Inflammasomes ignite innate immune defense in response to infectious pathogens and noninfectious dangers, primarily through sensors composed of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing (NLR) family proteins. NLRP6 is an inflammasome sensor that plays critical roles in regulating intestinal inflammation, and its overactivation is linked to autoinflammatory diseases such as inflammatory bowel disease. However, how NLRP6 is maintained in an inhibited structure is unknown. Here we report two cryogenic-electron microscopy structures of human NLRP6 monomer in the adenosine-5'-triphosphate (ATP)/NBD-bound (twisted conformation) and unbound (extended conformation) states. The ATP-binding event connects and compacts the NACHT subdomains and the LRR domain, thus maintaining NLRP6 in an inhibitory conformation. NBD interacts directly with helical domain 1, winged-helix domain and helical domain 2, further contributing to the autoinhibition. Disruption of ATP binding and NBD interactions unleashes the NLRP6 inflammasome activation in the cellular study. The structural comparison between twisted and extended conformations reveals that the rearrangement of an NLRP6-specific acidic loop modulates NLRP6 activity. Although ATP-binding of NLRP6 and MCC950 (a potent NLRP3 inhibitor)-binding of NLRP3 share a similar interaction location in the structures, MCC950 does not inhibit NLRP6 in cells. Together, our data reveal the ATP-mediated cooperative inhibition mechanism of NLRP6 and provide insight into the therapeutic intervention of NLRP6-related autoinflammatory disorders.
Zhen Cui, Qi Sheng, Minsoo Son et al.· Nature Structural & Molecula...· 0 citations
L-Isoleucine is an essential branched-chain amino acid for livestock and poultry, supporting protein accretion and regulating energy metabolism, immune function, and stress resilience. A genetically stable L-isoleucine producer, Corynebacterium glutamicum cgl-Ile0, was obtained via biosensor-assisted ARTP mutagenesis, and produced 11.52 g/L L-isoleucine in a 5-L fermenter, with a yield of 0.11 g/g and a productivity of 0.24 g/L/h. Whole-genome resequencing revealed four mutations associated with the phenotype, including aspBV346I, asdP27E, brnEL87S, and brnFR28P. Structure-guided protein engineering of two rate-limiting enzymes—threonine dehydratase (TD) and acetohydroxyacid synthase (AHAS)—generated strain cgl-Ile1, increasing titer, yield, and productivity by 39.50%, 27.27%, and 37.50%, respectively, relative to strain cgl-Ile0. Subsequent modular optimization of L-isoleucine biosynthesis, oxaloacetate supply module, cofactor-supply module, and transport/export module yielded the final strain cgl-Ile8. Through optimization pH and dissolved oxygen, the titer, yield and productivity of L-isoleucine produced by strain cgl-Ile8 in a 5-L fermenter were 48.49 g/L, 0.31 g/g and 1.01 g/L/h, which were 4.21-, 2.82-, and 4.21-fold those of strain cgl-Ile0, respectively. Scale-up to a 50-L fermenter further increased the titer, yield and productivity to 50.12 g/L, 0.32 g/g and 1.04 g/L/h, respectively, representing the highest reported L-isoleucine titer in C. glutamicum to date. We developed an industrial L-isoleucine-producing C. glutamicum strain by integrating biosensor-guided ARTP mutagenesis, structure-guided protein engineering, and modular pathway rewiring, providing a practical and transferable framework for constructing GRAS amino-acid producers for animal nutrition.
Junkun Cao, Ming Huang, Qi Sheng et al.· Journal of Animal Science an...· 0 citations
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