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The ubiquitin conjugating enzyme UBE2W catalyzes non-canonical ubiquitination on the N-termini of proteins, although its substrate repertoire remains unclear. To identify endogenous N-terminally-ubiquitinated substrates, we discover four monoclonal antibodies that selectively recognize tryptic peptides with an N-terminal diglycine remnant, corresponding to sites of N-terminal ubiquitination. Importantly, these antibodies do not recognize isopeptide-linked diglycine (ubiquitin) modifications on lysine. We solve the structure of one such antibody bound to a Gly-Gly-Met peptide to reveal the molecular basis for its selective recognition. We use these antibodies in conjunction with mass spectrometry proteomics to map N-terminal ubiquitination sites on endogenous substrates of UBE2W. These substrates include UCHL1 and UCHL5, where N-terminal ubiquitination distinctly alters deubiquitinase (DUB) activity. This work describes an antibody toolkit for enrichment and global profiling of endogenous N-terminal ubiquitination sites, while revealing functionally relevant substrates of UBE2W., (© 2021. The Author(s).)

Staphylococcus aureus is a commensal bacterium that causes severe infections in soft tissue and the bloodstream. During infection, S. aureus manipulates host cell response to facilitate its own replication and dissemination. Here, we show that S. aureus significantly decreases the level of SUMOylation, an essential post-translational modification, in infected macrophages 24 h post-phagocytosis. The reduced level of SUMOylation correlates with a decrease in the SUMO-conjugating enzyme Ubc9. The over-expression of SUMO proteins in macrophages impaired bacterial intracellular proliferation and the inhibition of SUMOylation with ML-792 increased it. Together, these findings demonstrated for the first time the role of host SUMOylation response toward S. aureus infection.

Common fragile sites (CFSs) are specific breakage-prone genomic regions and are present frequently in cancer cells. The (E2-independent) E3 ubiquitin-conjugating enzyme FATS (fragile site-associated tumor suppressor) has antitumor activity in cancer cells, but the function of FATS in immune cells is unknown. Here, we report a function of FATS in tumor development via regulation of tumor immunity. Fats -/- mice show reduced subcutaneous B16 melanoma and H7 pancreatic tumor growth compared with WT controls. The reduced tumor growth in Fats -/- mice is macrophage dependent and is associated with a phenotypic shift of macrophages within the tumor from tumor-promoting M2-like to antitumor M1-like macrophages. In addition, FATS deficiency promotes M1 polarization by stimulating and prolonging NF-κB activation by disrupting NF-κB/IκBα negative feedback loops and indirectly enhances both CD4 + T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) adaptive immune responses to promote tumor regression. Notably, transfer of Fats -/- macrophages protects mice against B16 melanoma. Together, these data suggest that FATS functions as an immune regulator and is a potential target in cancer immunotherapy., (© 2021. The Author(s).)

African swine fever virus (ASFV) is an acute and persistent swine virus with a high economic burden that encodes multiple genes to evade host immune response. In this work, we have revealed that early viral protein UBCv1, the only known conjugating enzyme encoded by a virus, modulates innate immune and inflammatory signaling. Transient overexpression of UBCv1 impaired activation of NF-κB and AP-1 transcription factors induced by several agonists of these pathways. In contrast, activation of IRF3 and ISRE signaling upon stimulation with TRIFΔRIP, cGAS/STING or RIG-I-CARD remained unaltered. Experiments aimed at mapping UBCv1 inhibitory activity indicated that this viral protein acts upstream or at the level step of IKKβ. In agreement with this, UBCv1 was able to block p65 nuclear translocation upon cytokine stimulation, a key event in NF-ĸB signaling. Additionally, A549 stably transduced for UBCv1 showed a significant decrease in the levels of NF-ĸB dependent genes. Interestingly, despite the well-defined capacity of UBCv1 to conjugate ubiquitin chains, a mutant disabled for ubiquitylation activity retained similar immunomodulatory activity as the wild-type enzyme, suggesting that the two functions are segregated. Altogether these data suggest that ASFV UBCv1 manipulates the innate immune response targeting the NF-κB and AP-1 pathways and opens new questions about the multifunctionality of this enzyme.

Human interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokinelike protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Type I interferon (IFN) plays an essential role in the host innate immune responses. Several ubiquitin-conjugating enzyme (E2) family members were reported to regulate type I IFN production and host antiviral immune responses. However, the molecular mechanisms are still not fully understood. Here, we report that UBE2S acts as a negative regulator in the type I IFN signaling pathway. Ectopic expression of UBE2S inhibits host antiviral immune responses and enhances viral replications, whereas deficiency of UBE2S enhances host antiviral immune responses and suppresses viral replications both in vitro and in vivo. Inhibition of type І IFN production by UBE2S is independent on its E2 and E3 enzymic activity. Mechanistically, UBE2S interacts with TBK1 and recruits ubiquitin-specific protease 15 (USP15) to remove Lys63 (K63)-linked polyubiquitin chains of TBK1. Our findings reveal a role of the UBE2S-USP15-TBK1 axis in the regulation of host antiviral innate immune responses., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Harbin Veterinary Research Institute. Published by Elsevier Inc. All rights reserved.)

