The Met1-linked ubiquitin machinery in inflammation and infection.

Ubiquitination is an essential post-translational modification that regulates most cellular processes. The assembly of ubiquitin into polymeric chains by E3 ubiquitin ligases underlies the pleiotropic functions ubiquitin chains regulate. Ubiquitin chains assembled via the N-terminal methionine, termed Met1-linked ubiquitin chains or linear ubiquitin chains, have emerged as essential signalling scaffolds that regulate pro-inflammatory responses, anti-viral interferon responses, cell death and xenophagy of bacterial pathogens downstream of innate immune receptors. Met1-linked ubiquitin chains are exclusively assembled by the linear ubiquitin chain assembly complex, LUBAC, and are disassembled by the deubiquitinases OTULIN and CYLD. Genetic defects that perturb the regulation of Met1-linked ubiquitin chains causes severe immune-related disorders, illustrating their potent signalling capacity. Here, we review the current knowledge about the cellular machinery that conjugates, recognises, and disassembles Met1-linked ubiquitin chains, and discuss the function of this unique posttranslational modification in regulating inflammation, cell death and immunity to pathogens.

Envelope protein ubiquitination drives entry and pathogenesis of Zika virus.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

N6-Methyladenosine Modification Controls Circular RNA Immunity.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and viral genomes. Mammalian cells possess innate immunity to detect foreign circRNAs, but the molecular basis of self versus foreign identity in circRNA immunity is unknown. Here, we show that N6-methyladenosine (m6A) RNA modification on human circRNAs inhibits innate immunity. Foreign circRNAs are potent adjuvants to induce antigen-specific T cell activation, antibody production, and anti-tumor immunity in vivo, and m6A modification abrogates immune gene activation and adjuvant activity. m6A reader YTHDF2 sequesters m6A-circRNA and is essential for suppression of innate immunity. Unmodified circRNA, but not m6A-modified circRNA, directly activates RNA pattern recognition receptor RIG-I in the presence of lysine-63-linked polyubiquitin chain to cause filamentation of the adaptor protein MAVS and activation of the downstream transcription factor IRF3. CircRNA immunity has considerable parallel to prokaryotic DNA restriction modification system that transforms nucleic acid chemical modification into organismal innate immunity.

Identification of linear polyubiquitin chain immunoreactivity in tau pathology of Alzheimer's disease.

Characteristic neuropathological structures observed in Alzheimer's disease, such as neurofibrillary tangles and dystrophic neurites of senile plaques, are universally ubiquitinated. Eight linkage types of polyubiquitin chains are known, and each type of chain exerts different intracellular action. Among them, linear polyubiquitin chain was recently reported to be associated with the pathomechanism of neuronal cell death. We therefore generated a novel antibody that specifically recognizes linear polyubiquitin chain. We immunohistochemically examined the brains of patients with Alzheimer's disease. A subset of neurofibrillary tangles, dystrophic neurites of senile plaques, and neuropil threads were evident to be immunopositive for linear polyubiquitin chains, but their number was fewer than those recognized by the antibody against K48-linked polyubiquitin chains. Double immunofluorescence investigation showed that in certain neurofibrillary tangles, a part of K48-linked polyubiquitin-immunopositive structures were immunolabeled for linear polyubiquitin chains. Our results imply that linear polyubiquitination follows K48-linked polyubiquitination in abnormally accumulated tau proteins in Alzheimer's disease, and imply involvement in its neurodegeneration.

Interpreting the Language of Polyubiquitin with Linkage-Specific Antibodies and Mass Spectrometry.

Posttranslational modification of cellular proteins by ubiquitin serves a variety of functions. Among the multitude of ubiquitin substrates, ubiquitin itself is the most prevalent. For many years, the direct detection of polyubiquitin chains attached to cellular substrates was not practical, with cell biologists relegated to indirect approaches involving site-directed mutagenesis or in vitro biochemistry. Recent advances in two technologies-polyubiquitin linkage-specific antibodies and mass spectrometry proteomics, have overcome that limitation. Using one or both of these, the direct analysis of polyubiquitin chain linkages on cellular substrate proteins may be performed. This paper describes the complimentary nature of linkage-specific antibodies and mass spectrometry proteomics for the characterization of complex ubiquitin signals using lessons learned in early development of both technologies.

