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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.

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-β. 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. published

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.

The covalent attachment of the cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) to hundreds of substrate proteins leads to their rapid degradation by the 26 S proteasome independently of ubiquitylation. Here, we identify another function of FAT10, showing that it interferes with the activation of SUMO1/2/3 in vitro and down-regulates SUMO conjugation and the SUMO-dependent formation of promyelocytic leukemia protein (PML) bodies in cells. Mechanistically, we show that FAT10 directly binds to and impedes the activity of the heterodimeric SUMO E1 activating enzyme AOS1/UBA2 by competing very efficiently with SUMO for activation and thioester formation. Nevertheless, activation of FAT10 by AOS1/UBA2 does not lead to covalent conjugation of FAT10 with substrate proteins which relies on its cognate E1 enzyme UBA6. Hence, we report that one ubiquitin-like modifier (FAT10) inhibits the conjugation and function of another ubiquitin-like modifier (SUMO) by impairing its activation., FAT10 is an ubiquitin-like modifier that targets proteins to proteasomal degradation. Here, the authors show that FAT10 also regulates SUMO activation in vitro and in cells, providing evidence for functional crosstalk between two ubiquitin-like modifiers.

The endoplasmic reticulum (ER) is an organelle with high protein density and therefore prone to be damaged by protein aggregates. One proposed preventive measure is a pre-emptive quality control pathway that attenuates ER import during protein folding stress. ER resident proteins are targeted into the ER via signal peptides cleaved rapidly upon ER insertion by the ER signal peptidase. Here we show that the ER insertion and cleavage of the ER-targeting peptide of the prostate carcinoma antigen prostate stem cell antigen (PSCA) is retarded and strongly reduced when the proteasome is inhibited or genetically silenced. Also overexpression of the C-terminally extended ubiquitin variant Ub2-UBB+1 or oxidative stress attenuated signal peptide processing. Proteasome inhibition likewise protracted ER signal processing of the ER targeted hormone leptin and the MHC class I molecule H-2Dd. These findings, which are consistent with a pre-emptive ER quality control pathway, may explain why an immunodominant MHC class I peptide ligand of PSCA spanning its ER signal peptidase cleavage site is efficiently generated in the cytoplasm from PSCA precursors that fail to reach the ER.

Human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10) also called ubiquitin D (UBD) is a member of the ubiquitin-like modifier (ULM) family. The FAT10 gene is localized in the MHC class I locus and FAT10 protein expression is mainly restricted to cells and organs of the immune system. In all other cell types and tissues, FAT10 expression is highly inducible by the pro-inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF). Besides ubiquitin, FAT10 is the only ULM which directly targets its substrates for degradation by the 26S proteasome. This poses the question as to why two ULMs sharing the proteasome-targeting function have evolved and how they differ from each other. This Review summarizes the current knowledge of the special structure of FAT10 and highlights its differences from ubiquitin. We discuss how these differences might result in differential outcomes concerning proteasomal degradation mechanisms and non-covalent target interactions. Moreover, recent insights about the structural and functional impact of FAT10 interacting with specific non-covalent interaction partners are reviewed.

The revelation that the human major histocompatibility complex (MHC) class I locus encodes a ubiquitin-like protein designated HLA-F adjacent transcript 10 (FAT10) or ubiquitin D (UBD) has attracted increasing attention to the function of this protein. Interestingly, the pro-inflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF) α synergize to strongly induce FAT10 expression, thereby suggesting a role of FAT10 in the immune response. Recent reports that FAT10 downregulates type I interferon production while it upregulates IFN-γ pose mechanistic questions on how FAT10 differentially regulates interferon induction. Several covalent and non-covalent binding partners of FAT10 involved in signal transduction pathways leading to IFN synthesis have been identified. After introducing FAT10, we review here recent insights into how FAT10 affects proteins in the interferon pathways, like the virus-responsive pattern recognition receptor RIG-I, the ubiquitin ligase ZNF598, and the deubiquitylating enzyme OTUB1. Moreover, we outline the consequences of FAT10 deficiency on interferon synthesis and viral expansion in mice and human cells. We discuss the need for covalent isopeptide linkage of FAT10 to the involved target proteins and the concomitant targeting for proteasomal degradation. After years of investigating the elusive biological functions of this fascinating ubiquitin-like modifier, we review the emerging evidence for a novel role of FAT10 in interferon regulation.