Hematopoietic development occurs in the bone marrow, and this process begins with hematopoietic stem cells (HSCs). Ubc9 is a unique E2-conjugating enzyme required for SUMOylation, an evolutionarily conserved post-translational modification system. We herein show that a conditional Ubc9 deletion in the hematopoietic system caused decreased thymus weight and reduced lymphocyte to myeloid cell ratio. Importantly, Ubc9 deletion in the hematopoietic system only selectively impaired the development of common lymphoid progenitors (CLPs) in the bone marrow and perturbed their potential to differentiate into lymphocytes, thereby decreasing the number of T/B cells in the periphery. Ubc9 was found to be required for CLP viability, and therefore, Ubc9 deficiency rendered CLPs to undergo apoptosis and attenuated their proliferation. Thus, Ubc9 plays a critical role in the regulation of CLP function during hematopoietic development in the bone marrow., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Brucella is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. Brucella 's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between Brucella melitensis 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. Brucella 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and Brucella 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, Brucella 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in B. melitensis 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of Brucella 16M ( p < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of Brucella in macrophages at the late stage of infection. Collectively, our results demonstrate that B. melitensis 16M may use the VirB IV secretion system of Brucella to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells., Competing Interests: The authors declare no conflicts of interest., (© 2019 The Korean Society of Veterinary Science.)

SUMOylation, the covalent attachment of the small ubiquitin-like modifier (SUMO) to target proteins, is emerging as a key modulator of eukaryotic immune function. In plants, a SUMO1/2-dependent process has been proposed to control the deployment of host defense responses. The molecular mechanism underpinning this activity remains to be determined, however. Here we show that increasing nitric oxide levels following pathogen recognition promote S-nitrosylation of the Arabidopsis SUMO E2 enzyme, SCE1, at Cys139. The SUMO-conjugating activities of both SCE1 and its human homolog, UBC9, were inhibited following this modification. Accordingly, mutation of Cys139 resulted in increased levels of SUMO1/2 conjugates, disabled immune responses, and enhanced pathogen susceptibility. Our findings imply that S-nitrosylation of SCE1 at Cys139 enables NO bioactivity to drive immune activation by relieving SUMO1/2-mediated suppression. The control of global SUMOylation is thought to occur predominantly at the level of each substrate via complex local machineries. Our findings uncover a parallel and complementary mechanism by suggesting that total SUMO conjugation may also be regulated directly by SNO formation at SCE1 Cys139. This Cys is evolutionary conserved and specifically S-nitrosylated in UBC9, implying that this immune-related regulatory process might be conserved across phylogenetic kingdoms., Competing Interests: The authors declare no conflict of interest.

Pevonedistat (MLN4924), a specific NEDD8-activating enzyme inhibitor, has been considered as a promising treatment for glioblastoma, which is currently in Phase I/II clinical trials. On the other hand, inhibition of neddylation pathway substantially upregulates the expression of T cell negative regulator programmed death-ligand 1 (PD-L1), which might account for the potential resistance via evasion of immune surveillance checkpoints. Whether administration of anti-PD-L1 enhances the efficacy of pevonedistat through a cytotoxic T cell-dependent mechanism in glioblastoma needs to be investigated. Here, we report that depletion of neddylation pathway key enzymes markedly elevates PD-L1 expression in glioblastoma cancer cells. Consistently, neddylation inhibitor pevonedistat significantly enhances PD-L1 expression in both glioblastoma cancer cell lines and animal models. Mechanistically, pevonedistat increases PD-L1 mRNA levels mainly through inhibiting Cullin1-F-box and WD repeat domain-containing 7 E3 ligase activity and accumulating c-MYC proteins, a direct transcriptional activator of PD-L1 gene expression. In addition, inhibition of Cullin3 activity by pevonedistat also blocks PD-L1 protein degradation. Importantly, pevonedistat attenuates T cell killing through PD-L1 induction, and blockade of PD-L1 restores the sensitivity of pevonedistat-treated glioblastoma cancer cells to T cell killing. The combination of pevonedistat and anti-PD-L1 therapy compared to each agent alone significantly increased the therapeutic efficacy in vivo. Our study demonstrates inhibition of neddylation pathway suppresses cancer-associated immunity and provides solid evidence to support the combination of pevonedistat and PD-L1/programmed cell death protein 1 immune checkpoint blockade as a potential therapeutic strategy to treat glioblastoma., (© 2019 UICC.)

Sepsis is a bi-phasic inflammatory disease that threatens approximately 30 million lives and claims over 14 million annually, yet little is known regarding the molecular switches and pathways that regulate this disease. Here, we have described ABCF1, an ATP-Binding Cassette (ABC) family member protein, which possesses an E2 ubiquitin enzyme activity, through which it controls the Lipopolysaccharide (LPS)- Toll-like Receptor-4 (TLR4) mediated gram-negative insult by targeting key proteins for K63-polyubiquitination. Ubiquitination by ABCF1 shifts the inflammatory profile from an early phase MyD88-dependent to a late phase TRIF-dependent signaling pathway, thereby regulating TLR4 endocytosis and modulating macrophage polarization from M1 to M2 phase. Physiologically, ABCF1 regulates the shift from the inflammatory phase of sepsis to the endotoxin tolerance phase, and modulates cytokine storm and interferon-β (IFN-β)-dependent production by the immunotherapeutic mediator, SIRT1. Consequently, ABCF1 controls sepsis induced mortality by repressing hypotension-induced renal circulatory dysfunction., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n , which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage - Sca-1 + c-Kit + CD150 + CD48 - HSPC subset (LSK CD150 + CD48 - cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n /Ubc13 deletion from Stat3 -deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150 + CD48 - cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation., Competing Interests: The authors declare no conflict of interest.

