E3 ligase HECTD3 promotes RNA virus replication and virus-induced inflammation via K33-linked polyubiquitination of PKR.

Uncontrolled viral replication and excessive inflammation are the main causes of death in the host infected with virus. Hence inhibition of intracellular viral replication and production of innate cytokines, which are the key strategies of hosts to fight virus infections, need to be finely tuned to eliminate viruses while avoid harmful inflammation. The E3 ligases in regulating virus replication and subsequent innate cytokines production remain to be fully characterized. Here we report that the deficiency of the E3 ubiquitin-protein ligase HECTD3 results in accelerated RNA virus clearance and reduced inflammatory response both in vitro and in vivo. Mechanistically, HECTD3 interacts with dsRNA-dependent protein kinase R (PKR) and mediates Lys33-linkage of PKR, which is the first non-proteolytic ubiquitin modification for PKR. This process disrupts the dimerization and phosphorylation of PKR and subsequent EIF2α activation, which results in the acceleration of virus replication, but promotes the formation of PKR-IKK complex and subsequent inflammatory response. The finding suggests HECTD3 is the potential therapeutic target for simultaneously restraining RNA virus replication and virus-induced inflammation once pharmacologically inhibited.

RNA-binding protein ZCCHC4 promotes human cancer chemoresistance by disrupting DNA-damage-induced apoptosis.

RNA-binding proteins (RBPs) play important roles in cancer development and treatment. However, the tumor-promoting RBPs and their partners, which may potentially serve as the cancer therapeutic targets, need to be further identified. Here, we report that zinc finger CCHC domain-containing protein 4 (ZCCHC4) is of aberrantly high expression in multiple human cancer tissues and is associated with poor prognosis and chemoresistance in patients of hepatocellular carcinoma (HCC), pancreatic cancer and colon cancer. ZCCHC4 promotes chemoresistance of HCC cells to DNA-damage agent (DDA) both in vitro and in vivo. HCC cell deficiency of ZCCHC4 reduces tumor growth in vivo and intratumoral interference of ZCCHC4 expression obviously enhances the DDA-induced antitumor effect. Mechanistically, ZCCHC4 inhibits DNA-damage-induced apoptosis in HCC cells by interacting with a new long noncoding RNA (lncRNA) AL133467.2 to hamper its pro-apoptotic function. Also, ZCCHC4 blocks the interaction between AL133467.2 and γH2AX upon DDA treatment to inhibit apoptotic signaling and promote chemoresistance to DDAs. Knockout of ZCCHC4 promotes AL133467.2 and γH2AX interaction for enhancing chemosensitivity in HCC cells. Together, our study identifies ZCCHC4 as a new predictor of cancer poor prognosis and a potential target for improving chemotherapy effects, providing mechanistic insights to the roles of RBPs and their partners in cancer progression and chemoresistance.

Graphene quantum dots rescue angiogenic retinopathy via blocking STAT3/Periostin/ERK signaling.

BACKGROUND: Pathological retinal angiogenesis resulting from a variety of ocular diseases including oxygen induced retinopathy, diabetic retinopathy and ocular vein occlusion, is one of the major reasons for vision loss, yet the therapeutic option is limited. Multiple nanoparticles have been reported to alleviate angiogenic retinopathy. However, the adverse effect cannot be ignored due to the relatively large scale. Graphene quantum dots (GQDs) have shown potential in drug delivery and have been proved biocompatible. In this study, Graphene quantum dots are extensively investigated for their application in angiogenic retinopathy therapy. RESULTS: We showed that GQDs were biocompatible nanomaterials in vitro and in vivo. The nanoparticles have a dose-dependent inhibitory effect on proliferation, migration, tube formation and sprouting of human umbilical vein endothelial cells (HUVECs). Further data show that GQDs could inhibit pathological retinal neovascularization in an oxygen-induced retinopathy (OIR) model. The data of RNA sequencing suggested that periostin is involved in this process. GQDs inhibit the expression of periostin via STAT3, and further regulated cell cycle-related protein levels through ERK pathway. The signaling pathway was conformed in vivo using OIR mouse model. CONCLUSIONS: The present study indicated that GQDs could be a biocompatible anti-angiogenic nanomedicine in the treatment of pathological retinal neovascularization via disrupting periostin/ERK pathway and subsequent cell cycle.

