Ming-Ming Hu

TRIM32 Protein Modulates Type I Interferon Induction and Cellular Antiviral Response by Targeting MITA/STING Protein for K63-linked Ubiquitination

Background: MITA is an adapter protein critically involved in virus-triggered type I IFN induction and cellular antiviral response. Results: The E3 ligase TRIM32 targets MITA for K63-linked ubiquitination and knockdown of TRIM32 inhibits virus-triggered type I IFN induction. Conclusion: TRIM32-mediated K63-linked ubiquitination of MITA is important for cellular antiviral response. Significance: These findings provide insights on the mechanisms of regulation of cellular antiviral response. Viral infection activates several transcription factors including NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. MITA (also called STING) is a critical adaptor protein that links virus-sensing receptors to IRF3 activation upon infection by both RNA and DNA pathogens. Here we show that the E3 ubiquitin ligase tripartite motif protein 32 (TRIM32) ubiquitinated MITA and dramatically enhanced MITA-mediated induction of IFN-β. Overexpression of TRIM32 potentiated virus-triggered IFNB1 expression and cellular antiviral response. Consistently, knockdown of TRIM32 had opposite effects. TRIM32 interacted with MITA, and was located at the mitochondria and endoplasmic reticulum. TRIM32 targeted MITA for K63-linked ubiquitination at K20/150/224/236 through its E3 ubiquitin ligase activity, which promoted the interaction of MITA with TBK1. These findings suggest that TRIM32 is an important regulatory protein for innate immunity against both RNA and DNA viruses by targeting MITA for K63-linked ubiquitination and downstream activation.

TRIM4 modulates type I interferon induction and cellular antiviral response by targeting RIG-I for K63-linked ubiquitination

RIG-I is a pivotal cytoplasmic sensor that recognizes different species of viral RNAs. This recognition leads to activation of the transcription factors NF-κB and IRF3, which collaborate to induce type I interferons (IFNs) and innate antiviral response. In this study, we identified the TRIM family protein TRIM4 as a positive regulator of RIG-I-mediated IFN induction. Overexpression of TRIM4 potentiated virus-triggered activation of IRF3 and NF-κB, as well as IFN-β induction, whereas knockdown of TRIM4 had opposite effects. Mechanistically, TRIM4 associates with RIG-I and targets it for K63-linked polyubiquitination. Our findings demonstrate that TRIM4 is an important regulator of the virus-induced IFN induction pathways by mediating RIG-I for K63-linked ubiquitination.

RNF26 Temporally Regulates Virus-Triggered Type I Interferon Induction by Two Distinct Mechanisms

Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms.

Sumoylation Promotes the Stability of the DNA Sensor cGAS and the Adaptor STING to Regulate the Kinetics of Response to DNA Virus

During viral infection, sensing of cytosolic DNA by the cyclic GMP-AMP synthase (cGAS) activates the adaptor protein STING and triggers an antiviral response. Little is known about the mechanisms that determine the kinetics of activation and deactivation of the cGAS-STING pathway, ensuring effective but controlled innate antiviral responses. Here we found that the ubiquitin ligase Trim38 targets cGas for sumoylation in uninfected cells and during the early phase of viral infection. Sumoylation of cGas prevented its polyubiquitination and degradation. Trim38 also sumoylated Sting during the early phase of viral infection, promoting both Sting activation and protein stability. In the late phase of infection, cGas and Sting were desumoylated by Senp2 and subsequently degraded via proteasomal and chaperone-mediated autophagy pathways, respectively. Our findings reveal an essential role for Trim38 in the innate immune response to DNA virus and provide insight into the mechanisms that ensure optimal activation and deactivation of the cGAS-STING pathway.

TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

Significance Infection of pathogenic microbes induces the body to produce cytokines, which are mediators of inflammation. TNFα and IL-1β are two important proinflammatory cytokines that trigger a series of cellular reactions, leading to induction of downstream genes and inflammation. Understanding how the cellular reactions are triggered by the proinflammatory cytokines is important for deciphering the molecular mechanisms of inflammation. In this study, we identified a protein called TRIM38, which negatively regulates TNFα- and IL-1β–triggered cellular reactions by causing degradation of TAB2/3, two cellular components essential for TNFα- and IL-1β–triggered cellular response. This study reveals a mechanism by which cells keep the inflammatory response in check to avoid excessive harmful immune response and may provide clues on treatments of inflammation. TNFα and IL-1β are two proinflammatory cytokines that play critical roles in many diseases, including rheumatoid arthritis and infectious diseases. How TNFα- and IL-1β–mediated signaling is finely tuned is not fully elucidated. Here, we identify tripartite-motif protein 38 (TRIM38) as a critical negative regulator of TNFα- and IL-1β–triggered signaling. Overexpression of TRIM38 inhibited activation of NF-κB and induction of downstream cytokines following TNFα and IL-1β stimulation, whereas knockdown or knockout of TRIM38 had the opposite effects. TRIM38 constitutively interacted with critical components TGF-β–activated kinase 1 (TAK1)-binding protein 2/3 (TAB2/3) and promoted lysosome-dependent degradation of TAB2/3 independent of its E3 ubiquitin ligase activity. Consistently, deficiency of TRIM38 resulted in abolished translocation of TAB2 to the lysosome, increased level of TAB2 in cells, and enhanced activation of TAK1 after TNFα and IL-1β stimulation. We conclude that TRIM38 negatively regulates TNFα- and IL-1β–induced signaling by mediating lysosome-dependent degradation of TAB2/3, two critical components in TNFα- and IL-1β–induced signaling pathways. Our findings reveal a previously undiscovered mechanism by which cells keep the inflammatory response in check to avoid excessive harmful immune response triggered by TNFα and IL-1β.

