FBXO38 regulates macrophage polarization to control the development of cancer and colitis.

Macrophages are highly plastic cells that differentially regulate multiple pathological conditions, including cancer and autoimmune diseases. In response to various stimuli, macrophages activate different intrinsic signaling pathways and polarize into distinct macrophage subsets. We aimed to identify key new effectors that could control macrophage polarization and impact the development of cancer or colitis. Following treatment with the supernatants of tumor cells, macrophages showed an upregulation in Fbxo38 expression. Subsequently, we further identified that FBXO38 promotes macrophage immunosuppressive function by upregulating the expression of M2-like genes via MAPK and IRF4 signaling without affecting M1-like macrophage polarization. Deletion of Fbxo38 in macrophages was found to block tumor development and protect against DSS-induced colitis. Considering the distinct regulation of tumor development by FBXO38 in T cells and macrophages, we suggest that a comprehensive understanding of FBXO38 function in different cell types is critical for its further translational usage.

WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response.

RIG-I-MAVS signaling pathway plays a crucial role in defending against pathogen infection and maintaining immune balance. Upon detecting viral RNA, RIG-I triggers the formation of prion-like aggregates of the adaptor protein MAVS, which then activates the innate antiviral immune response. However, the mechanisms that regulate the aggregation of MAVS are not yet fully understood. Here, we identified WDR77 as a MAVS-associated protein, which negatively regulates MAVS aggregation. WDR77 binds to MAVS proline-rich region through its WD2-WD3-WD4 domain and inhibits the formation of prion-like filament of recombinant MAVS in vitro. In response to virus infection, WDR77 is recruited to MAVS to prevent the formation of its prion-like aggregates and thus downregulate RIG-I-MAVS signaling in cells. WDR77 deficiency significantly potentiates the induction of antiviral genes upon negative-strand RNA virus infections, and myeloid-specific Wdr77-deficient mice are more resistant to RNA virus infection. Our findings reveal that WDR77 acts as a negative regulator of the RIG-I-MAVS signaling pathway by inhibiting the prion-like aggregation of MAVS to prevent harmful inflammation.

The Endoplasmic Reticulum ATP13A1 is Essential for MAVS-Mediated Antiviral Innate Immunity.

RIG-I-MAVS signaling pathway is essential for efficient innate immune response against virus infection. Though many components have been identified in RIG-I pathway and it can be partially reconstituted in vitro, detailed mechanisms involved in cells are still unclear. Here, a genome-wide CRISPR-Cas9 screen is performed using an engineered cell line IFNB-P2A-GSDMD-N, and ATP13A1, a putative dislocase located on the endoplasmic reticulum, is identified as an important regulator of RIG-I pathway. ATP13A1 deficiency abolishes RIG-I-mediated antiviral innate immune response due to compromised MAVS stability and crippled signaling potency of residual MAVS. Moreover, it is discovered that MAVS is subject to protease-mediated degradation in the absence of ATP13A1. As homozygous Atp13a1 knockout mice result in developmental retardation and embryonic lethality, Atp13a1 conditional knockout mice are generated. Myeloid-specific Atp13a1-deficient mice are viable and susceptible to RNA virus infection. Collectively, the findings reveal that ATP13A1 is indispensable for the stability and activation of MAVS and a proper antiviral innate immune response.

Human STING Is Regulated by an Autoinhibitory Mechanism for Type I Interferon Production.

Stimulator of interferon genes (STING) plays a pivotal role in type I interferon-mediated innate immune response to the cytoplasmic detection of aberrant DNA. STING is a membrane protein localized in endoplasmic reticulum (ER), which upon stimulation translocates to Golgi apparatus and activates downstream signaling cascades. However, the mechanism regulating STING activity and significance of its intracellular traffic are not completely understood. Here we identify a novel region of human STING comprising thirteen residues within its C-terminal tail (CTT) for downstream nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation. We also discover that STING CTT fragment can activate downstream signaling regardless of its ER localization. In addition, we reveal that ligand-binding domain (LBD) in the middle of STING binds and confers autoinhibition to its CTT for both NF-κB- and interferon regulatory factor 3-activation. Furthermore, STING LBD can inhibit the interferon-stimulating activity of STING CTT in trans and demonstrate a dominant negative effect on endogenous STING for interferon induction. We thus uncover an important autoinhibitory mechanism modulating STING activity.