The transmembrane endoplasmic reticulum-associated E3 ubiquitin ligase TRIM13 restrains the pathogenic-DNA-triggered inflammatory response.

The endoplasmic reticulum (ER)-localized stimulator of interferon genes (STING) is the core adaptor for the pathogenic-DNA-triggered innate response. Aberrant activation of STING causes autoinflammatory and autoimmune diseases, raising the concern about how STING is finely tuned during innate response to pathogenic DNAs. Here, we report that the transmembrane domain (TM)-containing ER-localized E3 ubiquitin ligase TRIM13 (tripartite motif containing 13) is required for restraining inflammatory response to pathogenic DNAs. TRIM13 deficiency enhances pathogenic-DNA-triggered inflammatory cytokine production, inhibits DNA virus replication, and causes age-related autoinflammation. Mechanistically, TRIM13 interacts with STING via the TM and catalyzes Lys6-linked polyubiquitination of STING, leading to decelerated ER exit and accelerated ER-initiated degradation of STING. STING deficiency reverses the enhanced innate anti-DNA virus response in TRIM13 knockout mice. Our study delineates a potential strategy for controlling the homeostasis of STING by transmembrane ER-associated TRIM13 during the pathogenic-DNA-triggered inflammatory response.

cGAS-like receptors: ancient catchers of viral nucleic acids.

Living organisms have evolved conserved 'catchers' for detecting and handling molecular patterns from invading viruses. Slavik et al. and Holleufer et al. recently identified cyclic GMP-AMP (cGAMP) synthase (cGAS)-like receptors that detect double-stranded viral RNA in Drosophila. cGAS-like receptors constitute a new expanding family of pattern recognition receptors (PRRs) for viral nucleic acids.

TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO.

Sensing of pathogenic nucleic acids by pattern recognition receptors (PRR) not only initiates anti-microbe defense but causes inflammatory and autoimmune diseases. E3 ubiquitin ligase(s) critical in innate response need to be further identified. Here we report that the tripartite motif-containing E3 ubiquitin ligase TRIM41 is required to innate antiviral response through facilitating pathogenic nucleic acids-triggered signaling pathway. TRIM41 deficiency impairs the production of inflammatory cytokines and type I interferons in macrophages after transfection with nucleic acid-mimics and infection with both DNA and RNA viruses. In vivo, TRIM41 deficiency leads to impaired innate response against viruses. Mechanistically, TRIM41 directly interacts with BCL10 (B cell lymphoma 10), a core component of CARD proteins-BCL10 - MALT1 (CBM) complex, and modifies the Lys63-linked polyubiquitylation of BCL10, which, in turn, hubs NEMO for activation of NF-κB and TANK-binding kinase 1 (TBK1) - interferon regulatory factor 3 (IRF3) pathways. Our study suggests that TRIM41 is the potential universal E3 ubiquitin ligase responsible for Lys63 linkage of BCL10 during innate antiviral response, adding new insight into the molecular mechanism for the control of innate antiviral response.

Publisher Correction: K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8+ T cell activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Small GTPase RBJ promotes cancer progression by mobilizing MDSCs via IL-6.

RBJ has been identified to be dysregulated in gastrointestinal cancer and promotes tumorigenesis and progression by mediating nuclear accumulation of active MEK1/2 and sustained activation of ERK1/2. Considering that nuclear accumulation and constitutive activation of MEK/ERK not only promotes tumor progression directly, but also induces chronic inflammation, we wonder whether and how RBJ impairs host immune-surveillance via chronic inflammation and consequently supports tumor progression. Here, we report that higher expression of RBJ in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. The forced expression of RBJ promotes tumor growth and metastasis both in vitro and in vivo. In addition, more accumulation of immune suppressive cells but less antitumor immune cell subpopulations were found in spleen and tumor tissue derived from RBJ force-expressed tumor-bearing mice. Furthermore, forced RBJ expression significantly promotes tumor cell production of pro-inflammatory cytokine IL-6 by constitutive activating MEK/ERK signaling pathway. Accordingly, RBJ knockdown significantly decreases tumor growth and metastasis in vitro and in vivo, with markedly reduced production of IL-6. Administration of anti-IL-6 neutralizing antibody could reduce MDSCs accumulation in tumor tissue in vivo. Therefore, our results demonstrate that RBJ-mediated nuclear constitutive activation of ERK1/2 leads to persistent production of IL-6 and increase of MDSCs recruitment, contributing to promotion of tumor growth and metastasis. These results suggest that RBJ contributes to tumor immune escape, maybe serving a potential target for design of antitumor drug.

