Immuno- and constitutive proteasomes do not differ in their abilities to degrade ubiquitinated proteins.

Immunoproteasomes are alternative forms of proteasomes that have an enhanced ability to generate antigenic peptides. Recently, Seifert and colleagues reported surprising observations concerning the functions of immunoproteasomes and cellular responses to interferon-γ: (1) that immunoproteasomes degrade ubiquitinated proteins faster than the constitutive proteasomes, (2) that polyubiquitin conjugates accumulate after interferon-γ treatment but then are preferentially degraded by immunoproteasomes, and (3) that immunoproteasome deficiency causes the formation of inclusions and more severe experimental autoimmune encephalomyelitis (EAE). In contrast, we find that polyubiquitin conjugates do not transiently accumulate following IFNγ-treatment and that immunoproteasomes do not prevent the formation of intracellular inclusions or protect against EAE. Furthermore, purified 26S constitutive and immunoproteasomes bind ubiquitin conjugates similarly and degrade them at similar rates. We conclude that, although immunoproteasomes can increase the generation of peptides appropriate for MHC class I presentation, they do not degrade ubiquitinated proteins more efficiently than constitutive particles.

Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity.

Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit ß5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of ß5c/X but not ß5i, thereby explaining the selectivity of PR-957 for ß5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.

scTEA-db: a comprehensive database of novel terminal exon isoforms identified from human single cell transcriptomes.

The usage of alternative terminal exons results in messenger RNA (mRNA) isoforms that differ in their 3' untranslated regions (3' UTRs) and often also in their protein-coding sequences. Alternative 3' UTRs contain different sets of cis-regulatory elements known to regulate mRNA stability, translation and localization, all of which are vital to cell identity and function. In previous work, we revealed that ∼25 percent of the experimentally observed RNA 3' ends are located within regions currently annotated as intronic, indicating that many 3' end isoforms remain to be uncovered. Also, the inclusion of not yet annotated terminal exons is more tissue specific compared to the already annotated ones. Here, we present the single cell-based Terminal Exon Annotation database (scTEA-db, www.scTEA-db.org) that provides the community with 12 063 so far not yet annotated terminal exons and associated transcript isoforms identified by analysing 53 069 publicly available single cell transcriptomes. Our scTEA-db web portal offers an array of features to find and explore novel terminal exons belonging to 5538 human genes, 110 of which are known cancer drivers. In summary, scTEA-db provides the foundation for studying the biological role of large numbers of so far not annotated terminal exon isoforms in cell identity and function.

Proteasome composition in immune cells implies special immune­cell­specific immunoproteasome function.

Immunoproteasomes are a special class of proteasomes, which can be induced with IFN-γ in an inflammatory environment. In recent years, it became evident that certain immune cell types constitutively express high levels of immunoproteasomes. However, information regarding the basal expression of proteolytically active immunoproteasome subunits in different types of immune cells is still rare. Hence, we quantified standard proteasome subunits (ß1c, ß2c, ß5c) and immunoproteasome subunits (LMP2, MECL-1, LMP7) in the major murine (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD11c+ dendritic cells, CD49d+ natural killer cells, Ly-6G+ neutrophils) and human immune cell (CD4+ T cells, CD8+ T cells, CD19+ B cells, CD1c+CD141+ myeloid dendritic cells, CD56+ natural killer cells, granulocytes) subsets. The different human immune cell types were isolated from peripheral blood and the murine immune cell subsets from spleen. We found that proteasomes of most immune cell subsets mainly consist of immunoproteasome subunits. Our data will serve as a reference and guideline for immunoproteasome expression and imply a special role of immunoproteasomes in immune cells.

The Significant Role of PA28αß in CD8+ T Cell-Mediated Graft Rejection Contrasts with Its Negligible Impact on the Generation of MHC-I Ligands.

The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αß). In particular, several immunogenic peptides have been reported to be PA28αß-dependent. In contrast, we did not observe a major impact of PA28αß on the generation of different major histocompatibility complex (MHC) classI ligands. PA28αß-knockout mice infected with the lymphocytic choriomeningitis virus (LCMV) or vaccinia virus showed a normal cluster of differentiation (CD) 8 response and viral clearance. However, we observed that the adoptive transfer of wild-type cells into PA28αß-knockout mice led to graft rejection, but not vice versa. Depletion experiments showed that the observed rejection was mediated by CD8+ cytotoxic T cells. These data indicate that PA28αß might be involved in the development of the CD8+ T cell repertoire in the thymus. Taken together, our data suggest that PA28αß is a crucial factor determining T cell selection and, therefore, impacts graft acceptance.