USP38 critically promotes asthmatic pathogenesis by stabilizing JunB protein.

Th2 immune response is critical for allergic asthma pathogenesis. Molecular mechanisms for regulating Th2 immunity are still not well understood. Here we report that the ubiquitin-specific protease USP38 is crucial for Th2-mediated allergic asthma. TCR stimulation up-regulated the USP38 level, and USP38 in turn mediated the protein stabilization of JunB, a transcription factor specific for Th2 development. Consequently, USP38 was specifically required for TCR-induced production of Th2 cytokines and Th2 development both in vitro and in vivo, and USP38-deficient mice were resistant to asthma pathogenesis induced by OVA or HDM. Mechanistically, USP38 directly associated with JunB, deubiquitinated Lys-48-linked poly-ubiquitination of JunB, and consequently blocked TCR-induced JunB turnover. USP38 represents the first identified deubiquitinase specifically for Th2 immunity and the associated asthma.

The Met1-Linked Ubiquitin Machinery: Emerging Themes of (De)regulation.

The linear ubiquitin chain assembly complex, LUBAC, is the only known mammalian ubiquitin ligase that makes methionine 1 (Met1)-linked polyubiquitin (also referred to as linear ubiquitin). A decade after LUBAC was discovered as a cellular activity of unknown function, there are now many lines of evidence connecting Met1-linked polyubiquitin to NF-κB signaling, cell death, inflammation, immunity, and cancer. We now know that Met1-linked polyubiquitin has potent signaling functions and that its deregulation is connected to disease. Indeed, mutations and deficiencies in several factors involved in conjugation and deconjugation of Met1-linked polyubiquitin have been implicated in immune-related disorders. Here, we discuss current knowledge and recent insights into the role and regulation of Met1-linked polyubiquitin, with an emphasis on the mechanisms controlling the function of LUBAC.

Innate immune signaling in Drosophila is regulated by transforming growth factor ß (TGFß)-activated kinase (Tak1)-triggered ubiquitin editing.

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 TGFß-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.

USP38 Inhibits Type I Interferon Signaling by Editing TBK1 Ubiquitination through NLRP4 Signalosome.

TBK1 is a component of the type I interferon (IFN) signaling pathway, yet the mechanisms controlling its activity and degradation remain poorly understood. Here we report that USP38 negatively regulates type I IFN signaling by targeting the active form of TBK1 for degradation in vitro and in vivo. USP38 specifically cleaves K33-linked poly-ubiquitin chains from TBK1 at Lys670, and it allows for subsequent K48-linked ubiquitination at the same position mediated by DTX4 and TRIP. Knockdown or knockout of USP38 increases K33-linked ubiquitination, but it abrogates K48-linked ubiquitination and degradation of TBK1, thus enhancing type I IFN signaling. Our findings identify an essential role for USP38 in negatively regulating type I IFN signaling, and they provide insights into the mechanisms by which USP38 regulates TBK1 ubiquitination through the NLRP4 signalosome.

The Matrix Protein of Nipah Virus Targets the E3-Ubiquitin Ligase TRIM6 to Inhibit the IKKε Kinase-Mediated Type-I IFN Antiviral Response.