The neddylation pathway belongs post-translational modifications and plays important roles in regulating viral infection and replication. To address the relationship of influenza A virus with the neddylation modification pathway, we demonstrate that IAV infection in A549 cells can activate the neddylation modification pathway to increase virus growth and enhance the expression of pro-inflammatory cytokines to increase pathogenicity. The pre-treatment of Nedd8-activating enzyme subunit 1 (NAE1)-specific inhibitor, MLN4924, interferes with Nedd8 conjugation and NF-κB activity. MLN4924 exhibited pronounced antiviral activity against different subtypes of influenza A virus, including classical H1N1 (PR8), H9N2 subtype, and pandemic H1N1 2009 (pdmH1N1) viruses. Through the inhibition of the CRL/NF-κB pathway, MLN4924 could significantly suppress the expression levels of pro-inflammatory cytokines induced by IAVs. These findings suggest that MLN4924 can be developed as a novel antiviral therapy for influenza infection for anti-viral efficacy and anti-inflammation activity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Protein sumoylation is a highly dynamic and reversible post-translational modification, involving covalently conjugation of the small ubiquitin-like modifier (SUMO) to the lysine residue of the target protein. Similar to ubiquitination, sumoylation is catalyzed by E1, E2 and several E3 ligases. However, sumoylation usually does not cause protein degradation but alter the target function through diverse mechanisms. Increasing evidences have shown that sumoylation plays pivotal roles in the pathogenesis of human diseases, including neuron degeneration, cancer and heart disease, etc. We and others have shown that sumoylation is critically implicated in mouse eye development. However, the expression of sumoylation machinery has not been characterized in normal and pathogenic retina. Worldwide, age-related macular degeneration (AMD) is the leading cause of irreversible blindness in aged person. In the present study, we investigated the expression of the major sumoylation enzymes in normal mice and sodium iodateinduced AMD mouse model., Methods: Four-week-old C57BL/6J mice were used in our experiment. A sterile 1% NaIO3 solution was freshly prepared in PBS from solid NaIO3. Experimental mice were injected with 70 mg/kg NaIO3, and similar volumes of PBS as control. Eyes were enucleated and immersion in FAA fixation overnight and processed for eye cross-sections. After fixation, cross sections eyes were dehydrated, embedded in paraffin, and 6 mm transverse sections were cut using the rotary microtome. Then paraffin sections were stained with hematoxylin and eosin (H&E), and mouse retinal thickness was observed to assess the histopathologic changes., Results: Significantly declined RNA levels of E1, E2 and E3 ligase PIAS1 in NaIO3-injected mouse RPE one day-post treatment. Consistently, the protein level of PIAS1 was also decreased at this time point. At the late stage of treatment (three days post-injection), significantly reduced expression of E1 enzyme SAE1/UBA2 was detected in NaIO3-injected mouse retinas. In the contrary, dramatically increased E3 ligase RanBP2 was found in the injected-retinas., Conclusion: Together, our results demonstrated for the first time the dynamic expression of sumoylation pathway enzymes during the progression of retina degeneration induced by oxidative stress. Dynamic expression of E1, E2 and E3 enzymes were found during the time course of RPE and retina degeneration, which revealed the potential regulatory roles of sumoylation in AMD pathogenesis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila the Imd pathway detects Gram-negative bacterial infections through recognition of diaminopimelic acid (DAP)-type peptidoglycan and activation of the NF-κB precursor Relish, which drives robust antimicrobial peptide gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and Lys-63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. Here, we demonstrate that Imd is rapidly Lys-63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 and Ubc13-Uev1a, in conjunction with the E3 ligase Diap2. Lys-63-ubiquitination activates the TGFβ-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of Lys-63 chains and the addition of Lys-48 polyubiquitin. This ubiquitin-editing process results in the proteasomal degradation of Imd, which we propose functions to restore homeostasis to the Drosophila immune response., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Innate immunity plays a pivotal role in virus infection. RIG-I senses viral RNA and initiates an effective innate immune response for type I interferon production. To transduce RIG-I-mediated antiviral signalling, a mitochondrial protein MAVS forms prion-like aggregates to activate downstream kinases and transcription factors. However, the activation mechanism of RIG-I is incompletely understood. Here we identify two ubiquitin enzymes Ube2D3 and Ube2N through chromatographic purification as activators for RIG-I on virus infection. We show that together with ubiquitin ligase Riplet, Ube2D3 promotes covalent conjugation of polyubiquitin chains to RIG-I, while Ube2N preferentially facilitates production of unanchored polyubiquitin chains. In the presence of these polyubiquitin chains, RIG-I induces MAVS aggregation directly on the mitochondria. Our data thus reveal two essential polyubiquitin-mediated mechanisms underlying the activation of RIG-I and MAVS for triggering innate immune signalling in response to viral infection in cells.