TRIM38 Negatively Regulates TLR3/4-Mediated Innate Immune and Inflammatory Responses by Two Sequential and Distinct Mechanisms

Tripartite motif (TRIM)38 is an E3 ubiquitin ligase that was reported to regulate signaling in innate immune and inflammatory responses in certain cell lines. In this study, we show that Trim38 deficiency markedly increased TLR3- and TLR4-mediated induction of type I IFNs and proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in immune cells and in vivo. Trim38 deficiency also caused the mice to be more susceptible to death triggered by polyinosinic-polycytidylic acid, LPS, and Salmonella typhimurium. Mechanistically, TRIM38 catalyzed K48-linked polyubiquitination of the TLR3/4 adapter protein TIR domain–containing adapter-inducing IFN-β at K228 and promoted its proteasomal degradation in immune cells. Moreover, Trim38 was highly induced by type I IFNs, which then negatively regulated TNF-α/IL-1β signaling in IFN-β–primed immune cells, but not unprimed immune cells, by mediating degradation of Tab2 in a lysosomal-dependent process. These results suggest that Trim38 negatively regulates TLR3/4-mediated innate immune and inflammatory responses by two sequential and distinct mechanisms. This study increases our understanding of how the innate immune response is initiated during the early phase of infection to defend against microbial invasion and is efficiently terminated during the late phase to prevent excessive and harmful inflammatory responses.

Death-associated protein kinase 1 is an IRF3/7-interacting protein that is involved in the cellular antiviral immune response

Interferon regulatory factor (IRF) 7 has been demonstrated to be a master regulator of virus-induced type I interferon production (IFN), and it plays a central role in the innate immune response against viruses. Here, we identified death-associated protein kinase 1 (DAPK1) as an IRF7-interacting protein by tandem affinity purification (TAP). Viral infection induced DAPK1–IRF7 and DAPK1–IRF3 interactions and overexpression of DAPK1 enhanced virus-induced activation of the interferon-stimulated response element (ISRE) and IFN-β promoters and the expression of the IFNB1 gene. Knockdown of DAPK1 attenuated the induction of IFNB1 and RIG-I expression triggered by viral infection or IFN-β, and they were enhanced by viral replication. In addition, viral infection or IFN-β treatment induced the expression of DAPK1. IFN-β treatment also activated DAPK1 by decreasing its phosphorylation level at serine 308. Interestingly, the involvement of DAPK1 in virus-induced signaling was independent of its kinase activity. Therefore, our study identified DAPK1 as an important regulator of the cellular antiviral response.

Innate immunity to RNA virus is regulated by temporal and reversible sumoylation of RIG-I and MDA5

Sensing of viral RNA by the cytosolic receptors RIG-I and melanoma differentiation-associated gene 5 (MDA5) leads to innate antiviral response. How RIG-I and MDA5 are dynamically regulated in innate antiviral response is not well understood. Here, we show that TRIM38 positively regulates MDA5- and RIG-I–mediated induction of downstream genes and acts as a SUMO E3 ligase for their dynamic sumoylation at K43/K865 and K96/K888, respectively, before and after viral infection. The sumoylation of MDA5 and RIG-I suppresses their K48-linked polyubiquitination and degradation in uninfected or early-infected cells. Sumoylation of the caspase recruitment domains of MDA5 and RIG-I is also required for their dephosphorylation by PP1 and activation upon viral infection. At the late phase of viral infection, both MDA5 and RIG-I are desumoylated by SENP2, resulting in their K48-linked polyubiquitination and degradation. These findings suggest that dynamic sumoylation and desumoylation of MDA5 and RIG-I modulate efficient innate immunity to RNA virus and its timely termination.

Multifaceted roles of TRIM38 in innate immune and inflammatory responses

The tripartite motif-containing (TRIM) proteins represent the largest E3 ubiquitin ligase family. The multifaceted roles of TRIM38 in innate immunity and inflammation have been intensively investigated in recent years. TRIM38 is essential for cytosolic RNA or DNA sensor-mediated innate immune responses to both RNA and DNA viruses, while negatively regulating TLR3/4- and TNF/IL-1β-triggered inflammatory responses. In these processes, TRIM38 acts as an E3 ubiquitin or SUMO ligase, which targets key cellular signaling components, or as an enzymatic activity-independent regulator. This review summarizes recent advances that highlight the critical roles of TRIM38 in the regulation of proper innate immune and inflammatory responses.

MSX1 Modulates RLR-Mediated Innate Antiviral Signaling by Facilitating Assembly of TBK1-Associated Complexes

Recognition of viral dsRNA by the retinoic acid–inducible gene-1–like receptors (RLRs) triggers signaling cascades that lead to activation of the TBK1 kinase and transcription factor IFN regulatory factor 3, induction of downstream antiviral genes, and innate antiviral responses. In this study, we identified muscle segment homeobox1 (MSX1) as an important modulator of RLR-mediated signaling pathways. Knockdown or knockout of MSX1 significantly impaired Sendai virus–triggered activation of TBK1 and IFN regulatory factor 3, induction of downstream antiviral genes, and cellular antiviral responses. Interestingly, MSX1 was translocated from the nucleus to cytoplasm, particularly mitochondria upon infection of Sendai virus. Biochemcially, MSX1 was important for assembly of TBK1/IKK-related kinase-associated protein 1/TNFR-associated factor-associated NF-κB activator complexes. Our results suggest that MSX1 is an important component of RLR-mediated signaling and reveal mechanisms on innate immune responses against RNA viruses.