NLRP1 and CARD8 Inflammasomes: Assembly, Regulation and Stress Sensing

摘要 核苷酸结合结构域(NBD)样和富含亮氨酸重复序列(LRR)的蛋白(NLR) 在细胞生物学中发挥不同的功能，调节一系列广泛的基本生物学 从免疫信号到胚胎发育的途径。在豁免权方面，几个NLR形式 被称为炎症体的超分子蛋白质信号复合体。炎性小体激活 Caspase-1是一种炎性蛋白酶，负责处理细胞因子白细胞介素1β(IL-1β)和 致孔蛋白Gasdermin D可促进嗜热细胞死亡。一个这样的炎症者- 形成蛋白NLRP1直接激活，以响应细胞内病原体和 作为模式识别受体的内源性多肽酶DPP9的抑制作用 和一个信号复合体。虽然大多数NLR共享公共域体系结构，但 NLRP1的多功能性和调节需要额外的结构组件。 除了NbD和LRR结构域之外，人NLRP1还含有一个N- 末端比林结构域(PYD)和一个罕见的功能发现结构域(FIIND)，后面跟着一个caspase 激活和招募结构域(CARD)位于其C-末端。唯一的另一种具有 FIND是CARD8，也被证明可以形成炎症体，导致细胞因子 分泌物和细胞死亡。从力学上讲，NLRP1和CARD8结构域是结构性的 催化自身蛋白降解切割，导致形成两个非共价结合的 多肽：自抑性N-末端片段和炎性C-末端片段。 此外，二肽基肽酶8或9(DPP8/9)结合并抑制NLRP1和CARD8，以及 DPP8/9小分子抑制剂诱导NLRP1和CARD8激活 已了解的路径。在这个应用中，我们建议阐明激活和调节 NLRP1和CARD8使用结构指导的方法。我们将确定NLRP1的结构 或CARD8与DPP8或DPP9的复合体，WT和突变体。我们将对结构进行分析 并进行额外的生化和细胞生物学实验来验证我们的假设。 在一个中心目标中，我们提出NLRP1和CARD8是内源的应激传感器 细胞调节失调，在不必要的炎症和疾病中发挥重要作用。

Abstract Nucleotide-binding domain (NBD)-like and leucine-rich repeat (LRR)-containing proteins (NLRs) perform diverse functions in cellular biology, mediating a broad set of fundamental biological pathways from immune signaling to embryonic development. In immunity, several NLRs form supramolecular protein signaling complexes called inflammasomes. Inflammasomes activate caspase-1, an inflammatory protease that processes the cytokine interleukin-1β (IL-1β) and the pore-forming protein gasdermin D to potentiate pyroptotic cell death. One such inflammasome- forming protein, NLRP1, is directly activated in response to intracellular pathogens and the inhibition of DPP9, an endogenous peptidase, serving as both a pattern recognition receptor and a signaling complex. While most NLRs share a common domain architecture, the multifunctionality and regulation of NLRP1 requires additional structural components. In addition to the characteristic NBD and LRR domains, human NLRP1 contains an N- terminal pyrin domain (PYD) and a rare function-to-find domain (FIIND) followed by a caspase activation and recruitment domain (CARD) on its C-terminus. The only other protein with a FIIND is CARD8, which has also been shown to form inflammasomes, leading to cytokine secretion and cell death. Mechanistically, the NLRP1 and CARD8 FIIND domains constitutively catalyze autoproteolytic cleavage, leading to the formation of two noncovalently associated peptides: an autoinhibitory N-terminal fragment and an inflammatory C-terminal fragment. Additionally, Dipeptidyl peptidase 8 or 9 (DPP8/9) binds and inhibits NLRP1 and CARD8, and small molecule inhibitors of DPP8/9 induces NLRP1 and CARD8 activation through some poorly understood pathway. In this application, we propose to elucidate the activation and regulation of NLRP1 and CARD8 using a structure-guided approach. We will determine structures of NLRP1 or CARD8 in complex with DPP8 or DPP9, both WT and mutants. We will analyze the structures and perform additional biochemical and cellular biological experiments to test our hypotheses. In one central aim, we propose that NLRP1 and CARD8 are stress sensors for endogenous cellular dysregulation and play important roles in unwanted inflammation and diseases.