Hong-Bing Shu

TRADD-TRAF2 and TRADD-FADD Interactions Define Two Distinct TNF Receptor 1 Signal Transduction Pathways

Tumor necrosis factor (TNF) can induce apoptosis and activate NF-kappa B through signaling cascades emanating from TNF receptor 1 (TNFR1). TRADD is a TNFR1-associated signal transducer that is involved in activating both pathways. Here we show that TRADD directly interacts with TRAF2 and FADD, signal transducers that activate NF-kappa B and induce apoptosis, respectively. A TRAF2 mutant lacking its N-terminal RING finger domain is a dominant-negative inhibitor of TNF-mediated NF-kappa B activation, but does not affect TNF-induced apoptosis. Conversely, a FADD mutant lacking its N-terminal 79 amino acids is a dominant-negative inhibitor of TNF-induced apoptosis, but does not inhibit NF-kappa B activation. Thus, these two TNFR1-TRADD signaling cascades appear to bifurcate at TRADD.

TNF-Dependent Recruitment of the Protein Kinase RIP to the TNF Receptor-1 Signaling Complex

The death domain of tumor necrosis factor (TNF) receptor-1 (TNFR1) triggers distinct signaling pathways leading to apoptosis and NF-kappa B activation through its interaction with the death domain protein TRADD. Here, we show that TRADD interacts strongly with RIP, another death domain protein that was shown previously to associate with Fas antigen. We also show that RIP is a serine-threonine kinase that is recruited by TRADD to TNFR1 in a TNF-dependent process. Overexpression of the intact RIP protein induces both NF-kappa B activation and apoptosis. However, expression of the death domain of RIP Induces apoptosis, but potently inhibits NF-kappa B activation by TNF. These results suggest that distinct domains of RIP participate in the TNF signaling cascades leading to apoptosis and NF-kappa B activation.

The Adaptor Protein MITA Links Virus-Sensing Receptors to IRF3 Transcription Factor Activation

Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

Reduced Competitiveness of Autoantigen-Engaged B Cells due to Increased Dependence on BAFF

Peripheral autoantigen binding B cells are poorly competitive with naive B cells for survival and undergo rapid cell death. However, in monoclonal Ig-transgenic mice lacking competitor B cells, autoantigen binding B cells can survive for extended periods. The basis for competitive elimination of autoantigen binding B cells has been unknown. Here we demonstrate that autoantigen binding B cells have increased dependence on BAFF for survival. In monoclonal Ig-transgenic mice, each autoantigen binding B cell receives elevated amounts of BAFF, exhibiting increased levels of NFkappaB p52 and of the prosurvival kinase Pim2. When placed in a diverse B cell compartment, BAFF receptor engagement and signaling are reduced and the autoantigen binding cells are unable to protect themselves from Bim and possibly other death-promoting factors induced by chronic BCR signaling. These findings indicate that under conditions where BAFF levels are elevated, autoantigen-engaged cells will be rescued from rapid competitive elimination, predisposing to the development of autoimmune disease.

Requirement for Casper (c-FLIP) in Regulation of Death Receptor–Induced Apoptosis and Embryonic Development

Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.

TALL-1 is a novel member of the TNF family that is down-regulated by mitogens.

Members of the tumor necrosis factor (TNF) family play important roles in modulation of immune responses. We describe the identification and cloning of a novel TNF family member that has been designated as TALL‐1. TALL‐1 is a 285‐amino acid type II transmembrane protein. Its carboxy terminus shares ∼35% sequence identity with the recently identified APRIL and ∼20–25% with TNF, FasL, TRAIL, and lymphotoxin‐α, suggesting that TALL‐1 and APRIL belong to a subfamily of the TNF family of ligands. Northern blot analysis suggests that TALL‐1 is expressed abundantly in peripheral blood leukocytes and weakly in spleen but is barely detectable in all other tissues examined. Reverse transcriptase‐polymerase chain reaction analysis indicates that TALL‐1 is specifically expressed in monocytes and macrophages but is undetectable in T and B lymphocytes. Furthermore, TALL‐1 expression is dramatically down‐regulated by phorbol myristate acetate/ionomycin. J. Leukoc. Biol. 65: 680–683; 1999.

Casper Is a FADD- and Caspase-Related Inducer of Apoptosis

Caspases are cysteine proteases that play a central role in apoptosis. Caspase-8 may be the first enzyme of the proteolytic cascade activated by the Fas ligand and tumor necrosis factor (TNF). Caspase-8 is recruited to Fas and TNF receptor-1 (TNF-R1) through interaction of its prodomain with the death effector domain (DED) of the receptor-associating FADD. Here we describe a novel 55 kDa protein, Casper, that has sequence similarity to caspase-8 throughout its length. However, Casper is not a caspase since it lacks several conserved amino acids found in all caspases. Casper interacts with FADD, caspase-8, caspase-3, TRAF1, and TRAF2 through distinct domains. When overexpressed in mammalian cells, Casper potently induces apoptosis. A C-terminal deletion mutant of Casper inhibits TNF- and Fas-induced cell death, suggesting that Casper is involved in these apoptotic pathways.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptors Signal NF-κB and JNK Activation and Apoptosis through Distinct Pathways

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that interacts with several receptors, including TRAIL-R1, TRAIL-R2, and TRAIL-R4. TRAIL-R1 and TRAIL-R2 can induce apoptosis of cancer cells and activate the transcription factor NF-κB. TRAIL-R4 can activate NF-κB and protect cells from TRAIL-induced apoptosis. Here we show that TRAIL-R1-, TRAIL-R2-, and TRAIL-R4-induced NF-κB activation are mediated by a TRAF2-NIK-IκB kinase α/β signaling cascade but is MEKK1 independent. TRAIL receptors also activate the protein kinase JNK. JNK activation by TRAIL-R1 is mediated by a TRAF2-MEKK1-MKK4 but not the TRAF2-NIK/IκB kinase α/β signaling pathway. We also show that activation of NF-κB or overexpression of TRAIL-R4 does not protect TRAIL-R1-induced apoptosis. Moreover, inhibition of NF-κB by IκBα sensitizes cells to tumor necrosis factor- but not TRAIL-induced apoptosis. These findings suggest that TRAIL receptors induce apoptosis, NF-κB and JNK activation through distinct signaling pathways, and activation of NF-κB is not sufficient for protecting cells from TRAIL-induced apoptosis.

The Ubiquitin Ligase RNF5 Regulates Antiviral Responses by Mediating Degradation of the Adaptor Protein MITA

Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

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.