The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients' sera, proving the importance of this antigen in host immunity and diagnostics.

Cytosolic inflammasomes are supramolecular complexes that are formed in response to intracellular pathogens and danger signals. However, as to date, the detailed description of a homotypic caspase recruitment domain (CARD) interaction between NLRP1 and ASC has not been presented. We found the CARD–CARD interaction between purified NLRP1CARD and ASCCARD experimentally and the filamentous supramolecular complex formation in an in vitro proteins solution. Moreover, we determined a high-resolution crystal structure of the death domain fold of the human ASCCARD. Mutational and structural analysis revealed three conserved interfaces of the death domain superfamily (Type I, II, and III), which mediate the assembly of the NLRP1CARD/ASCCARD complex. In addition, we validated the role of the three major interfaces of CARDs in assembly and activation of NLRP1 inflammasome in vitro. Our findings suggest a Mosaic model of homotypic CARD interactions for the activation of NLRP1 inflammasome. The Mosaic model provides insights into the mechanisms of inflammasome assembly and signal transduction amplification.

Murine caspase-11 is the centerpiece of the non-canonical inflammasome pathway that can respond to intracellular LPS and induce pyroptosis. Caspase-11 contains two components, an N-terminal caspase recruitment domain (CARD) and a C-terminal catalytic domain. The aggregation of caspase-11 is thought to promote the auto-processing and activation of caspase-11. However, the activation mechanism of caspase-11 remains unclear. In this study, we purified the caspase-11 CARD fused to an MBP tag and found it tetramerizes in solution. Crystallographic analysis reveals an extensive hydrophobic interface formed by the H1–2 helix mediating homotypic CARD interactions. Importantly, mutations of the helix H1–2 hydrophobic residues abolished the tetramerization of MBP-tagged CARD in solution and failed to induce pyroptosis in cells. Our study provides the first evidence of the homotypic interaction mode for an inflammatory caspase by crystal model. This finding demonstrates that the tetramerization of the N-terminal CARD can promote releasing of the catalytic domain auto-inhibition, leading to the caspase-11 activation.

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Death receptor 3 (DR3) (a.k.a. tumor necrosis factor receptor superfamily 25) plays a key role in the immune system by activating nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling pathway. Here we present the crystal structures of human and mouse DR3 intracellular death domain (DD) at 2.7 and 2.5 Å resolutions, respectively. The mouse DR3 DD adopts a classical six‐helix bundle structure while human DR3 DD displays an extended fold. Though there is one‐amino‐acid difference in the linker between maltose‐binding protein (MBP) tag and DR3 DD, according to our self‐interaction analysis, the hydrophobic interface discovered in MBP–hDR3 DD crystal structure is responsible for both hDR3 DD and mDR3 DD homotypic interaction. Furthermore, our biochemical analysis indicates that the sequence variation between human and mouse DR3 DD does not affect its structure and function. Small‐angle X‐ray scattering analysis shows the averaged solution structures of both human and mouse MBP‐DR3 DD are the combination of different conformations with different proportion. Through switching to the open conformation, DR3 DD could improve the interaction with downstream element TNFR‐associated death domain (TRADD). Here we propose an activation‐dependent structural rearrangement model: the DD region is folded as the six‐helix bundles in the resting state, while upon extracellular ligand engagement, it switches to the open conformation, which facilitates its self‐association and the recruitment of TRADD. Our results provide detailed insights into the architecture of DR3 DD and the molecular mechanism of activation. Databases All refined structure coordinates as well as the corresponding structure factors have been deposited in the PDB under the accession codes http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5YGS, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5YEV, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5YGP, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZNY, http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5ZNZ.

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Background and Aims: The innate-like mucosa-associated invariant T (MAIT) cells are enriched in human liver and have been linked to human HCC. However, their contributions to the progression of HCC are controversial due to the heterogeneity of MAIT cells, and new MAIT cell subsets remain to be explored. Approach and Results: Combining single cell RNA sequencing (scRNA-seq) and flow cytometry analysis, we performed phenotypic and functional studies and found that FOXP3+ CXCR3+ MAIT cells in HCC patients were regulatory MAIT cells (MAITregs) with high immunosuppressive potential. These MAITregs were induced under Treg-inducing condition and predominantly from FOXP3− CXCR3+ MAIT cells, which displayed mild Treg-related features and represented a pre-MAITreg reservoir. In addition, the induction and function of MAITregs were promoted by β1 adrenergic receptor signaling in pre-MAITregs and MAITregs, respectively. In HCC patients, high proportion of the intratumoral MAITregs inhibited antitumor immune responses and was associated with poor clinical outcomes. Conclusions: Together, we reveal an immunosuppressive subset of MAIT cells in HCC patients that contributes to HCC progression, and propose a control through neuroimmune crosstalk.

The NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates the NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that different ASC preference in three types of inflammasomes, namely the ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that signal transduction of the NLRP1 inflammasome relies on multiple interaction surfaces at its CARD domain. Finally, our results partly explain how mutations in NLRP1 lead to its constitutive activation in auto-inflammatory diseases. In conclusion, our study not only reveals how CARD8 downregulates the NLRP1 inflammasome activation, but also provides insights into the assembly mechanisms of CARD-containing inflammasomes.