ISG12a promotes immunotherapy of HBV-associated hepatocellular carcinoma through blocking TRIM21/AKT/ß-catenin/PD-L1 axis.

Hepatitis B virus (HBV) infection generally elicits weak type-I interferon (IFN) immune response in hepatocytes, covering the regulatory effect of IFN-stimulated genes. In this study, low level of IFN-stimulated gene 12a (ISG12a) predicted malignant transformation and poor prognosis of HBV-associated hepatocellular carcinoma (HCC), whereas high level of ISG12a indicated active NK cell phenotypes. ISG12a interacts with TRIM21 to inhibit the phosphorylation activation of protein kinase B (PKB, also known as AKT) and ß-catenin, suppressing PD-L1 expression to block PD-1/PD-L1 signaling, thereby enhancing the anticancer effect of NK cells. The suppression of PD-1-deficient NK-92 cells on HBV-associated tumors was independent of ISG12a expression, whereas the anticancer effect of PD-1-expressed NK-92 cells on HBV-associated tumors was enhanced by ISG12a and treatments of atezolizumab and nivolumab. Thus, tumor intrinsic ISG12a promotes the anticancer effect of NK cells by regulating PD-1/PD-L1 signaling, presenting the significant role of innate immunity in defending against HBV-associated HCC.

PZR suppresses innate immune response to RNA viral infection by inhibiting MAVS activation in interferon signaling mediated by RIG-I and MDA5.

RNA viral infections seriously endanger human health. Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2) suppresses innate immunity against influenza A virus, and pharmacological inhibition of SHP2 provokes hepatic innate immunity. SHP2 binds and catalyzes tyrosyl dephosphorylation of protein zero-related (PZR), but the regulatory effect of PZR on innate immune response to viral infection is unclear. In this study, the transcription and protein level of PZR in host cells were found to be decreased with RNA viral infection, and high level of PZR was uncovered to inhibit interferon (IFN) signaling mediated by RIG-I and MDA5. Through localizing in mitochondria, PZR targeted and interacted with MAVS (also known as IPS-1/VISA/Cardif), suppressing the aggregation and activation of MAVS. Specifically, Y263 residue in ITIM is critical for PZR to exert immunosuppression under RNA viral infection. Moreover, the recruited SHP2 by PZR that modified with tyrosine phosphorylation under RNA viral infection might inhibit phosphorylation activation of MAVS. In conclusion, PZR and SHP2 suppress innate immune response to RNA viral infection through inhibiting MAVS activation. This study reveals the regulatory mechanism of PZR-SHP2-MAVS signal axis on IFN signaling mediated by RIG-I and MDA5, which may provide new sight for developing antiviral drugs.

Establishment of cell culture model and humanized mouse model of chronic hepatitis B virus infection.

IMPORTANCE: Approximately 257 million people worldwide have been infected with hepatitis B virus (HBV), and HBV infection can cause chronic hepatitis, cirrhosis, and even liver cancer. The lack of suitable and effective infection models has greatly limited research in HBV-related fields for a long time, and it is still not possible to discover a method to completely and effectively remove the HBV genome. We have constructed a hepatocellular carcinoma cell line, HLCZ01, that can support the complete life cycle of HBV. This model can mimic the long-term stable infection of HBV in the natural state and can replace primary human hepatocytes for the development of human liver chimeric mice. This model will be a powerful tool for research in the field of HBV.

Carboxypeptidase A4 negatively regulates HGS-ETR1/2-induced pyroptosis by forming a positive feedback loop with the AKT signalling pathway.

