Zhonghe Zhai

ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization

We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non–self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

Cytochrome c release and caspase activation during menadione-induced apoptosis in plants

We report here the detection of the release of cytochrome c from mitochondria into the cytosol during menadione‐induced apoptosis in tobacco protoplasts. Western blot analysis indicated that the caspase specific inhibitors AC‐DEVD‐CHO (Ac‐Asp‐Glu‐Val‐Asp‐aldehyde) and AC‐YVAD‐CHO (N‐acetyl‐Try‐Val‐Ala‐aspartinal) inhibited the degradation of a caspase 3 specific substrate PARP (poly(ADP‐ribose) polymerase), and they had no effect on the release of cytochrome c. Further study showed that menadione could not induce apoptosis of mouse liver nuclei in tobacco cytosol extract containing no mitochondria. However, when cytochrome c or mitochondria was added into the cytosol extract, apoptosis of mouse liver nuclei and the degradation of PARP could both be detected. The results provide strong evidence that menadione can induce apoptosis in tobacco protoplasts via the release of cytochrome c from mitochondria into the cytosol.

A20 is a potent inhibitor of TLR3- and Sendai virus-induced activation of NF-κB and ISRE and IFN-β promoter

Toll‐like receptor 3 (TLR3) recognizes dsRNA generated during viral infection and activation of TLR3 results in induction of type I interferons (IFNs) and cellular anti‐viral response. TLR3 is associated with a TIR domain‐containing adapter protein TRIF, which activates distinct downstream pathways leading to activation of NF‐κB and ISRE sites in the promoters of type I IFNs. We show here that A20, a NF‐κB‐inducible zinc finger protein that has been demonstrated to be an inhibitor of TNF‐induced NF‐κB activation and a physiological suppressor of inflammatory response, potently inhibited TLR3‐ and Sendai virus‐mediated activation of ISRE and NF‐κB and IFN‐β promoter in reporter gene assays. A20 also inhibited TRIF‐, but not its downstream signaling components TBK1‐, IKKβ‐, and IKKε‐mediated activation of ISRE and NF‐κB and IFN‐β promoter. Moreover, A20 interacted with TRIF in co‐immunoprecipitation experiments. Finally, expression of A20 could be induced at protein level by Sendai virus infection. These data suggest that A20 targets TRIF to inhibit TLR3‐mediated induction of IFN‐β transcription and functions as a feedback negative regulator for TLR3 signaling and cellular anti‐viral response.

SIKE is an IKKε/TBK1‐associated suppressor of TLR3‐ and virus‐triggered IRF‐3 activation pathways

Viral infection or TLR3 engagement causes activation of the transcription factors IRF‐3 and NF‐κB, which collaborate to induce transcription of type I IFN genes. IKKε and TBK1 are two IKK‐related kinases critically involved in virus‐ and TLR3‐triggered activation of IRF‐3. We identified a protein termed SIKE (for Suppressor of IKKε) that interacts with IKKε and TBK1. SIKE is associated with TBK1 under physiological condition and dissociated from TBK1 upon viral infection or TLR3 stimulation. Overexpression of SIKE disrupted the interactions of IKKε or TBK1 with TRIF, RIG‐I and IRF‐3, components in virus‐ and TLR3‐triggered IRF‐3 activation pathways, but did not disrupt the interactions of TRIF with TRAF6 and RIP, components in TLR3‐triggered NF‐κB activation pathway. Consistently, overexpression of SIKE inhibited virus‐ and TLR3‐triggered interferon‐stimulated response elements (ISRE) but not NF‐κB activation. Knockdown of SIKE potentiated virus‐ and TLR3‐triggered ISRE but not NF‐κB activation. Moreover, overexpression of SIKE inhibited IKKε‐ and TBK1‐mediated antiviral response. These findings suggest that SIKE is a physiological suppressor of IKKε and TBK1 and plays an inhibitory role in virus‐ and TLR3‐triggered IRF‐3 but not NF‐κB activation pathways.

Negative regulation of MDA5- but not RIG-I-mediated innate antiviral signaling by the dihydroxyacetone kinase

Viral infection leads to activation of the transcription factors interferon regulatory factor-3 and NF-κB, which collaborate to induce type I IFNs. The RNA helicase proteins RIG-I and MDA5 were recently identified as two cytoplasmic viral RNA sensors that recognize different species of viral RNAs produced during viral replication. In this study, we identified DAK, a functionally unknown dihydroacetone kinase, as a specific MDA5-interacting protein. DAK was associated with MDA5, but not RIG-I, under physiological conditions. Overexpression of DAK inhibited MDA5- but not RIG-I- or TLR3-mediated IFN-β induction. Overexpression of DAK also inhibited cytoplasmic dsRNA and SeV-induced activation of the IFN-β promoter, whereas knockdown of endogenous DAK by RNAi activated the IFN-β promoter, and increased cytoplasmic dsRNA- or SeV-triggered activation of the IFN-β promoter. In addition, overexpression of DAK inhibited MDA5- but not RIG-I-mediated antiviral activity, whereas DAK RNAi increased cytoplasmic dsRNA-triggered antiviral activity. These findings suggest that DAK is a physiological suppressor of MDA5 and specifically inhibits MDA5- but not RIG-I-mediated innate antiviral signaling.