For efficient replication, viruses have developed mechanisms to evade innate immune responses, including the antiviral type-I interferon (IFN-I) system. Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family (genus Henipavirus), is known to encode for four P gene-derived viral proteins (P/C/W/V) with IFN-I antagonist functions. Here we report that NiV matrix protein (NiV-M), which is important for virus assembly and budding, can also inhibit IFN-I responses. IFN-I production requires activation of multiple signaling components including the IκB kinase epsilon (IKKε). We previously showed that the E3-ubiquitin ligase TRIM6 catalyzes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, and activate IKKε for induction of IFN-I mediated antiviral responses. Using co-immunoprecipitation assays and confocal microscopy we show here that the NiV-M protein interacts with TRIM6 and promotes TRIM6 degradation. Consequently, NiV-M expression results in reduced levels of unanchored K48-linked polyubiquitin chains associated with IKKε leading to impaired IKKε oligomerization, IKKε autophosphorylation and reduced IFN-mediated responses. This IFN antagonist function of NiV-M requires a conserved lysine residue (K258) in the bipartite nuclear localization signal that is found in divergent henipaviruses. Consistent with this, the matrix proteins of Ghana, Hendra and Cedar viruses were also able to inhibit IFNß induction. Live NiV infection, but not a recombinant NiV lacking the M protein, reduced the levels of endogenous TRIM6 protein expression. To our knowledge, matrix proteins of paramyxoviruses have never been reported to be involved in innate immune antagonism. We report here a novel mechanism of viral innate immune evasion by targeting TRIM6, IKKε and unanchored polyubiquitin chains. These findings expand the universe of viral IFN antagonism strategies and provide a new potential target for development of therapeutic interventions against NiV infections.

K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8(+) T cell activation.

The key molecular mechanisms that control signaling via T cell antigen receptors (TCRs) remain to be fully elucidated. Here we found that Nrdp1, a ring finger-type E3 ligase, mediated Lys33 (K33)-linked polyubiquitination of the signaling kinase Zap70 and promoted the dephosphorylation of Zap70 by the acidic phosphatase-like proteins Sts1 and Sts2 and thereby terminated early TCR signaling in CD8(+) T cells. Nrdp1 deficiency significantly promoted the activation of naive CD8(+) T cells but not that of naive CD4(+) T cells after engagement of the TCR. Nrdp1 interacted with Zap70 and with Sts1 and Sts2 and connected K33 linkage of Zap70 to Sts1- and Sts2-mediated dephosphorylation. Our study suggests that Nrdp1 terminates early TCR signaling by inactivating Zap70 and provides new mechanistic insights into the non-proteolytic regulation of TCR signaling by E3 ligases.

Ubiquitin-specific protease 2b negatively regulates IFN-ß production and antiviral activity by targeting TANK-binding kinase 1.

TANK-binding kinase 1 (TBK1) is essential for IFN regulatory factor 3 activation and IFN-ß production downstream of various innate receptors. However, how TBK1 activation is terminated is not well defined. In this study, we identified ubiquitin-specific protease (USP) 2b as a new negative regulator for TBK1 activation. Overexpression of USP2b inhibited retinoic acid-inducible gene-I-mediated IFN-ß signaling; in contrast, knockdown of USP2b expression by small interfering RNA enhanced retinoic acid-inducible gene-I-mediated IFN-ß signaling. Coimmunoprecipitation experiments demonstrated that USP2b interacted with TBK1. As a deubiquitinating enzyme, USP2b was demonstrated to cleave K63-linked polyubiquitin chains from TBK1 to inhibit TBK1 kinase activity. Consistent with the inhibitory roles of USP2b on TBK1 activation, knockdown of USP2b significantly inhibited the replication of vesicular stomatitis virus, whereas overexpression of USP2b resulted in enhanced replication of vesicular stomatitis virus. Therefore, our findings demonstrated that USP2b deubiquitinates K63-linked polyubiquitin chains from TBK1 to terminate TBK1 activation and negatively regulate IFN-ß signaling and antiviral immune response.

Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response.

UNLABELLED: Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE: RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.

The endoplasmic reticulum acts as a platform for ubiquitylated components of nuclear factor κB signaling.