Pyroptosis, a mode of inflammatory cell death, has recently gained significant attention. However, the underlying mechanism remains poorly understood. HGS-ETR1/2 is a humanized monoclonal antibody that can bind to DR4/5 on the cell membrane and induce cell apoptosis by activating the death receptor signalling pathway. In this study, by using morphological observation, fluorescence double staining, LDH release and immunoblot detection, we confirmed for the first time that HGS-ETR1/2 can induce GSDME-mediated pyroptosis in hepatocellular carcinoma cells. Our study found that both inhibition of the AKT signalling pathway and silencing of CPA4 promote pyroptosis, while the overexpression of CPA4 inhibits it. Furthermore, we identified a positive regulatory feedback loop is formed between CPA4 and AKT phosphorylation. Specifically, CPA4 modulates AKT phosphorylation by regulating the expression of the AKT phosphatase PP2A, while inhibition of the AKT signalling pathway leads to a decreased transcription and translation levels of CPA4. Our study reveals a novel mechanism of pyroptosis induced by HGS-ETR1/2, which may provide a crucial foundation for future investigations into cancer immunotherapy.

3ß-hydroxysteroid-Δ24 reductase dampens anti-viral innate immune responses by targeting K27 ubiquitination of MAVS and STING.

IMPORTANCE: The precise regulation of the innate immune response is essential for the maintenance of homeostasis. MAVS and STING play key roles in immune signaling pathways activated by RNA and DNA viruses, respectively. Here, we showed that DHCR24 impaired the antiviral response by targeting MAVS and STING. Notably, DHCR24 interacts with MAVS and STING and inhibits TRIM21-triggered K27-linked ubiquitination of MAVS and AMFR-triggered K27-linked ubiquitination of STING, restraining the activation of MAVS and STING, respectively. Together, this study elucidates how one cholesterol key enzyme orchestrates two antiviral signal transduction pathways.

Serpin-1a and serpin-6 regulate the Toll pathway immune homeostasis by synergistically inhibiting the Spätzle-processing enzyme CLIP2 in silkworm, Bombyx mori.

The Toll receptor signaling pathway is an important innate immune response of insects to pathogen infection; its extracellular signal transduction involves serine protease cascade activation. However, excessive or constitutive activation of the Toll pathway can be detrimental. Hence, the balance between activation and inhibition of the extracellular protease cascade must be tightly regulated to achieve favorable outcomes. Previous studies have shown that serpins-serine protease inhibitors-negatively regulate insect innate immunity by inhibiting extracellular protease cascade signaling. Although the roles of serpins in insect innate immunity are well described, the physiological mechanisms underlying their synergistic effects remain poorly understand. Here, we characterize the molecular mechanism by which serpin-1a and serpin-6 synergistically maintain immune homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Through in vitro biochemical assays and in vivo bioassays, we demonstrate that clip-domain serine protease 2 (CLIP2), as the Toll cascade-activating terminal protease, is responsible for processing proSpätzle1 to induce the expression of antimicrobial peptides. Further biochemical and genetic analyses indicate that constitutively expressed serpin-1a and inducible serpin-6 synergistically target CLIP2 to maintain homeostasis of the silkworm Toll pathway under physiological and pathological conditions. Taken together, this study provides new insights into the precise regulation of Toll cascade activation signals in insect innate immune responses and highlights the importance and complexity of insect immune homeostasis regulation.

The dual functions of KDM7A in HBV replication and immune microenvironment.

KDM7A (lysine demethylase 7A, also known as JHDM1D) is a histone demethylase, it is mainly involved in the intracellular post-translational modifications process. Recently, it has been proved that the histone demethylase members can regulate the replication of hepatitis B virus (HBV) and the expression of key molecules in the Janus-activated kinase-signal transducer and activator of the transcription (JAK/STAT) signaling pathway by chromatin modifying mechanisms. In our study, we identify novel roles of KDM7A in HBV replication and immune microenvironment through two subjects: pathogen and host. On the one hand, KDM7A is highly expressed in HBV-infected cells and promotes HBV replication in vitro and in vivo. Moreover, KDM7A interacts with HBV covalently closed circular DNA and augments the activity of the HBV core promoter. On the other hand, KDM7A can remodel the immune microenvironment. It inhibits the expression of interferon-stimulated genes (ISGs) through the IFN-γ/JAK2/STAT1 signaling pathway in both hepatocytes and macrophages. Further study shows that KDM7A interacts with JAK2 and STAT1 and affects their methylation. In general, we demonstrate the dual functions of KDM7A in HBV replication and immune microenvironment, and then we propose a new therapeutic target for HBV infection and immunotherapy. IMPORTANCE Histone lysine demethylase KDM7A can interact with covalently closed circular DNA and promote the replication of hepatitis B virus (HBV). The IFN-γ/JAK2/STAT1 signaling pathway in macrophages and hepatocytes is also downregulated by KDM7A. This study provides new insights into the mechanism of HBV infection and the remodeling of the immune microenvironment.

Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis.

How neuronal signaling affects brain myelination remains poorly understood. We show dysregulated neuronal RHEB-mTORC1-DLK1 axis impairs brain myelination. Neuronal Rheb cKO impairs oligodendrocyte differentiation/myelination, with activated neuronal expression of the imprinted gene Dlk1. Neuronal Dlk1 cKO ameliorates myelination deficit in neuronal Rheb cKO mice, indicating that activated neuronal Dlk1 expression contributes to impaired myelination caused by Rheb cKO. The effect of Rheb cKO on Dlk1 expression is mediated by mTORC1; neuronal mTor cKO and Raptor cKO and pharmacological inhibition of mTORC1 recapitulate elevated neuronal Dlk1 expression. We demonstrate that both a secreted form of DLK1 and a membrane-bound DLK1 inhibit the differentiation of cultured oligodendrocyte precursor cells into oligodendrocytes expressing myelin proteins. Finally, neuronal expression of Dlk1 in transgenic mice reduces the formation of mature oligodendrocytes and myelination. This study identifies Dlk1 as an inhibitor of oligodendrocyte myelination and a mechanism linking altered neuronal signaling with oligodendrocyte dysfunction.

Regulation of PKR-dependent RNA translation inhibition by TRIM21 upon virus infection or other stress.

The host always employs various ways to defend against viral infection and spread. However, viruses have evolved their own effective strategies, such as inhibition of RNA translation of the antiviral effectors, to destroy the host's defense barriers. Protein synthesis, commonly controlled by the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), is a basic cellular biological process among all species. In response to viral infection, in addition to inducing the transcription of antiviral cytokines by innate immunity, infected cells also inhibit the RNA translation of antiviral factors by activating the protein kinase R (PKR)-eIF2α signaling pathway. Regulation of innate immunity has been well studied; however, regulation of the PKR-eIF2α signaling pathway remains unclear. In this study, we found that the E3 ligase TRIM21 negatively regulates the PKR-eIF2α signaling pathway. Mechanistically, TRIM21 interacts with the PKR phosphatase PP1α and promotes K6-linked polyubiquitination of PP1α. Ubiquitinated PP1α augments its interaction with PKR, causing PKR dephosphorylation and subsequent translational inhibition release. Furthermore, TRIM21 can constitutively restrict viral infection by reversing PKR-dependent translational inhibition of various previously known and unknown antiviral factors. Our study highlights a previously undiscovered role of TRIM21 in regulating translation, which will provide new insights into the host antiviral response and novel targets for the treatment of translation-associated diseases in the clinic.

RasGRP1 promotes the acute inflammatory response and restricts inflammation-associated cancer cell growth.

An acute inflammatory response needs to be properly regulated to promote the elimination of pathogens and prevent the risk of tumorigenesis, but the relevant regulatory mechanism has not been fully elucidated. Here, we report that Ras guanine nucleotide-releasing protein 1 (RasGRP1) is a bifunctional regulator that promotes acute inflammation and inhibits inflammation-associated cancer. At the mRNA level, Rasgrp1 activates the inflammatory response by functioning as a competing endogenous RNA to specifically promote IL-6 expression by sponging let-7a. In vivo overexpression of the Rasgrp1 3' untranslated region enhances lipopolysaccharide-induced systemic inflammation and dextran sulphate sodium-induced colitis in Il6+/+ mice but not in Il6-/- mice. At the protein level, RasGRP1 overexpression significantly inhibits the tumour-promoting effect of IL-6 in hepatocellular carcinoma progenitor cell-like spheroids. Examination of the EGFR signalling pathway shows that RasGRP1 inhibits inflammation-associated cancer cell growth by disrupting the EGFR-SOS1-Ras-AKT signalling pathway. Tumour patients with high RasGRP1 expression have better clinical outcomes than those with low RasGRP1 expression. Considering that acute inflammation rarely leads to tumorigenesis, this study suggests that RasGRP1 may be an important bifunctional regulator of the acute inflammatory response and tumour growth.