Negative feedback regulation of cellular antiviral signaling by RBCK1-mediated degradation of IRF3

Viral infection causes host cells to produce type I interferons (IFNs), which are critically involved in viral clearance. Previous studies have demonstrated that activation of the transcription factor interferon regulatory factor (IRF)3 is essential for virus-triggered induction of type I IFNs. Here we show that the E3 ubiquitin ligase RBCC protein interacting with PKC1 (RBCK1) catalyzes the ubiquitination and degradation of IRF3. Overexpression of RBCK1 negatively regulates Sendai virus-triggered induction of type I IFNs, while knockdown of RBCK1 has the opposite effect. Plaque assays consistently demonstrate that RBCK1 negatively regulates the cellular antiviral response. Furthermore, viral infection leads to induction of RBCK1 and subsequent degradation of IRF3. These findings suggest that the cellular antiviral response is controlled by a negative feedback regulatory mechanism involving RBCK1-mediated ubiquitination and degradation of IRF3.

REUL Is a Novel E3 Ubiquitin Ligase and Stimulator of Retinoic-Acid-Inducible Gene-I

RIG-I and MDA5 are cytoplasmic sensors that recognize different species of viral RNAs, leads to activation of the transcription factors IRF3 and NF-κB, which collaborate to induce type I interferons. In this study, we identified REUL, a RING-finger protein, as a specific RIG-I-interacting protein. REUL was associated with RIG-I, but not MDA5, through its PRY and SPRY domains. Overexpression of REUL potently potentiated RIG-I-, but not MDA5-mediated downstream signalling and antiviral activity. In contrast, the RING domain deletion mutant of REUL suppressed Sendai virus (SV)-induced, but not cytoplasmic polyI:C-induced activation of IFN-β promoter. Knockdown of endogenous REUL by RNAi inhibited SV-triggered IFN-β expression, and also increased VSV replication. Full-length RIG-I, but not the CARD domain deletion mutant of RIG-I, underwent ubiquitination induced by REUL. The Lys 154, 164, and 172 residues of the RIG-I CARD domain were critical for efficient REUL-mediated ubiquitination, as well as the ability of RIG-I to induce activation of IFN-β promoter. These findings suggest that REUL is an E3 ubiquitin ligase of RIG-I and specifically stimulates RIG-I-mediated innate antiviral activity.

Lamin B receptor plays a role in stimulating nuclear envelope production and targeting membrane vesicles to chromatin during nuclear envelope assembly through direct interaction with importin β

Lamin B receptor (LBR), a chromatin and lamin binding protein in the inner nuclear membrane, has been proposed to play a vital role in nuclear envelope (NE) assembly. But the specific role for LBR in NE assembly remains unknown. In the present study, we show that overexpression of LBR causes membrane overproduction, inducing NE invagination and membrane stack formation, and that these processes require the transmembrane domain of LBR. Biochemical analysis shows that the N-terminal domain of LBR directly interacts with importin β in a Ran sensitive and importin α independent manner. Using an in vitro NE assembly assay, we also demonstrate that blocking full length LBR binding sites on importin β, by the addition of the LBR N-terminal domain inhibits the recruitment of LBR-containing vesicles to importin β- or Ran-coated beads to form NE structure. Our results suggest that LBR is recruited to chromatin through direct interaction with importin β to contribute to the fusion of membrane vesicles and formation of the NE.

Identification of downstream genes up-regulated by the tumor necrosis factor family member TALL-1

TALL‐1 is a member of the tumor necrosis factor family that binds to BCMA, TACI, and BAFF‐R, three receptors mostly expressed by mature B lymphocytes. Previous studies have shown that the TALL‐1 signaling is critically involved in B cell proliferation, maturation, and progression of lupus‐like, autoimmune diseases. In this report, we performed cDNA subtractive hybridization experiments to identify downstream genes up‐regulated by TALL‐1. These experiments indicated that 10 genes, including interleukin (IL)‐10, lymphocyte activation gene‐1 (LAG‐1), GCP‐2, PBEF, ferritin, PIM‐2, TFG, CD27 ligand, DUSP5, and archain, were up‐regulated at the mRNA level by TALL‐1 stimulation in B lymphoma RPMI‐8226 cells and/or primary B lymphocytes. We also demonstrated that TALL‐1 activated transcription of IL‐10 and LAG‐1 in a nuclear factor‐κB‐dependent manner in reporter gene assays. Moreover, our findings indicated BAFF‐R, but not TACI, could dramatically up‐regulate IL‐10 secretion by RPMI‐8226 cells. The identification of TALL‐1‐up‐regulated genes will help explain the mechanisms of TALL‐1‐triggered biological and pathological effects and to identify molecular targets for intervention of lupus‐like autoimmune diseases.

The Ret Finger Protein Inhibits Signaling Mediated by the Noncanonical and Canonical IκB Kinase Family Members

IFN regulatory factor-3 is a transcription factor that is required for the rapid induction of type I IFNs in the innate antiviral response. Two noncanonical IκB kinase (IKK) family members, IKKε and TRAF family-associated NF-κB activator-binding kinase-1, have been shown to phosphorylate IFN regulatory factor-3 and are critically involved in virus-triggered and TLR3-mediated signaling leading to induction of type I IFNs. In yeast two-hybrid screens for potential IKKε-interacting proteins, we identified Ret finger protein (RFP) as an IKKε-interacting protein. Coimmunoprecipitation experiments indicated that RFP interacted with IKKε and TRAF family-associated NF-κB activator-binding kinase-1 as well as the two canonical IKK family members, IKKβ and IKKα. RFP inhibited activation of the IFN-stimulated response element and/or NF-κB mediated by the IKK family members and triggered by TNF, IL-1, polyinosinic-polycytidylic acid (ligand for TLR3), and viral infection. Moreover, knockdown of RFP expression by RNA interference-enhanced activation of IFN-stimulated response element and/or NF-κB triggered by polyinosinic-polycytidylic acid, TNF, and IL-1. Taken together, our findings suggest that RFP negatively regulates signaling involved in the antiviral response and inflammation by targeting the IKKs.