Astaxanthin Inhibits STING Carbonylation and Enhances Antiviral Responses.

STING-mediated DNA sensing pathway plays a crucial role in the innate antiviral immune responses. Clarifying its regulatory mechanism and searching STING agonists has potential clinical implications. Although multiple STING agonists have been developed to target cancer, there are few for the treatment of infectious diseases. Astaxanthin, a natural and powerful antioxidant, serves many biological functions and as a potential candidate drug for many diseases. However, how astaxanthin combats viruses and whether astaxanthin regulates the cyclic GMP-AMP synthase-STING pathway remains unclear. In this study, we showed that astaxanthin markedly inhibited HSV-1-induced lipid peroxidation and inflammatory responses and enhanced the induction of type I IFN in C57BL/6J mice and mouse primary peritoneal macrophages. Mechanistically, astaxanthin inhibited HSV-1 infection and oxidative stress-induced STING carbonylation and consequently promoted STING translocation to the Golgi apparatus and oligomerization, which activated STING-dependent host defenses. Thus, our study reveals that astaxanthin displays a strong antiviral activity by targeting STING, suggesting that astaxanthin might be a promising STING agonist and a therapeutic target for viral infectious diseases.

S-nitrosothiol homeostasis maintained by ADH5 facilitates STING-dependent host defense against pathogens.

Oxidative (or respiratory) burst confers host defense against pathogens by generating reactive species, including reactive nitrogen species (RNS). The microbial infection-induced excessive RNS damages many biological molecules via S-nitrosothiol (SNO) accumulation. However, the mechanism by which the host enables innate immunity activation during oxidative burst remains largely unknown. Here, we demonstrate that S-nitrosoglutathione (GSNO), the main endogenous SNO, attenuates innate immune responses against herpes simplex virus-1 (HSV-1) and Listeria monocytogenes infections. Mechanistically, GSNO induces the S-nitrosylation of stimulator of interferon genes (STING) at Cys257, inhibiting its binding to the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Alcohol dehydrogenase 5 (ADH5), the key enzyme that metabolizes GSNO to decrease cellular SNOs, facilitates STING activation by inhibiting S-nitrosylation. Concordantly, Adh5 deficiency show defective STING-dependent immune responses upon microbial challenge and facilitates viral replication. Thus, cellular oxidative burst-induced RNS attenuates the STING-mediated innate immune responses to microbial infection, while ADH5 licenses STING activation by maintaining cellular SNO homeostasis.

The UAF1-USP1 Deubiquitinase Complex Stabilizes cGAS and Facilitates Antiviral Responses.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) detects cytoplasmic microbial DNA and self-DNA from genomic instability, initiates innate immunity, and plays fundamental roles in defense against viruses and the development of various diseases. The cellular cGAS level determines the magnitude of the response to DNA. However, the underlying mechanisms of the control of cGAS stability, especially its feedback regulation during viral infection, remain largely unknown. In this study, we show that viral infection induces the expression of the UAF1-USP1 deubiquitinase complex in primary peritoneal macrophages (PMs) of C57BL/6J mice. UAF1-USP interacts with cGAS, selectively cleaves its K48-linked polyubiquitination, and thus stabilizes its protein expression in PMs and HEK293T cells. Concordantly, the UAF1-USP1 deubiquitinase complex enhances cGAS-dependent type I IFN responses in PMs. Uaf1 deficiency and ML323 (a specific inhibitor of UAF1-USP1 deubiquitinase complex) attenuates cGAS-triggered antiviral responses and facilitates viral replication both in vitro and in vivo. Thus, our study uncovers a positive feedback mechanism of cGAS-dependent antiviral responses and suggests the UAF1-USP1 complex as a potential target for the treatment of diseases caused by aberrant cGAS activation.

Polyamine metabolism controls B-to-Z DNA transition to orchestrate DNA sensor cGAS activity.

Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) synthase (cGAS) is a universal double-stranded DNA (dsDNA) sensor that recognizes foreign and self-DNA in the cytoplasm and initiates innate immune responses and has been implicated in various infectious and non-infectious contexts. cGAS binds to the backbone of dsDNA and generates the second messenger, cGAMP, which activates the stimulator of interferon genes (STING). Here, we show that the endogenous polyamines spermine and spermidine attenuated cGAS activity and innate immune responses. Mechanistically, spermine and spermidine induced the transition of B-form DNA to Z-form DNA (Z-DNA), thereby decreasing its binding affinity with cGAS. Spermidine/spermine N1-acetyltransferase 1 (SAT1), the rate-limiting enzyme in polyamine catabolism that decreases the cellular concentrations of spermine and spermidine, enhanced cGAS activation by inhibiting cellular Z-DNA accumulation; SAT1 deficiency promoted herpes simplex virus 1 (HSV-1) replication in vivo. The results indicate that spermine and spermidine induce dsDNA to adopt the Z-form conformation and that SAT1-mediated polyamine metabolism orchestrates cGAS activity.

Posttranslational ISGylation of NLRP3 by HERC enzymes facilitates inflammasome activation in models of inflammation.

The NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is a crucial component of the innate immune system that initiates inflammatory responses. Posttranslational modifications (PTMs) of NLRP3, including ubiquitination and phosphorylation, control inflammasome activation and determine the intensity of inflammation. However, the role of other PTMs in controlling NLRP3 inflammasome activation remains unclear. This study found that TLR priming induced NLRP3 ISGylation (a type of PTM in which ISG15 covalently binds to the target protein) to stabilize the NLRP3 protein. Viral infection, represented by SARS-COV-2 infection, and type I IFNs induced expression of ISG15 and the predominant E3 ISGylation ligases HECT domain- and RCC1-like domain-containing proteins (HERCs; HERC5 in humans and HERC6 in mice). HERCs promoted NLRP3 ISGylation and inhibited K48-linked ubiquitination and proteasomal degradation, resulting in the enhancement of NLRP3 inflammasome activation. Concordantly, Herc6 deficiency ameliorated NLRP3-dependent inflammation as well as hyperinflammation caused by viral infection. The results illustrate the mechanism by which type I IFNs responses control inflammasome activation and viral infection-induced aberrant NLRP3 activation. This work identifies ISGylation as a PTM of NLRP3, revealing a priming target that modulates NLRP3-dependent immunopathology.

Selenoprotein K enhances STING oligomerization to facilitate antiviral response.

Stimulator-of-interferon gene (STING) is a vital element of the innate immune system against DNA viruses. Optimal activation of STING is crucial for maintaining immune homeostasis and eliminating invading viruses, and the oligomerization of STING is an essential prerequisite for STING activation. However, the mechanism of cGAMP-induced STING oligomerization in ER remains unclear. Selenoproteins are crucial for various physiological processes. Here, we identified that the endoplasmic reticulum (ER)-located transmembrane selenoprotein K (SELENOK) was induced during virus infection and facilitated innate immune responses against herpes simplex virus-1 (HSV-1). Mechanistically, SELENOK interacts with STING in the ER and promotes STING oligomerization, which in turn promotes its translocation from the ER to the Golgi. Consequently, Selenok deficiency suppresses STING-dependent innate responses and facilitates viral replication in vivo. Thus, the control of STING activation by selenium-mediated SELENOK expression will be a priming therapeutic strategy for the treatment of STING-associated diseases.

Myristic acid as a checkpoint to regulate STING-dependent autophagy and interferon responses by promoting N-myristoylation.

Stimulator of interferon gene (STING)-triggered autophagy is crucial for the host to eliminate invading pathogens and serves as a self-limiting mechanism of STING-induced interferon (IFN) responses. Thus, the mechanisms that ensure the beneficial effects of STING activation are of particular importance. Herein, we show that myristic acid, a type of long-chain saturated fatty acid (SFA), specifically attenuates cGAS-STING-induced IFN responses in macrophages, while enhancing STING-dependent autophagy. Myristic acid inhibits HSV-1 infection-induced innate antiviral immune responses and promotes HSV-1 replication in mice in vivo. Mechanistically, myristic acid enhances N-myristoylation of ARF1, a master regulator that controls STING membrane trafficking. Consequently, myristic acid facilitates STING activation-triggered autophagy degradation of the STING complex. Thus, our work identifies myristic acid as a metabolic checkpoint that contributes to immune homeostasis by balancing STING-dependent autophagy and IFN responses. This suggests that myristic acid and N-myristoylation are promising targets for the treatment of diseases caused by aberrant STING activation.

TOB1 attenuates IRF3-directed antiviral responses by recruiting HDAC8 to specifically suppress IFN-ß expression.