TRIM21 Regulates Virus-Induced Cell Pyroptosis through Polyubiquitination of ISG12a.

Pyroptosis is a form of regulated cell death mediated by the gasdermin protein family. During virus infection, cell pyroptosis restricts viral replication. The mechanisms of the tripartite motif (TRIM) protein family and IFN-stimulated genes (ISGs) against viruses have been studied. The role of TRIMs and ISGs in pyroptosis remains unclear. In this study, we show that TRIM21 interacts with ISG12a in viral infection and facilitates its translocation into the mitochondria by promoting its ubiquitination, thereby causing caspase 3 activation. Gasdermin E (GSDME) is specifically cleaved by caspase 3 upon viral infection, releasing the GSDME N-terminal domain, perforating the cell membrane, and causing cell pyroptosis. Our study uncovers a new mechanism of TRIM21 and ISG12a in regulating virus-induced cell pyroptosis.

Heat Shock-Binding Protein 21 Regulates the Innate Immune Response to Viral Infection.

The induction of interferons (IFNs) plays an important role in the elimination of invading pathogens. Heat shock binding protein 21 (HBP21), first known as a molecular chaperone of HSP70, is involved in tumor development. Heat shock binding proteins have been shown to regulate diverse biological processes, such as cell cycle, kinetochore localization, transcription, and cilium formation. Their role in antimicrobial immunity remains unknown. Here, we found that HBP21 drives a positive feedback loop to promote IRF3-mediated IFN production triggered by viral infection. HBP21 deficiency significantly impaired the virus-induced production of IFN and resulted in greater susceptibility to viral infection both in vitro and in vivo. Mechanistically, HBP21 interacted with IRF3 and promoted the formation of a TBK1-IRF3 complex. Moreover, HBP21 abolished the interaction between PP2A and IRF3 to repress the dephosphorylation of IRF3. Analysis of HBP21 protein structure further confirmed that HBP21 promotes the activation of IRF3 by depressing the dephosphorylation of IRF3 by PP2A. Further study demonstrated that virus-induced phosphorylation of Ser85 and Ser153 of HBP21 itself is important for the phosphorylation and dimerization of IRF3. Our study identifies HBP21 as a new positive regulator of innate antiviral response, which adds novel insight into activation of IRF3 controlled by multiple networks that specify behavior of tumors and immunity. IMPORTANCE The innate immune system is the first-line host defense against microbial pathogen invasion. The physiological functions of molecular chaperones, involving cell differentiation, migration, proliferation and inflammation, have been intensively studied. HBP21 as a molecular chaperone is critical for tumor development. Tumor is related to immunity. Whether HBP21 regulates immunity remains unknown. Here, we found that HBP21 promotes innate immunity response by dual regulation of IRF3. HBP21 interacts with IRF3 and promotes the formation of a TBK1-IRF3 complex. Moreover, HBP21 disturbs the interaction between PP2A and IRF3 to depress the dephosphorylation of IRF3. Analysis of HBP21 protein structure confirms that HBP21 promotes the activation of IRF3 by blocking the dephosphorylation of IRF3 by PP2A. Interestingly, virus-induced Ser85 and Ser153 phosphorylation of HBP21 is important for IRF3 activation. Our findings add to the known novel immunological functions of molecular chaperones and provide new insights into the regulation of innate immunity.

Homeodomain proteins POU-M2, antennapedia and abdominal-B are involved in regulation of the segment-specific expression of the clip-domain serine protease gene CLIP13 in the silkworm, Bombyx mori.

Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development. Our previous studies indicated that CLIP13, an epidermis-specific gene, was involved in cuticle remodeling during molting and metamorphosis in the silkworm, Bombyx mori. However, the transcriptional regulatory mechanism and regulatory pathways of CLIP13 remained unclear. In the present study, we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm. At the transcriptional level, expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis; homeodomain transcription factors POU-M2, antennapedia (Antp), and abdominal-B (Abd-B) showed similar expression change trends as CLIP13 in the head capsule, thorax, and abdomen, respectively. Furthermore, results of cell transfection assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation demonstrated that POU-M2, Antp, and Abd-B were involved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter. RNA interference-mediated silencing of POU-M2, Antp, and Abd-B led to a decrease of CLIP13 expression in the head capsule, the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments, respectively. Consistent with CLIP13, 20E treatment significantly upregulated expression of POU-M2, Antp, and Abd-B in the silkworm epidermis. Taken together, these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2, Antp, and Abd-B in different regions of the silkworm epidermis during metamorphosis, thus affecting the molting process. Our findings provide new insight into the functions of homeodomain transcription factors in insect molting.

TRIM10 binds to IFN-α/ß receptor 1 to negatively regulate type I IFN signal transduction.

The type I interferon (IFN-I) system is important for antiviral and anticancer immunity. Prolonged activation of IFN/JAK/STAT signaling is closely associated with autoimmune diseases. TRIM10 dysfunction may be associated closely with certain autoimmune disorders. Here, we observed that the serum TRIM10 protein level is lower in patients with systemic lupus erythematosus than in healthy control subjects. We speculated the possible involvement of TRIM10-induced modulation of the IFN/JAK/STAT signaling pathway in systemic lupus erythematosus. In line with our hypothesis, TRIM10 inhibited the activation of JAK/STAT signaling pathway triggered by various stimuli. TRIM10 restricted the IFN-I/JAK/STAT signaling pathway, which was independent of its E3 ligase activity. Mechanistically, TRIM10 interacted with the intracellular domain of IFNAR1 and blocked the association of IFNAR1 with TYK2. These data suggest the possible TRIM10 suppresses IFN/JAK/STAT signaling pathway through blocking the interaction between IFNAR1 and TYK2. Targeting TRIM10 is a potential strategy for treating autoimmune diseases.

Rheb mediates neuronal-activity-induced mitochondrial energetics through mTORC1-independent PDH activation.

Neuronal activity increases energy consumption and requires balanced production to maintain neuronal function. How activity is coupled to energy production remains incompletely understood. Here, we report that Rheb regulates mitochondrial tricarboxylic acid cycle flux of acetyl-CoA by activating pyruvate dehydrogenase (PDH) to increase ATP production. Rheb is induced by synaptic activity and lactate and dynamically trafficked to the mitochondrial matrix through its interaction with Tom20. Mitochondria-localized Rheb protein is required for activity-induced PDH activation and ATP production. Cell-type-specific gain- and loss-of-function genetic models for Rheb reveal reciprocal changes in PDH phosphorylation/activity, acetyl-CoA, and ATP that are not evident with genetic or pharmacological manipulations of mTORC1. Mechanistically, Rheb physically associates with PDH phosphatase (PDP), enhancing its activity and association with the catalytic E1α-subunit of PDH to reduce PDH phosphorylation and increase its activity. Findings identify Rheb as a nodal point that balances neuronal activity and neuroenergetics via Rheb-PDH axis.

Identification, characterization, and expression analysis of clip-domain serine protease genes in the silkworm, Bombyx mori.