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, we identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. We showed that Dorsal is required to limit the size of the progenitor pool. Additionally, we showed that activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. We also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. We showed that Dorsal repressed Ush expression levels to promote differentiation, whereas Cactus maintained Ush levels to block differentiation. Additionally, we showed that another Toll antagonist, Lesswright, also maintained the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling.

UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ubiquitination is a key regulatory mechanism in the immune deficiency (IMD) pathway in Drosophila. In this study, we first developed a simple immunoblot method to identify components involved in this pathway. Considering the emerging roles of ubiquitin-conjugating enzymes (E2s) in determining ubiquitin chain types and ubiquitination speed, we screened for E2s required for IMD activation. We found that UbcD4, in addition to the previously reported E2s Effete and Bendless, was required for activation of the IMD pathway. RNAi-mediated knockdown of the UbcD4 ortholog, E2-25K/Ube2K, inhibited TNFα- and LPS-mediated activation of the NF-κB pathway, implying that UbcD4 and E2-25K/Ube2K play a conserved role as positive regulators in both pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine gammaherpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by interferon-γ (IFN-γ). Using a lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16l1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5 deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ, and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Double-stranded RNA-dependent protein kinase (PKR) is intimately involved in type 2 diabetes due to its role in insulin resistance in peripheral tissues and anti-proliferative effect on pancreatic β-cells. Activated PKR was found to inhibit β-cell proliferation, partially through accumulation of P53. However the molecular mechanisms underlying PKR-dependent upregulation of P53 remain unknown. The results of the present study showed that PKR can be specifically activated in PKR overexpressing β-cells by a low dosage of the previously synthesized compound 1H-benzimidazole1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride (BEPP), and this led to upregulation of P53 through sumoylation-dependent stability. Activated PKR was found to interact with sumo-conjugating enzyme Ubc9, and P53 sumoylation relies on a PKR-Ubc9 protein-protein interaction. Additionally, a ceramide signal was needed for PKR activation to be triggered by glucolipotoxicity and TNFα stimulation, and stabilization of P53 required endogenous ceramide accumulation. Glucolipotoxicity and pro-inflammatory cytokines therefore promote the sumoylation-dependent stability of P53 via the ceramide/PKR/Ubc9 signalling pathway that is involved in pancreatic β-cell proliferation inhibition in the development of type 2 diabetes., (Copyright © 2015. Published by Elsevier Ltd.)

RNA virus infection is recognized by the RIG-I family of receptors that activate the mitochondrial adaptor MAVS, leading to the clearance of viruses. Antiviral signalling activation requires strict modulation to avoid damage to the host from exacerbated inflammation. Insulin receptor tyrosine kinase substrate (IRTKS) participates in actin bundling and insulin signalling and its deficiency causes insulin resistance. However, whether IRTKS is involved in the regulation of innate immunity remains elusive. Here we show that IRTKS deficiency causes enhanced innate immune responses against RNA viruses. IRTKS-mediated suppression of antiviral responses depends on the RIG-I-MAVS signalling pathway. IRTKS recruits the E2 ligase Ubc9 to sumoylate PCBP2 in the nucleus, which causes its cytoplasmic translocation during viral infection. The sumoylated PCBP2 associates with MAVS to initiate its degradation, leading to downregulation of antiviral responses. Thus, IRTKS functions as a negative modulator of excessive inflammation.

The ubiquitin-ligating enzyme (E3) Itch plays a crucial role in the regulation of inflammation, and Itch deficiency leads to severe airway inflammation. However, the molecular mechanisms by which Itch function is regulated remain elusive. In this study, we found that nontypeable Haemophilus influenzae induces the association of Itch with Ndfip1. Both Itch(-/-) and Ndfip1(-/-) mice exhibited severe airway inflammation in response to nontypeable Haemophilus influenza, which was associated with elevated expression of proinflammatory cytokines. Ndfip1 enhanced Itch ligase activity and facilitated Itch-mediated Tak1 ubiquitination. Mechanistically, Ndfip1 facilitated recruitment of ubiquitin-conjugating enzyme (E2) UbcH7 to Itch. The N-terminal region of Ndfip1 binds to UbcH7, whereas the PY motif binds to Itch. Hence, Ndfip1 acts as an adaptor for UbcH7 and Itch. Reconstitution of full-length Ndfip1 but not the mutants that fail to interact with either UbcH7 or Itch, restored the defect in Tak1 ubiquitination and inhibited elevated proinflammatory cytokine expression by Ndfip1(-/-) cells. These results provide new mechanistic insights into how Itch function is regulated during inflammatory signaling, which could be exploited therapeutically in inflammatory diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Objectives: The diagnosis of extrapulmonary tuberculous infections and nontuberculous mycobacterial (NTM) infections is difficult because the symptoms are nonspecific and suitable specimens for bacterial culture are often not available. Recent publications reported the existence of autoantibodies in tuberculous infections. We screened for specific autoantibodies in mycobacterial infections., Methods: We screened four in 29 patients with active mycobacterial infections and different controls using protein array technology. We could identify autoantibodies against ubiquitin-fold modifier-conjugating enzyme 1 (Ufc1) and pleckstrin homology domain containing, family G (with RhoGef domain) member 2 (Plekhg2) in all four patients. Subsequently, we designed enzyme-linked immunosorbent assays (ELISAs) for the detection of autoantibodies binding to Ufc1 and Plekhg2., Results: Autoantibodies binding to Ufc1 and Plekhg2 were found in 19 of 29 patients (66%) with active mycobacterial infections. In comparison, we found these autoantibodies in one of 31 patients (3%) with successfully treated mycobacterial infections, in three of 40 (8%) HIV-infected patients not receiving combination antiretorviral therapy (cART) and in six of 134 (5%) blood donors. Interestingly, six of eight (75%) patients with HIV-associated B-cell non-Hodgkin lymphoma (B-NHL) at the onset of disease had autoantibodies against Ufc1 and Plekhg2, but none of nine (0%) patients after treatment of HIV-associated B-NHL, none of seven patients with non-HIV-associated B-NHL and 11 of 115 (10%) patients with other malignant diseases had autoantibodies against both proteins., Conclusions: In view of the high frequency of these autoantibodies, we postulate that they might be of potential use for additional diagnostics for mycobacterial infections, and further studies may shed light on the pathomechanisms of these two autoantibodies., (© 2014 British HIV Association.)

