Zhou Jingfan, Liu Chengrong, Wang Xin, Liu Zhenshan, Ming Zizhen, Wang Yonggang, Wang Chunxia, Liang Qiming
Institute of Pediatric Infection, Immunity, and Critical Care Medicine, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Shanghai Institute of Immunology, Department of Immunology and Microbiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
PLoS Pathog. 2025 Jan 24;21(1):e1012877. doi: 10.1371/journal.ppat.1012877. eCollection 2025 Jan.
Function-to-find domain (FIIND)-containing proteins, including NLRP1 and CARD8, are vital components of the inflammasome signaling pathway, critical for the innate immune response. These proteins exist in various forms due to autoproteolysis within the FIIND domain, resulting in full-length (FL), cleaved N-terminal (NT), and cleaved C-terminal (CT) peptides, which form autoinhibitory complexes in the steady state. However, the detailed mechanism remains elusive. Here, we found that both NLRP1 paralogs and CARD8 form two conserved autoinhibitory complexes involving NT-CT interactions and FL-CT interactions, but with distinct mechanisms. Specifically, the Linker3 region located between LRR and FIIND in murine NLRP1b (mNLRP1b) plays an essential role in forming the NT-CT autoinhibitory complexes, while the ZU5 of rat NLRP1 (rNLRP1) and CARD8 mediates their NT-CT interaction. In addition, we explored the involvement of the cellular protease dipeptidyl peptidases 9 (DPP9) in these complexes, revealing differential interactions and the significance of domain structure. Besides the FL-DPP9-CT complex, DPP9 interacts with NTs of mNLRP1b, rNLRP1, and CARD8 through their ZU5 subdomains, forming NT-DPP9-CT complex; however, DPP9 cannot bind to NTs of hNLRP1. Further functional assay indicated that although DPP9 is involved in the NT-CT complex of rodent NLRP1 and CARD8, it does not influence the inhibitory activity of NT on CT. Our study enhanced the understanding of the regulatory functions of FIIND-containing proteins in inflammasome autoinhibition and activation and underscored the complexity of their interactions within the immune response.
含功能待寻结构域（FIIND）的蛋白质，包括NLRP1和CARD8，是炎性小体信号通路的重要组成部分，对先天免疫反应至关重要。由于FIIND结构域内的自蛋白水解作用，这些蛋白质以多种形式存在，产生全长（FL）、N端裂解（NT）和C端裂解（CT）肽，它们在稳态下形成自抑制复合物。然而，具体机制仍不清楚。在这里，我们发现NLRP1旁系同源物和CARD8都形成了两种保守的自抑制复合物，涉及NT-CT相互作用和FL-CT相互作用，但机制不同。具体而言，小鼠NLRP1b（mNLRP1b）中位于LRR和FIIND之间的Linker3区域在形成NT-CT自抑制复合物中起关键作用，而大鼠NLRP1（rNLRP1）和CARD8的ZU5介导它们的NT-CT相互作用。此外，我们探讨了细胞蛋白酶二肽基肽酶9（DPP9）在这些复合物中的作用，揭示了不同的相互作用和结构域结构的重要性。除了FL-DPP9-CT复合物外，DPP9通过其ZU5亚结构域与mNLRP1b、rNLRP1和CARD8的NTs相互作用，形成NT-DPP9-CT复合物；然而，DPP9不能与hNLRP1的NTs结合。进一步的功能分析表明，尽管DPP9参与了啮齿动物NLRP1和CARD8的NT-CT复合物的形成，但它不影响NT对CT的抑制活性。我们的研究加深了对含FIIND蛋白质在炎性小体自抑制和激活中的调节功能的理解，并强调了它们在免疫反应中相互作用的复杂性。