The innate and adaptive immune responses involve the stimulation of nuclear factor κB (NF-κB) transcription factors through the Lys(63) (K(63))-linked ubiquitylation of specific components of NF-κB signaling pathways. We found that ubiquitylated components of the NF-κB pathway accumulated on the cytosolic leaflet of the endoplasmic reticulum (ER) membrane after the engagement of cell-surface, proinflammatory cytokine receptors or antigen receptors. Through mass spectrometric analysis, we found that the ER-anchored protein metadherin (MTDH) was a partner for these ubiquitylated activators of NF-κB and that it directly bound to K(63)-linked polyubiquitin chains. Knockdown of MTDH inhibited the accumulation of ubiquitylated NF-κB signaling components at the ER, reduced the extent of NF-κB activation, and decreased the amount of proinflammatory cytokines produced. Our observations highlight an unexpected facet of the ER as a key subcellular gateway for NF-κB activation.

MHC class I antigen presentation of DRiP-derived peptides from a model antigen is not dependent on the AAA ATPase p97.

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

T cell costimulation with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes.

Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.

The E3 ubiquitin ligase MARCH8 negatively regulates IL-1ß-induced NF-κB activation by targeting the IL1RAP coreceptor for ubiquitination and degradation.

The proinflammatory cytokine interleukin-1 (IL-1) signals via type I IL-1 receptor (IL-1RI) and IL-1 receptor accessory protein (IL1RAP), which leads to activation of the transcription factor NF-κB and induction of a range of downstream proteins involved in inflammatory and immune responses. Here, we identified the E3 ubiquitin ligase membrane-associated RING-CH (MARCH8) as a suppressor of IL-1ß-induced NF-κB- and MAPK-activation pathways. Overexpression of MARCH8 inhibits IL-1ß-induced NF-κB and MAPK activation, whereas knockdown of MARCH8 has the opposite effect. Mechanistically, MARCH8 interacts with IL1RAP and targets its Lys512 for K48-linked polyubiquitination and degradation. Our findings suggest that MARCH8-mediated polyubiquitination and degradation of IL1RAP is an important mechanism for negative regulation of IL-1ß-induced signaling pathways.

Using linkage-specific monoclonal antibodies to analyze cellular ubiquitylation.

Antibodies that specifically recognize polyubiquitin chains containing ubiquitins linked at a particular lysine residue are powerful tools for interrogating endogenous protein modifications. Here, we describe protocols for revealing K11-, K48-, and K63-linked polyubiquitin chains by western blotting, immunoprecipitation, or immunostaining.

Impairment of immunoproteasome function by ß5i/LMP7 subunit deficiency results in severe enterovirus myocarditis.

Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 forming an alternative proteasome isoform, the immunoproteasome (IP). The in vivo function of IPs in pathogen-induced inflammation is still a matter of controversy. IPs were mainly associated with MHC class I antigen processing. However, recent findings pointed to a more general function of IPs in response to cytokine stress. Here, we report on the role of IPs in acute coxsackievirus B3 (CVB3) myocarditis reflecting one of the most common viral disease entities among young people. Despite identical viral load in both control and IP-deficient mice, IP-deficiency was associated with severe acute heart muscle injury reflected by large foci of inflammatory lesions and severe myocardial tissue damage. Exacerbation of acute heart muscle injury in this host was ascribed to disequilibrium in protein homeostasis in viral heart disease as indicated by the detection of increased proteotoxic stress in cytokine-challenged cardiomyocytes and inflammatory cells from IP-deficient mice. In fact, due to IP-dependent removal of poly-ubiquitinylated protein aggregates in the injured myocardium IPs protected CVB3-challenged mice from oxidant-protein damage. Impaired NFκB activation in IP-deficient cardiomyocytes and inflammatory cells and proteotoxic stress in combination with severe inflammation in CVB3-challenged hearts from IP-deficient mice potentiated apoptotic cell death in this host, thus exacerbating acute tissue damage. Adoptive T cell transfer studies in IP-deficient mice are in agreement with data pointing towards an effective CD8 T cell immune. This study therefore demonstrates that IP formation primarily protects the target organ of CVB3 infection from excessive inflammatory tissue damage in a virus-induced proinflammatory cytokine milieu.

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.