Interferon regulatory factor 3 (IRF3) is a key transcription factor required for the secretion of type I interferons (IFN-α/ß) and initiation of antiviral immune response. However, the negative feedback regulator of IRF3-directed antiviral response remains unknown. In this study, we demonstrated that viral infection induced the interaction of the transducer of ERBB2.1 (TOB1) with IRF3, which bound to the promoter region of Ifnb1 in macrophages. TOB1 inhibited Ifnb1 transcription by disrupting IRF3 binding and recruiting histone deacetylase 8 (HDAC8) to the Ifnb1 promoter region. Consequently, TOB1 attenuated IRF3-directed IFN-ß expression in virus-infected macrophages. Tob1 deficiency enhanced antiviral response and suppressed viral replication in vivo. Thus, we identified TOB1 as a feedback inhibitor of host antiviral innate immune response and revealed a mechanism underlying viral immune escape.

KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including RIG-I (encoded by Ddx58) and melanoma differentiation-associated gene 5 (MDA5) (encoded by Ifih1), are crucial for initiating antiviral responses. Endogenous retroviral elements (ERVs) are transposable elements derived from exogenous retroviruses that are integrated into the genome. KRAB-associated protein 1 (KAP1) is a key epigenetic suppressor of ERVs that protects cells from detrimental genome instability. Increased ERV transcripts are sensed by RLRs and trigger innate immune signaling. However, whether KAP1 directly controls RLRs activity remains unclear. In this study, we show that KAP1 attenuates RNA viral infection-induced type I IFNs and facilitates viral replication by inhibiting RIG-I/MDA5 expression in primary peritoneal macrophages (PMs) of C57BL/6J mice. Kap1 deficiency increases IFN-ß expression and inhibits vesicular stomatitis virus replication in C57BL/6J mice in vivo. Mechanistically, KAP1 binds to the promoter regions of Ddx58 and Ifih1 and promotes the establishment of repressive histone marks in primary PMs of C57BL/6J mice. Concordantly, KAP1 suppresses the expression of RIG-I and MDA5 at the transcriptional level in primary PMs of C57BL/6J mice. Our results establish that KAP1 epigenetically suppresses host antiviral responses by directly targeting RIG-1 and MDA5, thus facilitating the immune escape of RNA viruses.

TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.

Galectin-9 Targets NLRP3 for Autophagic Degradation to Limit Inflammation.

NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been implicated in a variety of inflammatory disorders, and its activation should be tightly controlled to avoid detrimental effects. NLRP3 protein expression is considered as the rate-limiting step for NLRP3 inflammasome activation. In this study, we show that galectin-9 (encoded by lgals9) attenuated NLRP3 inflammasome activation by promoting the protein degradation of NLRP3 in primary peritoneal macrophages of C57BL/6J mice. Lgals9 deficiency enhances NLRP3 inflammasome activation and promotes NLRP3-dependent inflammation in C57BL/6J mice in vivo. Mechanistically, galectin-9 interacts with NLRP3, promotes the formation of NLRP3/p62 (an autophagic cargo receptor, also known as SQSTM1) complex, and thus facilitates p62-dependent autophagic degradation of NLRP3 in primary peritoneal macrophages of C57BL/6J mice and HEK293T cells. Therefore, we identify galectin-9 as an "eat-me" signal for selective autophagy of NLRP3 and uncover the potential roles of galectins in controlling host protein degradation. Furthermore, our work suggests galectin-9 as a priming therapeutic target for the diseases caused by improper NLRP3 inflammasome activation.

RNF39 mediates K48-linked ubiquitination of DDX3X and inhibits RLR-dependent antiviral immunity.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are major cytosolic RNA sensors and play crucial roles in initiating antiviral innate immunity. Furthermore, RLRs have been implicated in multiple autoimmune disorders. Thus, RLR activation should be tightly controlled to avoid detrimental effects. "DEAD-box RNA helicase 3, X-linked" (DDX3X) is a key adaptor in RLR signaling, but its regulatory mechanisms remain unknown. Here, we show that the E3 ubiquitin ligase RNF39 inhibits RLR pathways through mediating K48-linked ubiquitination and proteasomal degradation of DDX3X. Concordantly, Rnf39 deficiency enhances RNA virus-triggered innate immune responses and attenuates viral replication. Thus, our results uncover a previously unknown mechanism for the control of DDX3X activity and suggest RNF39 as a priming intervention target for diseases caused by aberrant RLR activation.

UAF1 deubiquitinase complexes facilitate NLRP3 inflammasome activation by promoting NLRP3 expression.