Autophagy is a vital catabolic process for degrading bulky cytosolic contents, which cannot be resorbed via the proteasome. First described as a survival mechanism during nutrient starvation conditions, recent reports have demonstrated that autophagy supports metabolic functions of T cells at various stages of maturation and effector function. Autophagy is crucial for T-cell development at the precursor stage as self-renewability and quiescence of hematopoietic stem cells depend on autophagy of the mitochondria and the endoplasmic reticulum. Later, during development in the thymus, autophagy regulates peptide presentation in stromal cells and professional antigen-presenting cells, which mediate thymocyte selection. Furthermore, the metabolic changes when mature T cells enter the periphery and when they are activated are both dependent on autophagy. Lastly, autophagy prevents early aging and, thus, ensures maintenance of memory T cells.

Allogeneic haematopoietic stem cell transplantation (HSCT) represents the only curative therapy for the majority of bone marrow-derived cancers. Unfortunately, HSCT can result in serious complications such as graft-versus-host disease, graft failure and infection. In the last decade, there have been major advances in the understanding of the role of autophagy in many diseases and cellular processes. Recent findings have demonstrated a crucial role for autophagy in haematopoietic stem cell survival and function, antigen presentation, T-cell differentiation and response to cytokine stimulation. Given the critical requirement for each of these processes in HSCT and subsequent complications, it is surprising that the contribution of autophagy to HSCT per se is relatively unexplored. In addition, the increasing use of autophagy-modulating drugs in the clinic further highlights the need to understand the role of autophagy in allogeneic HSCT. This review will cover established and implicated roles of autophagy in HSCT, suggesting this pathway as an important therapeutic target for improving transplant outcomes.

The evolutionarily conserved catabolic process of autophagy involves the degradation of cytoplasmic components through lysosomal enzymes. Basal levels of autophagy maintain cellular homeostasis and under stress conditions high levels of autophagy are induced. It is often under such stress conditions that high levels of autophagy and cell death have been observed, leading to the idea that autophagy may act as an executioner of cell death. However the notion of autophagy as a cell death mechanism has been controversial and remains mechanistically undefined. There is now growing evidence that in specific contexts autophagy can indeed facilitate cell death. The pro-death role of autophagy is however complicated due to the extensive cross-talk between different signalling pathways. This review summarises the examples of where autophagy acts as a means of cell death and discusses the association of autophagy with the different cell death pathways.

