REN基因敲除HEK293细胞

Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson's disease. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoK. LysoK consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson's disease and reduces channel currents. Reduction in lysoK function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological α-synuclein. By contrast, the minor allele at rs3488217-another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson's disease-produces a gain-of-function in lysoK during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson's disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson's disease.

Ribonuclease (RNase) III Drosha initiates microRNA (miRNA) biogenesis. Emerging evidence suggests that Drosha modulates processes beyond miRNA biogenesis, indicative of the functional complexity of Drosha. Here, we show that Drosha facilitates activation of sterol regulatory element-binding protein 1 (SREBP1), the central player in triglyceride (TG) biosynthesis, in an RNase activity-independent manner. Mechanistically, cytoplasmic Drosha interacts with Sec31, a subunit of coat protein complex II (COPII), through its arginine/serine (RS)-rich domain. This interaction promotes COPII-mediated transport and activation of SREBP1. Moreover, Akt phosphorylates Drosha at Ser237 in the RS-rich domain, which is required for the Drosha-Sec31 interaction, SREBP1 processing, and TG accumulation in response to insulin. The Akt-Drosha-SREBP1 axis was hyperactivated in mice fed a high-fat, high-sugar diet, and hepatic Drosha deletion or administration of an interfering peptide that blocked Akt-mediated phosphorylation of Drosha ameliorated liver TG deposition and insulin resistance in this mouse model. Thus, our findings uncover a noncanonical function of Drosha involved in SREBP1 processing and lipogenesis.

Innate immunity is the first line to defend against pathogenic microorganisms, and Toll-like receptor (TLR)-mediated inflammatory responses are an essential component of innate immunity. However, the regulatory mechanisms of TLRs in innate immunity remain unperfected. We found that the expression of E3 ligase Ring finger protein 99 (RNF99) decreased significantly in peripheral blood monocytes from patients infected with Gram negative bacteria (G) and macrophages stimulated by TLRs ligands, indicating the role of RNF99. We also demonstrated for the first time, the protective role of RNF99 against LPS-induced septic shock and dextran sodium sulfate (DSS)-induced colitis using RNF99 knockout mice (RNF99) and bone marrow-transplanted mice. In vitro experiments revealed that RNF99 deficiency significantly promoted TLR-mediated inflammatory cytokine expression and activated the NF-κB and MAPK pathways in macrophages. Mechanistically, in both macrophages and HEK293 cell line with TLR4 stably transfection, RNF99 interacted with and degraded TAK1-binding protein (TAB) 2, a regulatory protein of the kinase TAK1, via the lysine (K)48-linked ubiquitin-proteasomal pathway on lysine 611 of TAB2, which further regulated the TLR-mediated inflammatory response. Overall, these findings indicated the physiological significance of RNF99 in macrophages in regulating TLR-mediated inflammatory reactions. It provided new insight into TLRs signal transduction, and offered a novel approach for preventing bacterial infections, endotoxin shock, and other inflammatory ills.

The ZIKA virus (ZIKV) evades the host immune response by degrading STAT2 through its NS5 protein, thereby inhibiting type I interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism underlying this process has remained elusive. In this study, we performed a genome-wide CRISPR/Cas9 screen, revealing that ZSWIM8 as the substrate receptor of Cullin3-RING E3 ligase is required for NS5-mediated STAT2 degradation. Genetic depletion of ZSWIM8 and CUL3 substantially impeded NS5-mediated STAT2 degradation. Biochemical analysis illuminated that NS5 enhances the interaction between STAT2 and the ZSWIM8-CUL3 E3 ligase complex, thereby facilitating STAT2 ubiquitination. Moreover, ZSWIM8 knockout endowed A549 and Huh7 cells with partial resistance to ZIKV infection and protected cells from the cytopathic effects induced by ZIKV, which was attributed to the restoration of STAT2 levels and the activation of IFN signaling. Subsequent studies in a physiologically relevant model, utilizing human neural progenitor cells, demonstrated that ZSWIM8 depletion reduced ZIKV infection, resulting from enhanced IFN signaling attributed to the sustained levels of STAT2. Our findings shed light on the role of ZIKV NS5, serving as the scaffold protein, reprograms the ZSWIM8-CUL3 E3 ligase complex to orchestrate STAT2 proteasome-dependent degradation, thereby facilitating evasion of IFN antiviral signaling. Our study provides unique insights into ZIKV-host interactions and holds promise for the development of antivirals and prophylactic vaccines.