The tyrosine kinase Src promotes phosphorylation of the kinase TBK1 to facilitate type I interferon production after viral infection.

Various pattern recognition receptors (PRRs) are activated in response to viral infection to stimulate the production of type I interferons (IFNs). However, central to the responses of all of these receptors is their activation of the kinase TBK1, which stimulates transcription by IFN regulatory factor 3 (IRF3). We investigated the mechanism by which the kinase activity of TBK1 is stimulated in response to viral infection. We found that the tyrosine kinase Src promoted the phosphorylation of TBK1 on Tyr179 upon viral infection of RAW264.7 macrophages. Mutation of Tyr179 to alanine resulted in impaired autophosphorylation of TBK1 at Ser172, which is required for TBK1 activation. The TBK1 Y179A mutant failed to rescue type I IFN production by virally infected RAW264.7 macrophages deficient in TBK1. Pharmacological inhibition of Src with AZD0530 and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of Src demonstrated that Src was critical for activating the TBK1-IRF3 pathway and stimulating type I IFN production. However, Src did not directly bind to recombinant TBK1 in vitro but instead bound to the proline-X-X-proline motifs within key PRR adaptor proteins, such as TRIF, MAVS, and STING, which formed complexes with TBK1 after PRR engagement. Together, our data suggest that Src is the major tyrosine kinase that primes TBK1 for autophosphorylation and activation, thus providing mechanistic insights into the regulation of TBK1 activity by various PRRs as part of the innate antiviral response.

Extracellular calcium elicits feedforward regulation of the Toll-like receptor-triggered innate immune response.

Despite the expanding knowledge on feedback regulation of Toll-like receptor (TLR) signaling, the feedforward regulation of TLR signaling for the proper innate response to invading microbes is not fully understood. Here, we report that extracellular calcium can coordinate the activation of the small GTPases Ras and Ras-proximate-1 (Rap1) upon TLR stimulation which favors activation of macrophages through a feedforward mechanism. We show that different doses of TLR agonists can trigger different levels of cytokine production, which can be potentiated by extracellular calcium but are impaired by the chelating reagent ethylene glycol tetraacetic acid (EGTA) or by knockdown of stromal interaction molecule 1 (STIM1). Upon TLR engagement, GTP-bound Ras levels are increased and GTP-bound Rap1 is decreased, which can be reversed by EGTA-mediated removal of extracellular calcium. Furthermore, we demonstrate that Rap1 knockdown rescues the inhibitory effects of EGTA on the TLR-triggered innate response. Examination of the TLR signaling pathway reveals that extracellular calcium may regulate the TLR response via feedforward activation of the extracellular signal-regulated kinase signaling pathway. Our data suggest that an influx of extracellular calcium, mediated by STIM1-operated calcium channels, may transmit the information about the intensity of extracellular TLR stimuli to initiate innate responses at an appropriate level. Our study may provide mechanistic insight into the feedforward regulation of the TLR-triggered innate immune response.

Lys29-linkage of ASK1 by Skp1-Cullin 1-Fbxo21 ubiquitin ligase complex is required for antiviral innate response.

Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1-Cul1-F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 (apoptosis signal-regulating kinase 1), and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response.

RNA editing by ADAR1 marks dsRNA as "self".

A paper recently published in Science reports that adenosine deaminase acting on RNA 1-dependent adenosine-to-inosine editing marks endogenous double strand RNA as self and prevents their immune recognition by cytosolic RNA sensor MDA5.