Autoantibodies can facilitate diagnostic and therapeutic means for type 1 diabetes (T1DM). We profiled autoantibodies from serum samples of 16 T1DM patients, 16 type 2 diabetic (T2DM) patients, and 27 healthy control subjects with normal glucose tolerance (NGT) by using protein microarrays containing 9,480 proteins. Two novel autoantibodies, anti-EEF1A1 and anti-UBE2L3, were selected from microarrays followed by immunofluorescence staining of pancreas. We then tested the validity of the candidates by ELISA in two independent test cohorts: 1) 95 adults with T1DM, 49 with T2DM, 11 with latent autoimmune diabetes in adults (LADA), 20 with Graves disease, and 66 with NGT and 2) 33 children with T1DM and 34 healthy children. Concentrations of these autoantibodies were significantly higher in T1DM patients than in NGT and T2DM subjects (P < 0.01), which was also confirmed in the test cohort of children (P < 0.05). Prevalence of anti-EEF1A1 and anti-UBE2L3 antibodies was 29.5% and 35.8% in T1DM, respectively. Of note, 40.9% of T1DM patients who lack anti-GAD antibodies (GADA) had anti-EEF1A1 and/or anti-UBE2L3 antibodies. These were also detected in patients with fulminant T1DM but not LADA. Our approach identified autoantibodies that can provide a new dimension of information indicative of T1DM independent of GADA and new insights into diagnosis and classification of T1DM., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Autophagy is a lysosomal degradation pathway that is important in cellular homeostasis. Prior work showed a key role for the autophagy related 5 (Atg5) in resistance to Toxoplasma gondii. Here we show that the cassette of autophagy proteins involved in the conjugation of microtubule-associated protein 1 light chain 3 (LC3) to phosphatidylethanolamine, including Atg7, Atg3, and the Atg12-Atg5-Atg16L1 complex play crucial roles in the control of T. gondii in vitro and in vivo. In contrast, pharmacologic modulation of the degradative autophagy pathway or genetic deletion of other essential autophagy genes had no substantial effects. Rather the conjugation system was required for targeting of LC3 and interferon-γ effectors onto the vacuolar membrane of T. gondii and its consequent disruption. These data suggest that the ubiquitin-like conjugation systems that reorganize intracellular membranes during canonical autophagy are necessary for proper targeting of immune effectors to the intracellular vacuole membranes utilized by pathogens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Coactivator activator (CoAA) is a dual-functional coregulator that regulates steroid receptor-mediated transcription and alternative splicing. Previously, we have shown that CoAA has tumor-suppressive potential in tumorigenic human kidney cells. Here, we uncover a molecular mechanism by which Sjogren syndrome-associated autoantigen (SSA), an estrogen receptor (ER) coactivator, induces MYC oncogene by removing repressive CoAA through E2-dependent degradation of CoAA and promotes G(1)/S transition of the cell cycle as well as anchorage-independent growth capability of breast cancer cells. We also show that E2 and ER enhance the E3 ligase activity of SSA to modulate CoAA through splicing isoform-selective ubiquitylation. We propose this as one potential molecular basis for the reduced tumor incidence in autoimmune disease patients and suggest SSA as a potential therapeutic target to treat breast cancer.

Taenia pisiformis larvae cause significant health problems to rabbits. At present, it is not known whether the recombinant antigen from the T. pisiformis oncosphere is able to confer protective immunity against T. pisiformis larval infection. The full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTpUbc2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against T. pisiformis infection. The full-length open reading frame of the TpUbc2 gene was 444 bp, and encoded a 16.63-kDa protein. Finally, rTpUbc2 was used to evaluate the ability to induce immunoprotective responses in rabbits. A 79.3-90.8% reduction (P < 0.01) in the recovery of larvae was observed in the experimental group compared to the control group. Specific anti-rTpUbc2 antibodies from immunized rabbits had significantly higher levels of IgG (P < 0.01) compared to the control group; however, no significant difference in IgA levels was found between groups (P > 0.05). Our data support the use of rTpUbc2 as a potential candidate to develop a vaccine against T. pisiformis larvae.

Ubiquitin-conjugated E2 enzyme (UBE2) is one of the main components of the proteasome degradation cascade. Previous studies have shown an increase of expression levels in individuals challenged to some pathogen organism such as virus and bacteria. The study was to characterize the immune response of UBE2 gene in the gastropod Concholepas concholepas through expression analysis and single nucleotide polymorphisms (SNP) discovery. Hence, UBE2 was identified from a cDNA library by 454 pyrosequencing, while SNP identification and validation were performed using De novo assembly and high resolution melting analysis. Challenge trials with Vibrio anguillarum was carried out to evaluate the relative transcript abundance of UBE2 gene from two to thirty-three hours post-treatment. The results showed a partial UBE2 sequence of 889 base pair (bp) with a partial coding region of 291 bp. SNP variation (A/C) was observed at the 546th position. Individuals challenged by V. anguillarum showed an overexpression of the UBE2 gene, the expression being significantly higher in homozygous individuals (AA) than (CC) or heterozygous individuals (A/C). This study contributes useful information relating to the UBE2 gene and its association with innate immune response in marine invertebrates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human ubiquitin-conjugating enzyme E2, also known as UbcH10, is defined as a cyclin-selective ubiquitin carrier protein and is essential for selective degradation of many short-lived proteins in eukaryotic cells. Recently more and more data show that UbcH10 could be a potential cancer biomarker. In this study, we have developed a monoclonal antibody (MAb) against UbcH10 using an expression recombinant protein. Hybridomas F001, F007, and F008 with high affinities belong to IgG1 subclass with κ light and are highly specific for UbcH10. Further experimentation showed that MAbs F001, F007, and F008 are suitable for the development of immunoassay core agents with sufficient sensitivity and specificity in vitro by Western-blot, immunofluorescence, and immunohistochemistry. These MAbs can be used as a tool for further investigation on functions related to the role of UbcH10 in tumorigenesis and development.

The novel human gene, designated ubiquitin-conjugating enzyme E2Q family member 1 (UBE2Q1) maps to chromosome 1q21.3. The gene has an open reading frame corresponding to 422 amino acids and contains a RWD domain and an E2 ubiquitin conjugating enzyme domain. Here, we investigated the expression levels of both mRNA and protein of UBE2Q1 gene in cancerous versus normal parts of breast specimens from 26 patients. Real-time PCR data showed that the relative expression level of UBE2Q1 mRNA was significantly greater in cancers than in non-cancerous tissues of breast specimens (Mean ± SEM, 0.064 ± 0.015 for cancers and 0.026 ± 0.01 for noncancerous tissues, P < 0.05 Mann-Whitney test). A rabbit polyclonal antibody was generated against an amino acid sequence predicted from the DNA sequence of UBE2Q1 gene. This antibody was used to perform Western blotting on 21 cases in our cohort of breast specimens. Thus, 13 (61.904%) of the cases showed an increase in the UBE2Q1 immunoreactivity in their cancerous tissues as compared with the corresponding normal tissues. This result along with the real-time PCR data shows that the novel human gene, UBE2Q1, is expressed in human breast and may have implications for pathogenesis of breast cancer.