Phenazine biosynthesis-like domain-containing protein (PBLD) has been reported to be involved in the development of many cancers. However, whether PBLD regulates innate immune responses and viral replication is unclear. In this study, although it was found that the activity of PBLD extends to other PRRs, we focused on the RLR pathway activated via the p53-USP4-MAVS axis in response to virus infections. We found that PBLD deubiquitinates and stabilizes MAVS through ubiquitin-specific protease 4 (USP4) to promote antiviral innate immunity. Mechanistically, PBLD activates the transcription of USP4 via the upregulation of p53. USP4, which is a MAVS-interacting protein, substantially stabilizes the MAVS protein by deconjugating K48-linked ubiquitination chains from the MAVS protein at Lys461 during RNA virus infection. Most intriguingly, RNA virus-infected primary macrophages (peritoneal macrophages, PMs, and bone marrow-derived macrophages, BMDMs) and internal organ cells (lung and liver) from PBLD-deficient mice suppress the IFN-I response and promote viral replication. Notably, PBLD-deficient mice are more susceptible to RNA virus infection than their wild-type littermates. Our findings highlight a unique function of PBLD in antiviral innate immunity through the p53-USP4-MAVS signaling, providing a preliminary basis for research on PBLD as a target molecule for treating RNA virus infection.

Histone deacetylase HDAC6 has been implicated in regulating antiviral innate immunity. However, its precise function in response to DNA virus infection remains elusive. Herein, we find that HDAC6 deficiency promotes the activation of cGAS-STING signaling and type I interferon (IFN) production, both in vitro and in vivo, resulting in a decrease in HSV-1 infection. Mechanistically, HDAC6 deacetylates tripartite motif protein 56 (TRIM56) at K110 in mice, thereby impairing the monoubiquitination cGAS and its DNA binding ability. Overexpression of TRIM56 K110Q protects mice against HSV-1 infection. Notably, different amino acids at position 110 of TRIM56 in human and mouse cause species-specific IFN responses. Further, we show that during early stages of HSV-1 infection, the viral protein US3 phosphorylates HDAC6 to inhibit the cGAS-mediated antiviral response. Our results suggest that HDAC6 inhibits cGAS activation through TRIM56 deacetylation in a species-specific manner.

The antiviral innate immunity is the first line of host defense against viral infection. Mitochondrial antiviral signaling protein (MAVS, also named Cardif/IPS-1/VISA) is a critical protein in RNA virus-induced antiviral signaling pathways. Our previous research suggested that E3 ubiquitin-protein ligases RING-finger protein (RNF90) negatively regulate cellular antiviral responses by targeting STING for degradation, though its role in RNA virus infection remains unknown. This study demonstrated that RNF90 negatively regulated RNA virus-triggered antiviral innate immune responses in RNF90-silenced PMA-THP1 cells, RNF90-deficient cells (including HaCaTs, MEFs, and BMDMs), and RNF90-deficient mice. However, RNF90 regulated RNA virus-triggered antiviral innate immune responses independent of STING. RNF90 promoted K48-linked ubiquitination of MAVS and its proteasome-dependent degradation, leading to the inhibition of innate immune responses. Altogether, our findings suggested a novel function and mechanism of RNF90 in antiviral innate immunity.

Influenza A virus (IAV) continuously poses a considerable threat to global health through seasonal epidemics and recurring pandemics. IAV RNA-dependent RNA polymerases (FluPol) mediate the transcription of RNA and replication of the viral genome. Searching for targets that inhibit viral polymerase activity helps us develop better antiviral drugs. Here, we identified heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) as an anti-influenza host factor. hnRNPAB interacts with NP of IAV to inhibit the interaction between PB1 and NP, which is dependent on the 5-amino-acid peptide of the hnRNPAB C-terminal domain (aa 318-322). We further found that the 5-amino-acid peptide blocks the interaction between PB1 and NP to destroy the FluPol activity. In vivo studies demonstrate that hnRNPAB-deficient mice display higher viral burdens, enhanced cytokine production, and increased mortality after influenza infection. These data demonstrate that hnRNPAB perturbs FluPol complex conformation to inhibit IAV infection, providing insights into anti-influenza defense mechanisms.