Parkin is an E3 ubiquitin ligase belonging to the RING-between-RING family. Mutations in the Parkin-encoding gene PARK2 are associated with familial Parkinson's disease. Here, we investigate the interplay between Parkin and the inflammatory cytokine-induced ubiquitin-like modifier FAT10. FAT10 targets hundreds of proteins for degradation by the 26S proteasome. We show that FAT10 gets conjugated to Parkin and mediates its degradation in a proteasome-dependent manner. Parkin binds to the E2 enzyme of FAT10 (USE1), auto-FAT10ylates itself, and facilitates FAT10ylation of the Parkin substrate Mitofusin2 in vitro and in cells, thus identifying Parkin as a FAT10 E3 ligase. On mitochondrial depolarization, FAT10ylation of Parkin inhibits its activation and ubiquitin-ligase activity causing impairment of mitophagy progression and aggravation of rotenone-mediated death of dopaminergic neuronal cells. In conclusion, FAT10ylation inhibits Parkin and mitophagy rendering FAT10 a likely inflammation-induced exacerbating factor and potential drug target for Parkinson's disease. published

Dendritic cell (DC) aggresome-like induced structures (DALIS) are protein aggregates of polyubiquitylated proteins that form transiently during DC maturation. DALIS scatter randomly throughout the cytosol and serve as antigen storage sites synchronising DC maturation and antigen presentation. Maturation of DCs is accompanied by the induction of the ubiquitin-like modifier FAT10 (also known as UBD), which localises to aggresomes, structures that are similar to DALIS. FAT10 is conjugated to substrate proteins and serves as a signal for their rapid and irreversible degradation by the 26S proteasome similar to, yet independently of ubiquitin, thereby contributing to antigen presentation. Here, we have investigated whether FAT10 is involved in the formation and turnover of DALIS, and whether proteins accumulating in DALIS can be modified through conjunction to FAT10 (FAT10ylated). We found that FAT10 localises to DALIS in maturing DCs and that this localisation occurs independently of its conjugation to substrates. Additionally, we investigated the DALIS turnover in FAT10-deficient and -proficient DCs, and observed FAT10-mediated disassembly of DALIS. Thus, we report further evidence that FAT10 is involved in antigen processing, which may provide a functional rationale as to why FAT10 is selectively induced upon DC maturation.

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. published

Numerous cellular processes are controlled by the proteasome, a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells, through regulated protein degradation. The immunoproteasome is a special type of proteasome which is inducible under inflammatory conditions and constitutively expressed in hematopoietic cells. MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome (IP), which is known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Furthermore, the immunoproteasome is involved in T cell expansion and inflammatory diseases. In recent years, targeting the immunoproteasome in cancer, autoimmune diseases, and transplantation proved to be therapeutically effective in preclinical animal models. However, the prime function of standard proteasomes and immunoproteasomes is the control of protein homeostasis in cells. To maintain protein homeostasis in cells, proteasomes remove proteins which are not properly folded, which are damaged by stress conditions such as reactive oxygen species formation, or which have to be degraded on the basis of regular protein turnover. In this review we summarize the latest insights on how the immunoproteasome influences protein homeostasis.

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. This article is protected by copyright. All rights reserved

The deubiquitylation of target proteins is mediated by deubiquitylating enzymes (DUB) such as OTUB1, which plays an important role in immune response, cell cycle progression, and DNA repair. Within these processes, OTUB1 reduces the ubiquitylation of target proteins in two distinct ways, either by using its catalytic DUB activity or in a noncatalytic manner by inhibiting the E2-conjugating enzyme. Here, we show that the ubiquitin-like modifier FAT10 regulates OTUB1 stability and functionality in different ways. Covalent FAT10ylation of OTUB1 resulted in its proteasomal degradation, whereas a noncovalent interaction stabilized OTUB1. We provide evidence that OTUB1 interacts directly with FAT10 and the E2-conjugating enzyme USE1. This interaction strongly stimulated OTUB1 DUB activity toward Lys-48–linked diubiquitin. Furthermore, the noncovalent interaction between FAT10 and OTUB1 not only enhanced its isopeptidase activity toward Lys-48–linked ubiquitin moieties but also strengthened its noncatalytic activity in reducing Lys-63 polyubiquitylation of its target protein TRAF3 (TNF receptor–associated factor 3). Additionally, the cellular clearance of overall polyubiquitylation by OTUB1 was strongly stimulated through the presence of FAT10. The addition of FAT10 also led to an increased interaction between OTUB1 and its cognate E2 UbcH5B, implying a function of FAT10 in the inhibition of polyubiquitylation. Overall, these data indicate that FAT10 not only plays a role in covalent modification, leading its substrates to proteasomal degradation, but also regulates the stability and functionality of target proteins by interacting in a noncovalent manner. FAT10 is thereby able to exert a major influence on ubiquitylation processes.

The major histocompatibility complex (MHC) class I restricted pathway of antigen processing allows the presentation of intracellular antigens to cytotoxic T lymphocytes. The proteasome is the main protease in the cytoplasm and the nucleus, which is responsible for the generation of most peptide ligands of MHC-I molecules. Peptides produced by the proteasome can be further trimmed or destroyed by numerous cytosolic or endoplasmic reticulum (ER) luminal proteases. Small molecule inhibitors are useful tools for probing the role of proteases in MHC class I antigen processing. Here, we describe different methods to test the impact of protease inhibitors in antigen presentation assays.