Immunoproteasome inhibition prevents progression of castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment. This study aims to explore the effect and underlying mechanisms of immunoproteasome inhibition, a novel immunotherapy, on the progression of CRPC. METHODS: The immunoproteasome subunit LMP7 was silenced by using gene knockout or inhibited by the epoxyketone inhibitor ONX 0914 in a mouse CRPC tumour graft model and in interferon-γ-pretreated human CRPC cell lines in vitro. RESULTS: CRPC tissues reveal a significant "tumour-elicited" Th17-type inflammatory response which induces immunoproteasome subunit expression. LMP7 deficiency in host mice or in CRPC tumour grafts had no effect on the "tumour-elicited" Th17-type inflammatory response and tumour progression. However, the selective LMP7 inhibitor ONX 0914 strongly suppressed the "tumour-elicited" Th17-type inflammatory response and CRPC tumour progression. Treatment of wild-type mice receiving LMP7-deficient CRPC tumour grafts with ONX 0914 further suggested that immunoproteasome inhibition prevents CRPC progression through suppressing IL-17-induced angiogenesis and epithelial-mesenchymal transition via inactivation of COX-2/VEGF-A signalling and ß-catenin/Snail signalling. Treatment of LMP7-deficient mice receiving wild-type CRPC tumour grafts with ONX 0914 and inhibition of LMP7 in PC3 and 22Rv.1 cells with ONX 0914 showed that immunoproteasome inhibition also prevents CRPC progression through inducing CRPC cell apoptosis via activation of the unfolded protein response. CONCLUSIONS: We define a critical role of the immunoproteasome in CRPC and propose immunoproteasome inhibition as a promising therapeutic approach to suppress CRPC progression.

Effective therapy of polymyositis in mice via selective inhibition of the immunoproteasome.

Polymyositis (PM) is a chronic autoimmune inflammatory myopathy resulting in muscle weakness. The limited approved therapies and their poor efficacy contribute to its comorbidity. We investigated the therapeutic use of ONX 0914 and KZR-616, selective inhibitors of the immunoproteasome, in C protein-induced myositis (CIM), a mouse model of PM that closely resembles the human disease. Diseased mice (day 13 postimmunization) were treated with 10 mg/kg ONX 0914, KZR-616, or vehicle on alternate days until day 28. Endpoints included muscle strength assessed by a grip strength meter, serum creatine kinase activity, histology, and immunohistochemistry analysis. Treatment with ONX 0914 or KZR-616 prevented the loss of grip strength in mice after CIM induction, while vehicle-treated animals displayed progressive muscle weakness. Immunoproteasome inhibition lowered PM-associated leukocyte infiltration of the muscle and prevented increased serum creatine kinase levels. LMP7-deficient mice were resistant to CIM induction, as they showed no alterations in grip strength or creatine kinase (CK) levels or muscular alterations. In conclusion, selective inhibition of the immunoproteasome displays therapeutic efficacy in a preclinical mouse model of PM with suppression of muscle inflammation and preservation of muscle strength. Positive results from this study support the rationale for using KZR-616 in clinical studies.

Immunoproteasome Upregulation Is Not Required to Control Protein Homeostasis during Viral Infection.

The prime function of proteasomes is the control of protein homeostasis in cells (i.e., the removal of proteins that are not properly folded, damaged by stress conditions like reactive oxygen species formation, or degraded on the basis of regular protein turnover). During viral infection, the standard proteasome is replaced by the so-called immunoproteasome (IP) in an IFN-γ-dependent manner. It has been proposed that the IP is required to protect cell viability under conditions of IFN-induced oxidative stress. In this study, we investigated the requirement for IP to cope with the enhanced need for protein degradation during lymphocytic choriomeningitis virus (LCMV) infection in mice lacking the IP subunit LMP7. We found that IP are upregulated in the liver but not in the spleen during LCMV infection, although the total proteasome content was not altered. The expression of standard proteasome subunits is not induced in LMP7-deficient mice, indicating that enhanced proteasomal activity is not required during viral infection. Furthermore, ubiquitin accumulation, apoptosis induction, and viral titers were similar in LCMV-infected mice lacking LMP7 compared with wild-type mice. Taken together, these data indicate that the IP is not required to regulate protein homeostasis during LCMV infection.

Three-Phase Motor Inverter and Current Sensing GaN Power IC.

A three-phase GaN-based motor inverter IC with three integrated phase current mirror sensors (sense-FETs or sense-HEMTs, 1200:1 ratio), a temperature sensor, and an amplifier is presented and experimentally operated. The three low-side currents are read out by virtual grounding transimpedance amplifiers. A modified summed DC current readout circuit using only one amplifier is also discussed. During continuous 24 V motor operation with space-vector pulse width modulation (SVPWM), the sensor signal is measured and a bidirectional measurement capability is verified. The measured risetime of the sensor signal is 51 ns, indicating around 7 MHz bandwidth (without intentional optimization for high bandwidth). The IC is operated up to 32 V on DC-biased semi-floating substrate to limit negative static back-gating of the high-side transistors to around -7% of the DC-link voltage. Analysis of the capacitive coupling from the three switch-nodes to the substrate is calculated for SVPWM based on capacitance measurement, resulting in four discrete semi-floating substrate voltage levels, which is experimentally verified. Integrated advanced power converter topologies with sensors improve the power density of power electronics applications, such as for low-voltage motor drive.