The maintenance of immune homeostasis requires regulatory T cells (Treg cells). Here we found that Treg cell–specific ablation of Ubc13, a Lys63 (K63)-specific ubiquitin-conjugating enzyme, caused aberrant T cell activation and autoimmunity. Although Ubc13 deficiency did not affect the survival of Treg cells or expression of the transcription factor Foxp3, it impaired the in vivo suppressive function of Treg cells and rendered them sensitive to the acquisition of T helper type 1 (TH1) cell– and interleukin 17 (IL-17)-producing helper T (TH17) cell–like effector phenotypes. This function of Ubc13 involved its downstream target, the kinase IKK. The Ubc13-IKK signaling axis controlled the expression of specific Treg cell effector molecules, including IL-10 and SOCS1. Collectively, our findings suggest that the Ubc13-IKK signaling axis regulates the molecular program that maintains Treg cell function and prevents Treg cells from acquiring inflammatory phenotypes.

The association between hyperinflammatory states and numerous diseases is widely recognized, but our understanding of the molecular strategies that have evolved to prevent uncontrolled activation of inflammatory responses remains incomplete. Here, we report a critical, nontranscriptional role of GPS2 as a guardian against hyperstimulation of the TNF-α-induced gene program. GPS2 cytoplasmic actions are required to specifically modulate RIP1 ubiquitylation and JNK activation by inhibiting TRAF2/Ubc13 enzymatic activity. In vivo relevance of GPS2 anti-inflammatory role is confirmed by inhibition of TNF-α target genes in macrophages and by improved insulin signaling in the adipose tissue of aP2-GPS2 transgenic mice. As the nontranscriptional role is complemented by GPS2 functioning as positive and negative cofactor for nuclear receptors, in vivo overexpression also results in elevated circulating level of Resistin and development of hepatic steatosis. Together, these studies define GPS2 as a molecular guardian required for precise control of inflammatory responses involved in immunity and homeostasis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ro52 (TRIM21) is an E3 ligase of the tripartite motif family that negatively regulates proinflammatory cytokine production by ubiquitinating transcription factors of the interferon regulatory factor family. Autoantibodies to Ro52 are present in patients with lupus and Sjögren's syndrome, but it is not known if these autoantibodies affect the function of Ro52. To address this question, the requirements for Ro52 E3 ligase activity were first analyzed in detail. Scanning a panel of E2 ubiquitin-conjugating enzymes, we found that UBE2D1-4 and UBE2E1-2 supported the E3 ligase activity of Ro52 and that the E3 ligase activity of Ro52 was dependent on its RING domain. We also found that the N-terminal extensions in the class III E2 enzymes affected their interaction with Ro52. Although the N-terminal extension in UBE2E3 made this E2 enzyme unable to function together with Ro52, the N-terminal extensions in UBE2E1 and UBE2E2 allowed for a functional interaction with Ro52. Anti-Ro52-positive patient sera and affinity-purified anti-RING domain autoantibodies inhibited the E3 activity of Ro52 in ubiquitination assays. Using NMR, limited proteolysis, ELISA, and Ro52 mutants, we mapped the interactions between Ro52, UBE2E1, and anti-Ro52 autoantibodies. We found that anti-Ro52 autoantibodies inhibited the E3 ligase activity of Ro52 by sterically blocking the E2/E3 interaction between Ro52 and UBE2E1. Our data suggest that anti-Ro52 autoantibodies binding the RING domain of Ro52 may be actively involved in the pathogenesis of rheumatic autoimmune disease by inhibiting Ro52-mediated ubiquitination.

Ubiquitination, the covalent attachment of ubiquitin molecules to proteins, is emerging as a widely utilized mechanism for rapidly regulating cell signaling. Recent studies indicate that ubiquitination plays potent roles in regulating a variety of signals in both innate and adaptive immune cells. Here, we will review recent studies of ubiquitin ligases, ubiquitin chain linkages, and ubiquitin binding proteins that highlight the diversity and specificity of ubiquitin dependent functions in immune cells. We will also review studies that shed light on how ubiquitination signals are integrated in cell-type-specific fashion to regulate the immune system in vivo.

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13(flox/flox)K5-Cre). At birth, the skin of the Ubc13(flox/flox)K5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13(flox/flox)K5-Cre mouse epidermis. In culture, Ubc13(flox/flox)K5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13(flox/flox) keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13(flox/flox)K5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.

Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.

