) alter proteasomal cleavage preference, optimise the generation of peptide ligands of MHC class I molecules, alter cytokine profile, influence T-helper cell differentiation, and play a role in T-cell survival. Small molecule inhibitors are useful tools for probing the role of the immunoproteasome in immune functions. Here, we describe different methods to characterise immunoproteasome-selective inhibitors. Thereby, we provide the methodology to analyse the specificity and cell permeability of immunoproteasome inhibitors, as well as to functionally investigate immunoproteasome inhibitors in antigen presentation.

The proteasome, a multicatalytic protease, is responsible for the degradation of intracellular proteins. Stimulation of cells with inflammatory cytokines, such as IFN-gamma, leads to the replacement of the constitutive catalytic proteasome subunits by the inducible subunits low molecular mass polypeptide (LMP)2 (beta1i), multicatalytic endopeptidase complex-like-1 (beta2i), and LMP7 (beta5i), which are required for the production of certain MHC class I-restricted T cell epitopes. In this study, we investigated the effect of immunoproteasomes on the development of dextran sulfate sodium-induced colitis. Colitis induction in LMP2-, LMP7-, and multicatalytic endopeptidase complex-like-1-deficient mice caused reduced weight loss compared with wild-type mice. Although colon lengths were shortened in wild-type mice, no reduction was observed in immunoproteasome-deficient mice. In accordance with this, proinflammatory cytokines, such as TNF-alpha and IL-1beta, were not upregulated in these mice. Blockage of LMP7 by a novel LMP7-selective inhibitor (PR-957) strongly reduced pathological symptoms of dextran sulfate sodium-induced colitis. Production of numerous cytokines in PR-957-treated mice was suppressed, resulting in reduced inflammation and tissue destruction. Taken together, these results demonstrate that an immunoproteasome-specific inhibitor can be used to attenuate autoimmune diseases like colitis.

The proteasome, a multicatalytic protease, is responsible for the generation of most MHC class I ligands. Bortezomib, a proteasome inhibitor, is clinically approved for treatment of multiple myeloma and mantle cell myeloma. In the present study, we investigated the effect of bortezomib on viral infection. Infection of bortezomib-treated mice with the lymphocytic choriomeningitis virus (LCMV) led to a decreased cytotoxic T cell response to several LCMV-derived CD8(+) T cell epitopes. Bortezomib treatment caused a reduced expansion of CD8(+) T lymphocytes and increased viral titers in LCMV-infected mice. Administration of bortezomib during expansion of CD8(+) T cells had no influence on the cytotoxic T cell response, suggesting that bortezomib interferes with priming of naive T cells. Indeed, determination of Ag load in spleen 4 days post infection, revealed a reduced presentation of LCMV-derived cytotoxic T cell epitopes on MHC class I molecules. In summary, we show that proteasome inhibition with bortezomib led to an increased susceptibility to viral infection, and demonstrate for the first time, that proteasome inhibitors can alter Ag processing in vivo.

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low-molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I-restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-gamma and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.