TRIM24 facilitates antiviral immunity through mediating K63-linked TRAF3 ubiquitination.

Ubiquitination is an essential mechanism in the control of antiviral immunity upon virus infection. Here, we identify a series of ubiquitination-modulating enzymes that are modulated by vesicular stomatitis virus (VSV). Notably, TRIM24 is down-regulated through direct transcriptional suppression induced by VSV-activated IRF3. Reducing or ablating TRIM24 compromises type I IFN (IFN-I) induction upon RNA virus infection and thus renders mice more sensitive to VSV infection. Mechanistically, VSV infection induces abundant TRIM24 translocation to mitochondria, where TRIM24 binds with TRAF3 and directly mediates K63-linked TRAF3 ubiquitination at K429/K436. This modification of TRAF3 enables its association with MAVS and TBK1, which consequently activates downstream antiviral signaling. Together, these findings establish TRIM24 as a critical positive regulator in controlling the activation of antiviral signaling and describe a previously unknown mechanism of TRIM24 function.

TRAF3IP3 mediates the recruitment of TRAF3 to MAVS for antiviral innate immunity.

RIG-I-MAVS antiviral signaling represents an important pathway to stimulate interferon production and confer innate immunity to the host. Upon binding to viral RNA and Riplet-mediated polyubiquitination, RIG-I promotes prion-like aggregation and activation of MAVS. MAVS subsequently induces interferon production by activating two signaling pathways mediated by TBK1-IRF3 and IKK-NF-κB respectively. However, the mechanism underlying the activation of MAVS downstream pathways remains elusive. Here, we demonstrated that activation of TBK1-IRF3 by MAVS-Region III depends on its multimerization state and identified TRAF3IP3 as a critical regulator for the downstream signaling. In response to virus infection, TRAF3IP3 is accumulated on mitochondria and thereby facilitates the recruitment of TRAF3 to MAVS for TBK1-IRF3 activation. Traf3ip3-deficient mice demonstrated a severely compromised potential to induce interferon production and were vulnerable to RNA virus infection. Our findings uncover that TRAF3IP3 is an important regulator for RIG-I-MAVS signaling, which bridges MAVS and TRAF3 for an effective antiviral innate immune response.

Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity.

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination.

Cbl Ubiquitin Ligases Control B Cell Exit from the Germinal-Center Reaction.

Selective expansion of high-affinity antigen-specific B cells in germinal centers (GCs) is a key event in antibody affinity maturation. GC B cells with improved affinity can either continue affinity-driven selection or exit the GC to differentiate into plasma cells (PCs) or memory B cells. Here we found that deleting E3 ubiquitin ligases Cbl and Cbl-b (Cbls) in GC B cells resulted in the early exit of high-affinity antigen-specific B cells from the GC reaction and thus impaired clonal expansion. Cbls were highly expressed in GC light zone (LZ) B cells, where they promoted the ubiquitination and degradation of Irf4, a transcription factor facilitating PC fate choice. Strong CD40 and BCR stimulation triggered the Cbl degradation, resulting in increased Irf4 expression and exit from GC affinity selection. Thus, a regulatory cascade that is centered on the Cbl ubiquitin ligases ensures affinity-driven clonal expansion by connecting BCR affinity signals with differentiation programs.

MLL5 suppresses antiviral innate immune response by facilitating STUB1-mediated RIG-I degradation.

Trithorax group protein MLL5 is an important epigenetic modifier that controls cell cycle progression, chromatin architecture maintenance, and hematopoiesis. However, whether MLL5 has a role in innate antiviral immunity is largely unknown. Here we show that MLL5 suppresses the RIG-I-mediated anti-viral immune response. Mll5-deficient mice infected with vesicular stomatitis virus show enhanced anti-viral innate immunity, reduced morbidity, and viral load. Mechanistically, a fraction of MLL5 located in the cytoplasm interacts with both RIG-I and its E3 ubiquitin ligase STUB1, which promotes K48-linked polyubiquitination and proteasomal degradation of RIG-I. MLL5 deficiency attenuates the RIG-I and STUB1 association, reducing K48-linked polyubiquitination and accumulation of RIG-I protein in cells. Upon virus infection, nuclear MLL5 protein translocates from the nucleus to the cytoplasm inducing STUB1-mediated degradation of RIG-I. Our study uncovers a previously unrecognized role for MLL5 in antiviral innate immune responses and suggests a new target for controlling viral infection.

