The Lysosomal Rag-Ragulator Complex Licenses RIPK1 and Caspase-8-mediated Pyroptosis by Yersinia.

Host cells initiate cell death programs to limit pathogen infection. Inhibition of transforming growth factor-ß-activated kinase 1 (TAK1) by pathogenic Yersinia in macrophages triggers receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent caspase-8 cleavage of gasdermin D (GSDMD) and inflammatory cell death (pyroptosis). A genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen to uncover mediators of caspase-8-dependent pyroptosis identified an unexpected role of the lysosomal FLCN-FNIP2-Rag-Ragulator supercomplex, which regulates metabolic signalling and the mechanistic target of rapamycin complex 1 (mTORC1). In response to Yersinia infection, FADD, RIPK1 and caspase-8 were recruited to Rag-Ragulator, causing RIPK1 phosphorylation and caspase-8 activation. Pyroptosis activation depended on Rag GTPase activity and lysosomal tethering of Rag-Ragulator, but not mTORC1. Thus, the lysosomal metabolic regulator Rag-Ragulator instructs the inflammatory response to Yersinia.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

Gasdermins: pore-forming activities and beyond.

Gasdermins (GSDMs) belong to a protein superfamily that is found only in vertebrates and consists of GSDMA, GSDMB, GSDMC, GSDMD, DFNA5 (a.k.a. GSDME) and DFNB59 (a.k.a. Pejvakin (PJVK)) in humans. Except for DFNB59, all members of the GSDM superfamily contain a conserved two-domain structure (N-terminal and C-terminal domains) and share an autoinhibitory mechanism. When the N-terminal domain of these GSDMs is released, it possesses pore-forming activity that causes inflammatory death associated with the loss of cell membrane integrity and release of inflammatory mediators. It has also been found that spontaneous mutations occurring in the genes of GSDMs have been associated with the development of certain autoimmune disorders, as well as cancers. Here, we review the current knowledge of the expression profile and regulation of GSDMs and the important roles of this protein family in inflammatory cell death, tumorigenesis and other related diseases.

Author Correction: CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development.

In Fig. 2a of this Technical Report originally published, the authors inadvertently used the same set of images for the 4B2N1 and 4B2N3 cells when preparing the figure. The three images (bright field, Oct4-EGFP and pCAG-mRFP) of 4B2N3 cells have now been replaced with the correct versions. The source data for the four cell lines in Fig. 2a, captured in the three independent experiments, have been deposited to Figshare (https://doi.org/10.6084/m9.figshare.7387607.v1), and the figure legends and Methods section have been amended to reflect this. Additionally, the unprocessed blots in Supplementary Fig. 7 corresponding to the top right 'WCL IB: Flag' panel of Fig. 7e were mistakenly duplicates of the unprocessed blots for the bottom left 'IP Flag IB: HA' panel of Fig. 7e, and all unprocessed blots for Supplementary Fig. 6 were mislabelled as blots corresponding to Supplementary Fig. 7. Supplementary Fig. 7 has now been updated to show the correct unprocessed blots for the bottom left 'IP Flag IB: HA' panel of Fig. 7e and to correct the labelling of the unprocessed blots corresponding to Supplementary Fig. 6.

The Cyclopeptide Astin C Specifically Inhibits the Innate Immune CDN Sensor STING.

cGAS-STING signaling is essential for innate immunity. Its misregulation promotes cancer or autoimmune and autoinflammatory diseases, and it is imperative to identify effective lead compounds that specifically downregulate the pathway. We report here that astin C, a cyclopeptide isolated from the medicinal plant Aster tataricus, inhibits cGAS-STING signaling and the innate inflammatory responses triggered by cytosolic DNAs. Moreover, mice treated with astin C are more susceptible to HSV-1 infection. Consistently, astin C markedly attenuates the autoinflammatory responses in Trex1-/- BMDM cells and in Trex1-/- mouse autoimmune disease model. Mechanistically, astin C specifically blocks the recruitment of IRF3 onto the STING signalosome. Collectively, this study characterizes a STING-specific small-molecular inhibitor that may be applied for potentially manipulating the STING-mediated clinical diseases.

CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development.