Immunoproteasome (IP) inhibition holds potential as a novel treatment option for various immune-mediated pathologies. The IP inhibitor ONX 0914 reduced T cell cytokine secretion and Th17 polarization and showed pre-clinical efficacy in a range of autoimmune disorders, transplant-allograft rejection, virus-mediated tissue damage, and colon cancer progression. However, the molecular basis of these effects has remained largely elusive. Here, we have analyzed the effects of ONX 0914 in primary human and mouse lymphocytes. ONX 0914-treatment impaired primary T cell activation in vitro and in vivo. IP inhibition reduced ERK-phosphorylation sustainment, while leaving NF-κB and other signaling pathways unaffected. Naïve T and B cells expressed nearly exclusively immuno- or mixed proteasomes but no standard proteasomes and IP inhibition but not IP-deficiency induced mild proteostasis stress, reduced DUSP5 expression and enhanced DUSP6 protein levels due to impaired degradation. However, accumulation of DUSP6 did not cause the reduced ERK-phosphorylation in a non-redundant manner. We show that broad-spectrum proteasome inhibition and immunoproteasome inhibition have distinct effects on T cell activation at the molecular level. Notably, ONX 0914-treated T cells recovered from proteostasis stress without apoptosis induction, apparently via Nrf1-mediated up-regulation of standard proteasomes. In contrast, B cells were more susceptible to apoptosis after ONX 0914-treatment. Our data thus provide mechanistic insights how IP inhibition functionally impedes T and B cells likely accounting for its therapeutic benefits. published

Antigen processing for direct presentation on MHC class I molecules is a multistep process requiring the concerted activity of several cellular complexes. The essential steps at the beginning of this pathway, namely protein synthesis at the ribosome and degradation via the proteasome, have been known for years. Nevertheless, there is a considerable lack of factors identified to function between protein synthesis and degradation during antigen processing. Here, we analyzed the impact of the chaperone BAG6 on MHC class I cell surface expression and presentation of virus-derived peptides. Although an essential role of BAG6 in antigen processing has been proposed previously, we found BAG6 to be dispensable in this pathway. Still, interaction of BAG6 and the model antigen tyrosinase was enhanced during proteasome inhibition pointing towards a role of BAG6 in antigen degradation. Redundant chaperone pathways potentially mask the contribution of BAG6 to antigen processing and presentation. published

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 Vβ-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.

The development of drugs directly interfering with neurodegeneration has proven to be astonishingly difficult. Alternative therapeutic approaches could result from a better understanding of the supportive function of glial cells for stressed neurons. Therefore, here, we investigated the mechanisms involved in the endogenous neuro-defensive activity of astrocytes. A well-established model of postmitotic human dopaminergic neurons (LUHMES cells) was used in the absence ('LUHMES' mono-culture) or presence ('co-culture') of astrocytes. Inhibition of the LUHMES proteasome led to proteotoxic (protein aggregates; ATF-4 induction) and oxidative (GSH-depletion; NRF-2 induction) stress, followed by neuronal apoptosis. The presence of astrocytes attenuated the neuronal stress response, and drastically reduced neurodegeneration. A similar difference between LUHMES mono- and co-cultures was observed, when proteotoxic and oxidative stress was triggered indirectly by inhibitors of mitochondrial function (rotenone, MPP+). Human and murine astrocytes continuously released glutathione (GSH) into the medium, and transfer of glia-conditioned medium was sufficient to rescue LUHMES, unless it was depleted for GSH. Also, direct addition of GSH to LUHMES rescued the neurons from inhibition of the proteasome. Both astrocytes and GSH blunted the neuronal ATF-4 response and similarly upregulated NRF-1/NFE2L1, a transcription factor counter-regulating neuronal proteotoxic stress. Astrocyte co-culture also helped to recover the neurons' ability to degrade aggregated poly-ubiquitinated proteins. Overexpression of NRF-1 attenuated the toxicity of proteasome inhibition, while knockdown increased toxicity. Thus, astrocytic thiol supply increased neuronal resilience to various proteotoxic stressors by simultaneously attenuating cell death-related stress responses, and enhancing the recovery from proteotoxic stress through upregulation of NRF-1. published

The 20S immunoproteasome (IP) is an interferon(IFN)-γ − and tumor necrosis factor (TNF) −inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits β1, β2, and β5 are replaced by their cytokine inducible homologues β1i (LMP2), β2i (MECL-1), and β5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-γ/TNF inducible factors: the 11S proteasome regulator PA28αβ and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28αβ for the CP and IP to be 12.2 nM +/− 2.8 nM and 15.3 nM +/− 2.7 nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28αβ did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-γ stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28αβ, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP.