Ubiquitin conjugating enzyme variants (Uev) Uev1 and Mms2 share >90% sequence identity but with distinct biological functions. Here, we report the monomeric and heterodimeric crystal structures of Uev1 and comparison with that of Mms2. Uev1 alone or in complex with Ubc13 is nearly identical with the corresponding Mms2 structures, except in one surface area containing 7/14 amino acid variations. To probe the biological significance of this unique region, we raised monoclonal antibodies specifically recognizing this region of Uev1, but not of Mms2. Epitope mapping and site-specific mutagenesis revealed at least two distinct epitopes within this region. These data collectively suggest the existence of cellular proteins capable of distinguishing Uev1 from Mms2 and directing the Ubc13-Uev complex to different pathways.

High-throughput studies to determine differential immune (humoral) response to diseases are becoming of increasing interest because the information they provide can help in early diagnosis as well as monitoring of therapeutics. Protein microarrays are a high-throughput and convenient technology that can be applied to the study of the humoral response. Proteins can be arrayed on slides and then probed with serum from different classes of patients to observe differences that may exist among autoantibodies that reflect differences in disease states. However, such studies may be difficult to interpret due to the weak overall signal response of such protein microarrays. We propose that this weak signal response is due to the physical positioning of the disease proteins that renders them sterically hindered from binding partners in the serum. In this study, we hypothesize that reducing the complexity and size of the disease proteins by chemical digestion using cyanogen bromide (CNBr) may enhance the overall signal from the humoral response and facilitate visualization of disease-specific responses in various classes of serum. A modified protein microarray methodology using CNBr digestion is presented here. The new workflow was applied to a set of 10 serum samples from healthy subjects, 10 from patients with chronic pancreatitis and 10 from patients diagnosed with pancreatic cancer and the results were compared to results obtained in the absence of CNBr digestion. CNBr digestion allowed the identification of 10 additional autoantibodies that responded to serum, 5 of which were unique to pancreatitis and cancer sera. This new methodology may increase the sensitivity of microarray studies measuring autoantibodies in serum.

The AP-1 family member JunB is a critical regulator of T cell function. JunB is a transcriptional activator of various cytokine genes, such as IL-2, IL-4, and IL-10; however, the post-translational modifications that regulate JunB activity in T cells are poorly characterized. We show here that JunB is conjugated with small ubiquitin-like modifier (SUMO) on lysine 237 in resting and activated primary T cells and T cell lines. Sumoylated JunB associated with the chromatin-containing insoluble fraction of cells, whereas nonsumoylated JunB was also in the soluble fraction. Blocking JunB sumoylation by mutation or use of a dominant-negative form of the SUMO-E2 Ubc-9 diminished its ability to transactivate IL-2 and IL-4 reporter genes. In contrast, nonsumoylable JunB mutants showed unimpaired activity with reporter genes controlled by either synthetic 12-O-tetradecanoylphorbol-13-acetate response elements or NF-AT/AP-1 and CD28RE sites derived from the IL-2 promoter. Ectopic expression of JunB in activated human primary CD4(+) T cells induced activation of the endogenous IL-2 promoter, whereas the nonsumoylable JunB mutant did not. Thus, our work demonstrates that sumoylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation.

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate anti-viral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination.

Ubiquitin conjugating enzyme functions as the second enzyme required for protein ubiquitination and plays an important role in ubiquitin transferring and substrate specific recognition. UBE2W, a newly described member of E2 family, was formerly reported probably involving in phototransduction or retinal degeneration in Drosophila. In this study, we report that murine UBE2W harbors a typical UBC domain and is highly conserved in different vertebrate homologues. GST-tagged UBE2W was expressed in E. coli BL21 (DE3) and purified with GST affinity chromatography. Using this antigen, we generated and further separated rabbit polyclonal antibody of UBE2W, of which the activity and specificity were confirmed by immunoblotting of transiently expressed myc-UBE2W fusion protein. Wide expression of UBE2W was found in brain, muscle, heart, lung, liver, spleen, kidney and testis of mouse with the generated antibody, indicating the functional importance of this novel protein. Furthermore, the UBE2W highly expression was confined to the adult testis and was developmental stage-specific.

The Rip2 kinase contains a caspase recruitment domain and has been implicated in the activation of the transcriptional factor NF-kappaB downstream of Toll-like receptors, Nod-like receptors, and the T cell receptor. Although Rip2 has been linked to Nod signaling, how Nod-Rip2 proteins mediate NF-kappaB activation has remained unclear. We find Rip2 required for Nod2-mediated NF-kappaB activation and to a lesser extent mitogen-activated protein kinase activation. We demonstrate that Rip2 and IkappaB kinase-gamma become stably polyubiquitinated upon treatment of cells with the NOD2 ligand, muramyl dipeptide. We also demonstrate a requirement for the E2-conjugating enzyme Ubc13, the E3 ubiquitin ligase Traf6, and the ubiquitin-activated kinase Tak1 in Nod2-mediated NF-kappaB activation. Rip2 polyubiquitination is also stimulated when macrophages are infected with live Mycobacterium tuberculosis but not when infected with heat-killed bacteria. Consistent with our data linking Rip2 to NOD and not Toll-like receptor signaling, M. tuberculosis-induced Rip2 polyubiquitination appears MyD88-independent. Collectively, these data reveal that the NOD2 pathway is ubiquitin-regulated and that Rip2 employs a ubiquitin-dependent mechanism to achieve NF-kappaB activation.

Although patients diagnosed with melanoma of