Evidence for an involvement of the ubiquitin-like modifier ISG15 in MHC class I antigen presentation.

The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-ß. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing.

Immunoproteasome Inhibition Selectively Kills Human CD14+ Monocytes and as a Result Dampens IL-23 Secretion.

MECL-1 (ß2i), LMP2 (ß1i), and LMP7 (ß5i) 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.

Recent insights how combined inhibition of immuno/proteasome subunits enables therapeutic efficacy.

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.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (ß1i), MECL-1 (ß2i), and LMP7 (ß5i). 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.

Immunoproteasome inhibition induces plasma cell apoptosis and preserves kidney allografts by activating the unfolded protein response and suppressing plasma cell survival factors.

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.

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the T cell repertoire.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin-like activities suggesting distinct functions in thymic T cell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP118-125 -specific T cells although the class I mediated presentation of GP118-125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7-deficient CD8+ T cells into RAG1-deficient mice we show that LMP7-deficient mice lacked GP118-125 -specific T cell precursors and that LMP7 was required in radioresistant cells - most likely thymic epithelial cells - to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP118-125 -specific T cell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic T cell repertoire.

On the role of the immunoproteasome in transplant rejection.

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.

Immunoproteasome inhibition prevents chronic antibody-mediated allograft rejection in renal transplantation.

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 (ß5i)-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.

The expression profile of the ubiquitin-like modifier FAT10 in immune cells suggests cell type-specific functions.

The TNF and IFN-γ-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) is most prominently expressed in immunological tissues but information regarding basal expression and inducibility of FAT10 in the different types of immune cells is still lacking. Hence, we investigated FAT10 mRNA expression in the major human and murine immune cell subsets, and FAT10 protein expression in human leukocytes. We isolated the different human leukocytes from peripheral blood and the murine immune cell subsets from spleen. The purified leukocytes were left untreated or stimulated with TNF and INF-γ or LPS to induce FAT10 followed by quantitative real-time PCR or western blot analysis. Basal expression of FAT10 mRNA and protein was generally low but strongly up-regulated by IFN-γ and TNF in all immune cell subsets. LPS treatment induced FAT10 expression marginally in human CD8+ T cells and murine granulocytes, but it increased Fat10 expression significantly in murine regulatory T cells. Yet, in human CD8+ T cells, natural killer cells, natural killer T cells, and dendritic cells, the FAT10 mRNA was expressed without induction. Similarly, murine macrophages, monocytes, and regulatory T cells expressed Fat10 in the absence of stimulation. In summary, our findings suggest particular functions of FAT10 in these cell types. Furthermore, we observed not only a cell type-specific but also a species-specific basal FAT10 expression profile. Our data will serve as a guideline for future investigations to further elucidate FAT10's role in the immune system.

Inhibition and deficiency of the immunoproteasome subunit LMP7 attenuates LCMV-induced meningitis.

In addition to antigen processing, immunoproteasomes were recently shown to exert functions influencing cytokine production by monocytes and T cells, T-helper cell differentiation, and T-cell survival. Moreover, selective inhibition of the immunoproteasome subunit LMP7 ameliorated symptoms of autoimmune diseases including CD4(+) T-cell mediated EAE. In this study, we show that LMP7 also plays a crucial role in the pathogenesis of lymphocytic choriomeningitis virus (LCMV)-induced meningitis mediated by CTLs. Mice lacking functional LMP7 display delayed and reduced clinical signs of disease accompanied by a strongly decreased inflammatory infiltration into the brain. Interestingly, we found that selective inhibition and genetic deficiency of LMP7 affect the pathogenesis of LCMV-induced meningitis in a distinct manner. Our findings support the important role of LMP7 in inflammatory disorders and suggest immunoproteasome inhibition as a novel strategy against inflammation-induced neuropathology in the CNS.

The Ubiquitin-like Modifier FAT10 Is Selectively Expressed in Medullary Thymic Epithelial Cells and Modifies T Cell Selection.

HLA-F adjacent transcript 10 (FAT10) is a cytokine-inducible ubiquitin-like modifier that is highly expressed in the thymus and directly targets FAT10-conjugated proteins for degradation by the proteasome. High expression of FAT10 in the mouse thymus could be assigned to strongly autoimmune regulator-expressing, mature medullary thymic epithelial cells, which play a pivotal role in negative selection of T cells. Also in the human thymus, FAT10 is localized in the medulla but not the cortex. TCR Vß-segment screening revealed a changed T cell repertoire in FAT10-deficient mice. Analysis of five MHC class I- and II-restricted TCR-transgenic mice demonstrated an altered thymic negative selection in FAT10-deficient mice. Furthermore, the repertoire of peptides eluted from MHC class I molecules was influenced by FAT10 expression. Hence, we identified FAT10 as a novel modifier of thymic Ag presentation and epitope-dependent elimination of self-reactive T cells, which may explain why the fat10 gene could recently be linked to enhanced susceptibility to virus-triggered autoimmune diabetes.