The 26S proteasome is the main protease in eukaryotes. Proteolysis occurs within the cylindrical 20S proteasome that is constitutively expressed in most tissues. However, three tissue-specific versions of the 20S proteasome have been discovered to date. The immunoproteasome is optimized to process antigens and it directs the differentiation of T helper (Th) cells. The thymoproteasome is selectively expressed in cortical epithelial cells of the thymus where it plays an essential role in the positive selection of T lymphocytes. Finally, the spermatoproteasome is found in the testes where it is required during spermatogenesis. Here, we outline how tissue-specific proteasomes adapt to functional needs in their respective tissues and how their selective inhibition may be used to interfere with autoimmune diseases and cancer.

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.

The Nobel prize has been awarded for the discovery of ubiquitin as a transferable signal for the degradation of proteins by the 26S proteasome. While isopeptide linkage of a protein with a single ubiquitin does not serve as a degradation signal for the proteasome, poly-ubiquitylation via several different lysine residues within ubiquitin leads to efficient proteasomal degradation. Ubiquitin-like modifiers have not been shown to directly mediate proteasomal degradation except for the cytokine inducible modifier HLA-F adjacent transcript 10 (FAT10), which consists of two ubiquitin-like domains. FAT10 ends with a free diglycine motif at its C-terminus which is required for isopeptide linkage to hundreds of different substrates. In contrast to ubiquitin, a single FAT10 suffices to bind to the 26S proteasome and to efficiently mediate proteasomal degradation in a ubiquitin-independent manner. Here we review the data on ubiquitin-independent degradation by FAT10, on how FAT10 is conjugated to its substrates, how FAT10 binds to the 26S proteasome, and how the ubiquitin-like (UBL)–ubiquitin-associated (UBA) protein NUB1L accelerates FAT10 mediated proteolysis. Finally, with a glimpse on recently identified substrates, we will discuss the currently emerging knowledge about the biological functions of FAT10. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

During the last years it has emerged that the ubiquitin-like modifier FAT10 is directly involved in cancer development. FAT10 expression is highly up-regulated by proinflammatory cytokines IFN-γ and TNF-α in all cell types and tissues and it was also found to be upregulated in many cancer types such as glioma, colorectal, liver or gastric cancer. While proinflammatory cytokines within the tumor microenvironment probably contribute to FAT10 overexpression, an increasing body of evidence argues that pro-malignant capacities of FAT10 itself largely underlie its broad and intense overexpression in tumor tissues. FAT10 thereby regulates pathways involved in cancer development such as the NF-κB- or Wnt-signaling. Moreover, FAT10 directly interacts with and influences downstream targets such as p53 or β-catenin, leading to enhanced survival, proliferation, invasion and metastasis formation of cancer cells but also of non-malignant cells. In this review we will provide an overview of the regulation of FAT10 expression as well as its function in carcinogenesis. published

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 Beta5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of Beta5c/X but not Beta5i, thereby explaining the selectivity of PR-957 for Beta5i. 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.

The chemokine receptor CCR7 is essential for lymphocyte and dendritic cell homing to secondary lymphoid organs. Owing to the ability to induce directional migration, CCR7 and its ligands CCL19 and CCL21 are pivotal for the regulation of the immune system. Here, we identify a novel function for receptor ubiquitylation in the regulation of the trafficking process of this G-protein-coupled seven transmembrane receptor. We discovered that CCR7 is ubiquitylated in a constitutive, ligand-independent manner and that receptor ubiquitylation regulates the basal trafficking of CCR7 in the absence of chemokine. Upon CCL19 binding, we show that internalized CCR7 recycles back to the plasma membrane via the trans-Golgi network. An ubiquitylation-deficient CCR7 mutant internalized normally after ligand binding, but inefficiently recycled in immune cells and was transiently retarded in the trans-Golgi network compartment of HEK293 transfectants. Finally, we demonstrate that the lack of CCR7 ubiquitylation profoundly impairs immune cell migration. Our results provide evidence for a novel function of receptor ubiquitylation in the regulation of CCR7 recycling and immunecell migration.

The analysis of cell types involved in cross-priming of particulate Ag is essential to understand and improve immunotherapies using microparticles. In this study, we show that murine splenic dendritic cells (DCs) as well as macrophages (MΦs) are able to efficiently endocytose poly(D,L-lactate-co-glycolate) acid (PLGA) microspheres (MS) and to cross-present encapsulated Ags in the context of MHC class I molecules in vitro. A comparison of purified CD8+ and CD8− DCs indicated that both DC subtypes are able to present OVA-derived epitopes on MHC class I and II in vitro. To determine the contribution of DCs and MΦs to cross-priming of PLGA MS in vivo, DCs were depleted in transgenic CD11c-DTR mice, and MΦs were depleted by clodronate liposomes in wild-type mice before immunizing mice with OVA-encapsulated MS. Our results show that the depletion of DCs or MΦs alone only led to minor differences in the OVA-specific immune responses. However, simultaneous depletion of DCs and MΦs caused a strong reduction of primed effector cells, indicating a redundancy of both cell populations for the priming of PLGA MS-encapsulated Ag. Finally, we analyzed PLGA MS trafficking to draining lymph nodes after s.c. injection. It was evident that fluorescent particles accumulated within draining lymph nodes over time. Further analysis of PLGA MS-positive lymphatic cells revealed that mainly CD8− DCs and MΦs contained MS. Moreover, immune responses in BATF3 knockout mice lacking CD8+ DCs were normal. The results presented in this work strongly suggest that in vivo cross-priming of PLGA MS-encapsulated Ag is performed by CD8− DCs and MΦs.