NOD-like receptor protein 3 (NLRP3) detects microbial infections or endogenous danger signals and activates the NLRP3 inflammasome, which has important functions in host defense and contributes to the pathogenesis of inflammatory diseases, and thereby needs to be tightly controlled. Deubiquitination of NLRP3 is considered a key step in NLRP3 inflammasome activation. However, the mechanisms by which deubiquitination controls NLRP3 inflammasome activation are unclear. Here, we show that the UAF1/USP1 deubiquitinase complex selectively removes K48-linked polyubiquitination of NLRP3 and suppresses its ubiquitination-mediated degradation, enhancing cellular NLRP3 levels, which are indispensable for subsequent NLRP3 inflammasome assembly and activation. In addition, the UAF1/USP12 and UAF1/USP46 complexes promote NF-κB activation, enhance the transcription of NLRP3 and proinflammatory cytokines (including pro-IL-1ß, TNF, and IL-6) by inhibiting ubiquitination-mediated degradation of p65. Consequently, Uaf1 deficiency attenuates NLRP3 inflammasome activation and IL-1ß secretion both in vitro and in vivo. Our study reveals that the UAF1 deubiquitinase complexes enhance NLRP3 and pro-IL-1ß expression by targeting NLRP3 and p65 and licensing NLRP3 inflammasome activation.

Redox homeostasis maintained by GPX4 facilitates STING activation.

Stimulator-of-interferon genes (STING) is vital for sensing cytosolic DNA and initiating innate immune responses against microbial infection and tumors. Redox homeostasis is the balance of oxidative and reducing reactions present in all living systems. Yet, how the intracellular redox state controls STING activation is unclear. Here, we show that cellular redox homeostasis maintained by glutathione peroxidase 4 (GPX4) is required for STING activation. GPX4 deficiency enhanced cellular lipid peroxidation and thus specifically inhibited the cGAS-STING pathway. Concordantly, GPX4 deficiency inhibited herpes simplex virus-1 (HSV-1)-induced innate antiviral immune responses and promoted HSV-1 replication in vivo. Mechanistically, GPX4 inactivation increased production of lipid peroxidation, which led to STING carbonylation at C88 and inhibited its trafficking from the endoplasmic reticulum (ER) to the Golgi complex. Thus, cellular stress-induced lipid peroxidation specifically attenuates the STING DNA-sensing pathway, suggesting that GPX4 facilitates STING activation by maintaining redox homeostasis of lipids.

USP1-UAF1 deubiquitinase complex stabilizes TBK1 and enhances antiviral responses.

Optimal activation of TANK-binding kinase 1 (TBK1) is crucial for initiation of innate antiviral immunity and maintenance of immune homeostasis. Although several E3 ubiquitin ligases have been reported to regulate TBK1 activation by mediating its polyubiquitination, the functions of deubiquitinase on TBK1 activity remain largely unclear. Here, we identified a deubiquitinase complex, which is formed by ubiquitin specific peptidase 1 (USP1) and USP1-associated factor 1 (UAF1), as a viral infection-induced physiological enhancer of TBK1 expression. USP1-UAF1 complex enhanced TLR3/4 and RIG-I-induced IFN regulatory factor 3 (IRF3) activation and subsequent IFN-ß secretion. Mechanistically, USP1 and UAF1 bound to TBK1, removed its K48-linked polyubiquitination, and then reversed the degradation process of TBK1. Furthermore, we found that ML323, a specific USP1-UAF1 inhibitor, attenuated IFN-ß expression and enhanced viral replication both in vitro and in vivo. Therefore, our results outline a novel mechanism for the control of TBK1 activity and suggest USP1-UAF1 complex as a potential target for the prevention of viral diseases.

The E3 Ubiquitin Ligase TRIM40 Attenuates Antiviral Immune Responses by Targeting MDA5 and RIG-I.

Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), including melanoma differentiation-associated gene 5 (MDA5) and RIG-I, are crucial for host recognition of non-self RNAs, especially viral RNA. Thus, the expression and activation of RLRs play fundamental roles in eliminating the invading RNA viruses and maintaining immune homeostasis. However, how RLR expression is tightly regulated remains to be further investigated. In this study, we identified a major histocompatibility complex (MHC)-encoded gene, tripartite interaction motif 40 (TRIM40), as a suppressor of RLR signaling by directly targeting MDA5 and RIG-I. TRIM40 binds to MDA5 and RIG-I and promotes their K27- and K48-linked polyubiquitination via its E3 ligase activity, leading to their proteasomal degradation. TRIM40 deficiency enhances RLR-triggered signaling. Consequently, TRIM40 deficiency greatly enhances antiviral immune responses and decreases viral replication in vivo. Thus, we demonstrate that TRIM40 limits RLR-triggered innate activation, suggesting TRIM40 as a potential therapeutic target for the control of viral infection.