Multiple truncated isoforms of MAVS prevent its spontaneous aggregation in antiviral innate immune signalling.

In response to virus infection, RIG-I-like receptors (RLRs) sense virus RNA and induce MAVS to form prion-like aggregates to further propagate antiviral signalling. Although monomeric MAVS recombinant protein can assemble into prion-like filaments spontaneously in vitro, endogenous MAVS in cells is prevented from aggregation until viral infection. The mechanism preventing cellular MAVS from spontaneous aggregation is unclear. Here we show that multiple N-terminal truncated isoforms of MAVS are essential in preventing full-length MAVS from spontaneous aggregation through transmembrane domain-mediated homotypic interaction. Without these shorter isoforms, full-length MAVS is prone to spontaneous aggregation and Nix-mediated mitophagic degradation. In the absence of N-terminally truncated forms, blocking Nix-mediated mitophagy stabilizes full-length MAVS, which aggregates spontaneously and induces the subsequent expression of type I interferon and other proinflammatory cytokines. Our data thus uncover an important mechanism preventing spontaneous aggregation of endogenous MAVS to avoid accidental activation of antiviral innate immune signalling.

Ube2D3 and Ube2N are essential for RIG-I-mediated MAVS aggregation in antiviral innate immunity.

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.

Mixed Lineage Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7).

Mixed lineage leukemia 5 (MLL5) protein is a trithorax family histone 3 lysine 4 (H3K4) methyltransferase that regulates diverse biological processes, including cell cycle progression, hematopoiesis and cancer. The mechanisms by which MLL5 protein stability is regulated have remained unclear to date. Here, we showed that MLL5 protein stability is cooperatively regulated by O-GlcNAc transferase (OGT) and ubiquitin-specific protease 7 (USP7). Depletion of OGT in cells led to a decrease in the MLL5 protein level through ubiquitin/proteasome-dependent proteolytic degradation, whereas ectopic expression of OGT protein suppressed MLL5 ubiquitylation. We further identified deubiquitinase USP7 as a novel MLL5-associated protein using mass spectrometry. USP7 stabilized the MLL5 protein through direct binding and deubiquitylation. Loss of USP7 induced degradation of MLL5 protein. Conversely, overexpression of USP7, but not a catalytically inactive USP7 mutant, led to decreased ubiquitylation and increased MLL5 stability. Co-immunoprecipitation and co-immunostaining assays revealed that MLL5, OGT and USP7 interact with each other to form a stable ternary complex that is predominantly located in the nucleus. In addition, upregulation of MLL5 expression was correlated with increased expression of OGT and USP7 in human primary cervical adenocarcinomas. Our results collectively reveal a novel molecular mechanism underlying regulation of MLL5 protein stability and provide new insights into the functional interplay among O-GlcNAc transferase, deubiquitinase and histone methyltransferase.

An autoinhibitory mechanism modulates MAVS activity in antiviral innate immune response.

In response to virus infection, RIG-I senses viral RNA and activates the adaptor protein MAVS, which then forms prion-like filaments and stimulates a specific signalling pathway leading to type I interferon production to restrict virus proliferation. However, the mechanisms by which MAVS activity is regulated remain elusive. Here we identify distinct regions of MAVS responsible for activation of transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These IRF3- and NF-κB-stimulating regions recruit preferential TNF receptor-associated factors (TRAFs) for downstream signalling. Strikingly, these regions' activities are inhibited by their respective adjacent regions in quiescent MAVS. Our data thus show that an autoinhibitory mechanism modulates MAVS activity in unstimulated cells and, on viral infection, individual regions of MAVS are released following MAVS filament formation to activate antiviral signalling cascades.

Structural basis for the prion-like MAVS filaments in antiviral innate immunity.

Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001.

MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.

In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. [corrected] We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade.

A novel acetylation of ß-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation.

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of ß-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of ß-tubulin localizes at the interface of α-/ß-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.

Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity.

RIG-I detects invading viral RNA and activates the transcription factors NF-kappaB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5'-triphosphate strongly activates the RIG-I-IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria, and cytosol. Activation of RIG-I requires not only RNA but also polyubiquitin chains linked through lysine 63 (K63) of ubiquitin. RIG-I binds specifically to K63-polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63-ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a ubiquitin sensor, and demonstrate that unanchored K63-polyubiquitin chains are signaling molecules in antiviral innate immunity.