When cells are stimulated with pro-inflammatory cytokines, most of their constitutively expressed proteasomes are replaced with immunoproteasomes, which increase the production of peptides for presentation on MHC class I molecules. In addition, cortical thymic epithelial cells selectively express a type of proteasome known as the thymoproteasome that is required for the positive selection of thymocytes. Here, we discuss how these specialized types of proteasome shape the T cell receptor repertoire of cytotoxic T lymphocytes and propose that immunoproteasomes have functions, in addition to antigen processing, that influence cytokine production and T cell differentiation, survival and function. We also discuss how inhibitors of immunoproteasomes can suppress undesired T cell responses in autoimmune diseases.

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3β. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.

Bacterial invasion of eukaryotic cells is counteracted by cell-autonomous innate immune mechanisms including xenophagy. The decoration of cytosolic bacteria by ubiquitylation and binding of galectin-8 leads to recruitment of autophagy adaptors like p62 (also known as SQSTM1), NDP52 (also known as CALCOCO2) and optineurin, which initiate the destruction of bacteria by xenophagy. Here, we show that the functionally barely characterized IFNγ- and TNFα-inducible ubiquitin-like modifier FAT10 (also known as ubiquitin D, UBD), which binds to the autophagy adaptor p62, but has not been shown to associate with pathogens before, is recruited to cytosolic Salmonella Typhimurium in human cells. FAT10-decorated S. Typhimurium were simultaneously decorated with ubiquitin, p62, NDP52 and the autophagy marker LC3B (MAP1LC3B). FAT10 colocalized with p62-positive microdomains on S. Typhimurium, whereas colocalization with NDP52 was only partial. A kinetic analysis revealed an early, but only transient, decoration of bacteria by FAT10, which resembled that of p62. Although bacterial replication was not detectably altered in FAT10-depleted or overexpressing cells in vitro , survival experiments revealed that NRAMP1-transgenic mice that were FAT10-deficient had a higher susceptibility to orally inoculated S. Typhimurium bacteria than NRAMP1-transgenic mice that were wild-type for FAT10. Taken together, our data suggest a role for FAT10 in the intracellular defense against bacteria.

When cells are stimulated with the cytokines IFN-gamma or TNF-alpha, the synthesis of three proteasome subunits LMP2 (beta1i), LMP7 (beta5i), and MECL-1 (beta2i) is induced. These subunits replace the three subunits delta (beta1), MB1 (beta5), and Z (beta2), which bear the catalytically active sites of the proteasome, during proteasome neosynthesis. The cytokine-induced exchanges of three active site subunits of a complex protease is unprecedented in biology and one may expect a strong functional driving force for this system to evolve. These cytokine-induced replacements of proteasome subunits are believed to favour the production of peptide ligands of major histocompatibility complex (MHC) class I molecules for the stimulation of cytotoxic T cells. Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be pivotal for the generation or destruction of several unique epitopes. In this review we discuss the recent data on LMP2/LMP7/MECL-1-dependent epitope generation and the functions of each of these subunit exchanges. We propose that these subunit exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not proteolytically generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues.

The cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) targets its substrates for degradation by the proteasome. FAT10 is conjugated to its substrates via the bispecific, ubiquitin-activating and FAT10-activating enzyme UBA6, the likewise bispecific conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By MS analysis, we found that USE1 undergoes self-FAT10ylation in cis, mainly at Lys323. Mutation of Lys323 to an arginine did not abolish auto-FAT10ylation of USE1, but every other lysine could instead be modified with FAT10. Similarly to bulk FAT10 substrates, FAT10ylation of USE1 accelerated its proteasomal degradation. Interestingly, the USE1–FAT10 conjugate continued to be an active E2 enzyme, because both FAT10 and ubiquitin could still be thioester-linked to the USE1–FAT10 conjugate. We therefore suggest that the major function of USE1 auto-FAT10ylation is to serve as a negative feedback mechanism to limit the conjugation of FAT10 upon its cytokine-mediated induction by reducing the amount of USE1 through proteasomal degradation of the USE1–FAT10 conjugate. Structured digital abstract •USE1 physically interacts with FAT10 by anti bait coimmunoprecipitation (View interaction) •USE1 physically interacts with FAT10 by anti tag coimmunoprecipitation (View interaction)