Clip-domain serine proteases (CLIPs), characterized by regulatory module clip domains, constitute an important serine protease family identified in insects and other arthropods. They participate in host immune response and embryonic development in a cascade-activated manner. Here, we present a genome-wide identification and expression analysis of CLIP genes in the silkworm, Bombyx mori. A total of 26 CLIP genes were identified in the silkworm genome. Bioinformatics analysis indicated that these CLIPs clustered into four subfamilies (CLIPA-D), and exhibit a close evolutionary relationship with CLIPs of Manduca sexta. Tissue expression profiling revealed that silkworm CLIP genes are mainly expressed in the integument, head, fat body, and hemocytes. Temporal expression profiles showed that 15 CLIP genes were predominantly expressed during the fifth-instar larval stage, early and later period of the pupal stage, and adult stage, whereas 10 CLIP genes were mainly expressed in the wandering stage and middle to later period of the pupal stage in the integument. Pathogens and 20-hydroxyecdysone (20E) induction analysis indicated that 14 CLIP genes were positively regulated by 20E, 9 were negatively regulated by 20E but positively regulated by pathogens, and 5 were positively regulated by both factors in the integument. Together, these results suggested that silkworm CLIP genes may play multiple functions in integument development, including melanization of new cuticle, molting and immune defense. Our data provide a comprehensive understanding of CLIP genes in the silkworm integument and lays a foundation for further functional studies of CLIP genes in the silkworm.

Silkworm serpin32 functions as a negative-regulator in prophenoloxidase activation.

The extracellular serine protease cascade is an essential component of insect humoral immunity. Serine protease inhibitors (serpins) play an important regulatory role in the process of insect immunity by regulating the serine protease cascade pathway. We aimed to clarify the function of Bmserpin32 in this study. First, we performed homologous sequence alignment and phylogenetic analysis of Bmserpin32. Bmserpin32 was found to share 64% amino acid sequence identity with Manduca sexta serpin7, an immunomodulatory protein. Bmserpin32 cDNA was cloned, and the recombinant Bmserpin32 protein was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity and gel filtration chromatography. The activity assay showed that Bmserpin32 had significant inhibitory activity against trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and site-directed mutagenesis combined with activity assays indicated that the cleavage site of Bmserpin32 is between Arg359 and Ile360. After infection with E. coli or Micrococcus luteus, the expression level of Bmserpin32 in immune-related tissues was significantly upregulated. In addition, Bmserpin32 could delay or inhibit the melanization of hemolymph by inhibiting the activation of prophenoloxidase in larval hemolymph. Furthermore, a physiological target of Bmserpin32 was identified as the clip protease, BmPAP3, an apparent ortholog of M. sexta propenoloxidase-activating protease-3. Our observations enable a better understanding of the physiological role of Bmserpin32 in regulating melanization in silkworm.

Proteomic analysis of Bombyx mori molting fluid: Insights into the molting process.

Molting is an essential biological process occurring multiple times throughout the life cycle of most Ecdysozoa. Molting fluids accumulate and function in the exuvial space during the molting process. In this study, we used liquid chromatography-tandem mass spectrometry to investigate the molting fluids to analyze the molecular mechanisms of molting in the silkworm, Bombyx mori. In total, 375 proteins were identified in molting fluids from the silkworm at 14-16h before pupation and eclosion, including 12 chitin metabolism-related enzymes, 35 serine proteases, 15 peptidases, and 38 protease inhibitors. Gene ontology analysis indicated that "catalytic" constitutes the most enriched function in the molting fluid. Gene expression patterns and bioinformatic analyses suggested that numerous enzymes are involved in the degradation of cuticle proteins and chitin. Protein-protein interaction network and activity analyses showed that protease inhibitors are involved in the regulation of multiple pathways in molting fluid. Additionally, many immune-related proteins may be involved in the immune defense during molting. These results provide a comprehensive proteomic insight into proteolytic enzymes and protease inhibitors in molting fluid, and will likely improve the current understanding of physiological processes in insect molting. BIOLOGICAL SIGNIFICANCE: Insect molting constitutes a dynamic physiological process. To better understand this process, we used LC-MS/MS to investigate the proteome of silkworm molting fluids and identified key proteins involved in silkworm molting. The biological processes of the old cuticle degradation pathway and immune defense response were analyzed in the proteome of silkworm molting fluid. We report that protease inhibitors serve as key factors in the regulation of the molting process. The proteomic results provide new insight into biological molting processes in insects.