Post-traumatic stress disorder symptoms in family caregivers of adult patients with acute leukemia from a dyadic perspective.

BACKGROUND: Acute leukemia is a fatal disease in adults that not only affects the patients who suffer from it but also their family caregivers. No studies have investigated post-traumatic stress disorder symptoms (PTSS) in family caregivers of adult patients with acute leukemia using a matched sample. The current study examined PTSS in adult patients with acute leukemia and their family caregivers and investigated the factors associated with caregivers' PTSS. METHODS: A total of 163 patient-caregiver dyads completed questionnaires assessing their PTSS, psychological resilience, and perceived social support. Hierarchical linear regression was used to explore the related factors of caregivers' PTSS. RESULTS: More caregivers than patients met caseness criteria for PTSS (36.8% vs. 18.4%, p < 0.001). Among caregivers, being more closely related to the patients (e.g., spouses and parents), having patients with higher PTSS and having lower psychological resilience were independently associated with more severe PTSS. CONCLUSIONS: Caregivers of acute leukemia patients had significantly more severe PTSS than did their patients. This study is the first to investigate PTSS among family caregivers of adult patients with acute leukemia and its related factors in a matched sample. More attention should be paid to the caregivers of patients with acute leukemia to minimize their PTSS and thus improve mental health of caregivers and reduce potential negative consequences for the patients themselves.

Histone demethylase RBP2 promotes malignant progression of gastric cancer through TGF-ß1-(p-Smad3)-RBP2-E-cadherin-Smad3 feedback circuit.

Some feedback pathways are critical in the process of tumor development or malignant progression. However the mechanisms through which these pathways are epigenetically regulated have not been fully elucidated. Here, we demonstrated that the histone demethylase RBP2 was crucial for TGF-ß1-(p-Smad3)-RBP2-E-cadherin-Smad3 feedback circuit that was implicated in malignant progression of tumors and its knockdown significantly inhibited gastric cancer (GC) metastasis both in vitro and in vivo. Mechanistically, RBP2 can directly bind to E-cadherin promoter and suppress its expression, facilitating EMT and distant metastasis of GC. RBP2 can also be induced by TGF-ß1, a key inducer of EMT, through phosphorylated Smad3 (p-Smad3) pathway in GC. The upregulated RBP2 can be recruited by p-smad3 to E-cadherin promoter and enhance its suppression, contributing to the promotion of metastasis of GC. In addition, the suppression of E-cadherin by RBP2 attenuated inhibition of Smad3 phosphorylation (exerted by E-cadherin), resulting further induction of RBP2 expression, and thus constituting positive feedback regulation during GC malignant progression. This TGF-ß1-(p-Smad3)-RBP2-E-cadherin-Smad3 feedback circuit may be a novel mechanism for GC malignant progression and suppression of RBP2 expression may serve as a new strategy for the prevention of tumor distant metastasis.

Histone demethylase RBP2 induced by Helicobactor Pylori CagA participates in the malignant transformation of gastric epithelial cells.

Gastric epithelial cell malignant transformation induced by Helicobactor Pylori contributes to tumor development, but the underlying mechanisms for this remain unclear. Here we demonstrate that RBP2, a newly identified histone demethylase, can be induced by CagA via PI3K/AKT-Sp1 pathway depending on AKT phosphorylation. Sp1 directly binds to RBP2 promoter and enhances its expression then the upregulated RBP2 significantly increases Cyclin D1 transcription, which contributes to gastric epithelial cell malignant transformation. Further data indicate that knockdown of endogenous RBP2 dominantly inhibits gastric cancer (GC) development both in vitro and in vivo. In conclusion, this CagA- PI3K/AKT-Sp1-RBP2-Cyclin D1 pathway may serve as a novel mechanism for gastric epithelial cell malignant transformation and then gastric cancer (GC). Therefore, RBP2 may link chronic inflammation to tumor development and its inhibition may have potential therapeutic advantages.