The 20 S proteasome is an endoprotease complex that preferentially cleaves peptides C-terminal of hydrophobic, basic, and acidic residues. Recently, we showed that these specific activities, classified as chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing (PGPH) activity, are differently affected by Ritonavir, an inhibitor of human immunodeficiency virus-1 protease. Ritonavir competitively inhibited the chymotrypsin-like activity, whereas the trypsin-like activity was enhanced. Here we demonstrate that the Ritonavir-mediated up-regulation of the trypsin-like activity is not affected by specific active site inhibitors of the chymo-trypsin-like and PGPH activity. Moreover, we show that the mutual regulation of chymotrypsin-like and PGPH activities by their substrates as described previously by a "cyclical bite-chew" model is not affected by selective inhibitors of the respective active sites. These data challenge the bite-chew model and suggest that effectors of proteasome activity can act by binding to non-catalytic sites. Accordingly, we propose a kinetic "two-site modifier" model that assumes that the substrate (or effector) may bind to an active site as well as to a second non-catalytic modifier site. This model appears to be valid as it describes the complex kinetic effects of Ritonavir very well. Since Ritonavir partially inhibits major histocompatibility complex class I restricted antigen presentation, the postulated modifier site may be required to coordinate the active centers of the proteasome for the production of class I peptide ligands.

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5–1 μM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89–168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5–1 μM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

Dendritic cells (DC) are highly specialized professional antigen presenting cells which are pivotal for the initiation and control of the cytotoxic T cell response. Upon stimulation by cytokines, bacteria, or CD40L DC undergo a maturation process from an antigen-receptive state to a state of optimal stimulation of T cells. We investigated the composition of proteasomes of DC derived from human peripheral blood monocytes before and after stimulation by CD40L, LPS, or proinflammatory cytokines (TNF-alpha + IL-6 + IL-1beta). Immunoprecipitation of proteasomes and analysis on two-dimensional gels revealed that during maturation the inducible proteasome subunits LMP2, LMP7, and MECL-1 are up-regulated and that the neosynthesis of proteasomes is switched exclusively to the production of immunoproteasomes containing these subunits. The proteasome regulator PA28 is markedly up-regulated in mature DC and in addition a so - far unidentified 21-kDa protein co-precipitates with the proteasome in LPS - stimulated DC. These changes in proteasome composition may be functionally linked to special properties of DC like MHC class I up-regulation or cross-priming. Our findings imply that the spectrum of class I-bound peptides may change after DC maturation which could be relevant for the design of DC - based vaccines.

The proteasome is the main nonlysosomal endoprotease in the cytoplasm and nucleus of all eukaryotic cells. It is responsible for the generation of most antigenic peptides as ligands for major histocompatibility complex (MHC) class I proteins. The proteasome hence qualifies as a target for modifying or silencing antigen processing and presentation to cytotoxic T cells, which are important players in transplant rejection and autoimmune disease. The authors summarize recent progress in the understanding of antigen processing by the proteasome and discuss the potential of novel and selective proteasome inhibitors as drugs for suppressing or modifying the cytotoxic immune response.

Proteasomes generate peptides bound by major histocompatibility complex (MHC) class I molecules. Avoiding proteasome inhibitors, which in most cases do not distinguish between individual active sites within the cell, we used a molecular genetic approach that allowed for the first time the in vivo analysis of defined proteasomal active sites with regard to their significance for antigen processing. Functional elimination of the δ/low molecular weight protein (LMP) 2 sites by substitution with a mutated inactive LMP2 T1A subunit results in reduced cell surface expression of the MHC class I H-2Ld and H-2Dd molecules. Surface levels of H-2Ld and H-2Dd molecules were restored by external loading with peptides. However, as a result of the active site mutation, MHC class I presentation of a 9-mer peptide derived from a protein of murine cytomegalovirus was enhanced about three- to fivefold. Our experiments provide evidence that the δ/LMP2 active site elimination limits the processing and presentation of several peptides, but may be, nonetheless, beneficial for the generation and presentation of others.

We have cloned the mouse PA28 proteasome activator cDNAs. Northern blot demonstrates high PA28 mRNA levels in liver, kidney and lung. mRNA levels are low in thymus, spleen and brain. In contrast, PA28 protein levels vary little between these tissues. Immunocytological analysis and cell fractionation experiments demonstrate that both subunits are almost equally distributed between the cytoplasm and the nucleus. Interestingly, PA28alpha spares nucleoli, while PA28beta is strongly enhanced in the nucleolus. This indicates for the first time that the PA28alpha and PA28beta subunits may serve nuclear functions which may be different from and independent of each other.

The eukaryotic 20S proteasome is known to associate with the IFNγ-inducible regulator PA28. We analyzed the kinetics of product generation by 20S proteasomes with and without PA28. In the absence of PA28, the 20S proteasome rapidly generates peptides that have been cleaved only once, while internal fragments accumulate only slowly. In the presence of PA28, products generated by two flanking cleavages appear immediately as main products while the generation of single-cleavage products is strongly reduced. Kinetic data support a PA28-induced, coordinated double-cleavage mechanism. In particular, degradation of peptides derived from mouse cytomegalovirus pp89 and JAK1 kinase in the presence of PA28 leads to strongly enhanced production of the respective major histocompatibility complex ligands and potential precursors. These results show that PA28 profoundly alters the cleavage mechanism of the proteasome and appears to optimize the generation of dominant T-cell epitopes.