CRISPR-mediated base editing can introduce single-nucleotide changes in the DNA of living cells. One intriguing application of base editing is to screen pivotal amino acids for protein function in vivo; however, it has not been achieved. Here, we report an enhanced third-generation base-editing system with extra nuclear localization sequences that can efficiently introduce a homozygous base mutation in embryonic stem cells. Meanwhile, we establish a strategy to generate base-mutant mice by injection of haploid embryonic stem cells carrying a constitutively expressed enhanced third-generation base-editing system (4B2N1) and single guide RNA into oocytes. Moreover, transfection of 4B2N1 cells with a single guide RNA library targeting the Dnd1 gene allows one-step generation of mutant mice with a base mutation. This enables the identification of four missense mutations that completely deplete primordial germ cells through disruption of DND1 protein stability and protein-protein interaction. Thus, our strategy provides an effective tool for in vivo screening of amino acids that are crucial for protein function.

UXT-V1 facilitates the formation of MAVS antiviral signalosome on mitochondria.

Virus infection induces the MAVS-TNFR-associated factor (TRAF) 3 signaling axis on mitochondria. It remains to elucidate the corresponding regulatory processes. In this study, we identify UXT-V1 as a novel TRAF3-binding protein. UXT-V1 is critical for the virus-induced activation of NF-κB and IFN regulatory factor 3. Reduction of UXT-V1 impairs the induction of IFN-ß and attenuates the host antiviral responses. The N-terminal TRAF-binding motif of UXT-V1 binds to the C-terminal TRAF domain of TRAF3, thus facilitating the interaction between TRAF3 and MAVS. Notably, TRAF3 and TNFR-associated death domain protein are recruited onto mitochondria upon virus infection. These translocations are blocked when knocking down UXT-V1. Thus, UXT-V1 represents a novel integral component of the MAVS signalosome on mitochondria, mediating the innate antiviral signal transduction.

Negative feedback regulation of NF-κB action by CITED2 in the nucleus.

NF-κB is a family of important transcription factors that modulate immunity, development, inflammation, and cancer. The biological activity of NF-κB is subjected to various spatial and temporal regulations. Bioinformatics analysis predicts that CITED2 is topologically close to NF-κB in the protein interaction networks. In this study, we show that ectopic expression or knockdown of CITED2 attenuates or potentiates, respectively, the expression of NF-κB-responsive genes. Mechanistically, CITED2 constitutively localizes inside the nucleus and interacts specifically with the coactivator p300. This prevents p65 from binding to p300, impairs p65 acetylation, and attenuates p65 binding to its cognate promoters. Furthermore, LPS induces CITED2 expression via NF-κB in macrophages. CITED2 sensitizes cells to TNF-α-induced apoptosis. Collectively, this study identifies CITED2 as a novel regulator of NF-κB in the nucleus, which reveals a negative feedback mechanism for NF-κB signaling.

UXT is a novel and essential cofactor in the NF-kappaB transcriptional enhanceosome.

As a latent transcription factor, nuclear factor kappaB (NF-kappaB) translocates from the cytoplasm into the nucleus upon stimulation and mediates the expression of genes that are important in immunity, inflammation, and development. However, little is known about how it is regulated inside the nucleus. By a two-hybrid approach, we identify a prefoldin-like protein, ubiquitously expressed transcript (UXT), that is expressed predominantly and interacts specifically with NF-kappaB inside the nucleus. RNA interference knockdown of UXT leads to impaired NF-kappaB activity and dramatically attenuates the expression of NF-kappaB-dependent genes. This interference also sensitizes cells to apoptosis by tumor necrosis factor-alpha. Furthermore, UXT forms a dynamic complex with NF-kappaB and is recruited to the NF-kappaB enhanceosome upon stimulation. Interestingly, the UXT protein level correlates with constitutive NF-kappaB activity in human prostate cancer cell lines. The presence of NF-kappaB within the nucleus of stimulated or constitutively active cells is considerably diminished with decreased endogenous UXT levels. Our results reveal that UXT is an integral component of the NF-kappaB enhanceosome and is essential for its nuclear function, which uncovers a new mechanism of NF-kappaB regulation.