Abnormal phosphorylation of the microtubule-associated protein tau in neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal lobar degeneration, is associated with disrupted axonal transport and synaptic dysfunction ultimately manifesting as histopathological lesions of protein aggregates. Glycogen synthase kinase 3β (GSK3β) may be critical for the pathological hyperphosphorylation of tau. Here, we examined the role of the proteasome-associated protein Nedd8 ultimate buster 1 (NUB1) in the neuropathogenic phosphorylation and aggregation of tau. We reveal that NUB1 interacted with both tau and GSK3β to disrupt their interaction, and abolished recruitment of GSK3β to tau inclusions. Moreover, NUB1 reduced GSK3β-mediated phosphorylation of tau and aggregation of tau in intracellular inclusions. Strikingly, NUB1 induced GSK3β degradation. Deletion of the NUB1 ubiquitin-like (UBL) domain did not impair the interaction with tau and GSK3β, and the ability to suppress the phosphorylation and aggregation of tau was not affected. However, the UBL motif was necessary for GSK3β degradation. Deletion of the NUB1 ubiquitin-associated (UBA) domain abrogated the ability of NUB1 to interact with and degrade GSK3β. Moreover, the UBA domain was required to suppress the aggregation of tau. Silencing of NUB1 in cells stabilized endogenous GSK3β and exacerbated tau phosphorylation. Thus, we propose that NUB1, by regulating GSK3β levels, modulates tau phosphorylation and aggregation, and is a key player in neurodegeneration associated with tau pathology. Moreover, NUB1 regulation of GSK3β could modulate numerous signalling pathways in which GSK3β is a centrally important effector.

The major histocompatibility complex (MHC) class I restricted pathway of antigen processing allows the presentation of intracellular antigens to cytotoxic T lymphocytes. The proteasome is the main protease in the cytoplasm and the nucleus, which is responsible for the generation of most peptide ligands of MHC-I molecules. Peptides produced by the proteasome can be further trimmed or destroyed by numerous cytosolic or endoplasmic reticulum (ER) lumenal proteases. Small molecule inhibitors are useful tools for probing the role of proteases in MHC class I antigen processing. Here, we describe different methods to test the impact of protease inhibitors in antigen presentation assays.

FAT10 is a ubiquitin-like modifier proposed to function in apoptosis induction, cell cycle control and NF-kB activation. Upon induction by pro-inflammatory cytokines, hundreds of endogenous substrates become covalently conjugated to FAT10 leading to their proteasomal degradation. Nevertheless, only three substrates have been identified so far to which FAT10 becomes covalently attached through a non-reducible isopeptide bond, and these are the FAT10-conjugating enzyme USE1 which auto-FAT10ylates itself in cis, the tumor suppressor p53 and the ubiquitin-activating enzyme UBE1 (UBA1). To identify additional FAT10 substrates and interaction partners, we used a new monoclonal FAT10-specific antibody to immunopurify endogenous FAT10 conjugates from interferon (IFN)cand tumor necrosis factor (TNF)alpha-stimulated cells for identification by mass spectrometry. In addition to two already known FAT10- interacting proteins, histone deacetylase 6 and UBA6, we identified 569 novel FAT10-interacting proteins involved in different functional pathways such as autophagy, cell cycle regulation, apoptosis and cancer. Thirty-one percent of all identified proteins werecategorized as putative covalently linked substrates. One of the identified proteins, the autophagosomal receptor p62/SQSTM1, was further investigated. p62 becomes covalently mono-FAT10ylated at several lysines, and FAT10 colocalizes with p62 in p62 bodies.Strikingly, FAT10ylation of p62 leads to its proteasomal degradation, and prolonged induction of endogenous FAT10 expression by proinflammatory cytokines leads to a decrease of endogenous p62. The elucidation of the FAT10 degradome should enable a better understanding of why FAT10 has evolved as an additional transferable tag for proteasomal degradation.

FAT10 is the only ubiquitin-like modifier that can target proteins for degradation by the proteasome in a ubiquitin-independent manner. The degradation of FAT10-linked proteins by the proteasome is strongly accelerated by the ubiquitin-like-ubiquitin-associated protein NEDD8 ultimate buster-1 long (NUB1L). Here we show how FAT10 and NUB1L dock with the 26S proteasome to initiate proteolysis. We identify the 26S proteasome subunit hRpn10/S5a as the receptor for FAT10, whereas NUB1L can bind to both Rpn10 and Rpn1/S2. Unexpectedly, FAT10 and NUB1L both interact with hRpn10 via the VWA domain. FAT10 degradation in yeast shows that human Rpn10 can functionally reconstitute Rpn10-deficient yeast and that the VWA domain of hRpn10 suffices to enable FAT10 degradation. Depletion of hRpn10 causes an accumulation of FAT10-conjugates also in human cells. In conclusion, we identify the VWA domain of hRpn10 as a receptor for ubiquitin-like proteins within the 26S proteasome and elucidate how FAT10 mediates efficient proteolysis by the proteasome.

Mutations in AIPL1 cause the inherited blindness Leber congenital amaurosis (LCA). AIPL1 has previously been shown to interact with NUB1, which facilitates the proteasomal degradation of proteins modified with the ubiquitin-like protein FAT10. Here we report that AIPL1 binds non-covalently to free FAT10 and FAT10ylated proteins and can form a ternary complex with FAT10 and NUB1. In addition, AIPL1 antagonised the NUB1-mediated degradation of the model FAT10 conjugate, FAT10-DHFR, and pathogenic mutations of AIPL1 were defective in inhibiting this degradation. While all AIPL1 mutants tested still bound FAT10-DHFR, there was a close correlation between the ability of the mutants to interact with NUB1 and their ability to prevent NUB1-mediated degradation. Interestingly, AIPL1 also co-immunoprecipitated the E1 activating enzyme for FAT10, UBA6, suggesting AIPL1 may have a role in directly regulating the FAT10 conjugation machinery. These studies are the first to implicate FAT10 in retinal cell biology and LCA pathogenesis, and reveal a new role of AIPL1 in regulating the FAT10 pathway.

Expression of a productive T-cell receptor (TCR) beta gene has profound consequences for T-cell development, preceding an increase in thymocyte number, appearance of CD4 and CD8 coreceptors and suppression of further TCR beta-gene rearrangement. Here Marcus Groettrup and Harald von Boehmer discuss the data obtained in various experimental models and describe a novel signal transducing receptor complex on pre-T cells, which may regulate the TCR beta-induced developmental changes.

Tissue inflammation is accompanied by the cytokine-mediated replacement of constitutive proteasomes by immunoproteasomes that finally leads to an optimized generation of MHC class I restricted epitopes for Ag presentation. The brain is considered an immunoprivileged organ, where both the special anatomy as well as active tolerance mechanisms repress the development of inflammatory responses and help to prevent immunopathological damage. We analyzed the immunoproteasome expression in the brain after an infection with lymphocytic choriomeningitis virus (LCMV) and could show that LCMV-infection of mice leads to the transcriptional induction of inducible proteasome subunits in the brain. However, compared with other organs, i.p. and even intracranial infection with LCMV only led to a faint expression of mature immunoproteasome in the brain and resulted in the accumulation of immunoproteasomal precursors. By immunohistology, we could identify microglia-like cells as the main producers of immunoproteasome, whereas in astrocytes immunoproteasome expression was almost exclusively restricted to nuclei. Neither the immunoproteasome subunits low molecular mass polypeptide 2 nor multicatalytic endopeptidase complex-like–1 were detected in neurons or oligodendrocytes. In vitro studies of IFN-γ–stimulated primary astrocytes suggested that the observed accumulation of immunoproteasomal precursor complexes takes place in this cell population. Functionally, the lack of immunoproteasomes protracted and lowered the severity of LCMV-induced meningitis in LMP7−/− mice suggesting a contribution of immunoproteasomes in microglia to exacerbate immunopathological damage. We postulate a posttranslationally regulated mechanism that prevents abundant and inappropriate immunoproteasome assembly in the brain and may contribute to the protection of poorly regenerating cells of the CNS from immunopathological destruction.

Immunoproteasomes containing the IFN-inducible subunits β1i (LMP2), β2i (MECL-1) and β5i (LMP7) alter proteasomal cleavage preference and optimize the generation of peptide ligands of MHC class I molecules. Here, we report on an unexpected new function of immunoproteasome subunits for the survival and expansion of CD4(+) and CD8(+) T cells during viral infection of mice. The effect of immunoproteasome subunit deficiency on T-cell survival upon adoptive transfer was most prominent for the lack of LMP7 followed by MECL-1 and LMP2. The survival of T cells in uninfected mice or the homeostatic expansion after transfer into RAG-2(-/-) mice was not affected by the lack of the immunosubunits. Lymphocytic choriomeningitis virus (LCMV)-specific CD8(+) T cells lacking LMP7 or MECL-1 started to divide after transfer into LCMV-infected mice but experienced a considerable cell loss within 2 days after transfer. We provide strong evidence that the loss of immunoproteasome-deficient T cells after transfer is not a consequence of graft rejection by the host, but instead is based on the requirement for immunoproteasomes for the survival of T cells in LCMV-infected mice. Therefore, the immunoproteasome may qualify as a potential new target for the suppression of undesired proinflammatory T-cell responses.