NLRP1 and CARD8 Inflammasomes: Assembly, Regulation and Stress Sensing

摘要 含核苷酸结合域（NBD）样和富含亮氨酸重复序列（LRR）的蛋白质（NLR） 在细胞生物学中发挥多种功能，介导一系列广泛的基本生物学过程。 从免疫信号传导到胚胎发育的途径。在免疫中，几个NLR形成 超分子蛋白质信号复合物称为炎性小体。炎性小体激活 半胱天冬酶-1，一种炎症蛋白酶，其处理细胞因子白细胞介素-1 β（IL-1β）和细胞因子白细胞介素-1 β。 孔形成蛋白gasdermin D，以增强热变性细胞死亡。一个这样的炎性小体- 形成蛋白NLRP 1直接响应于细胞内病原体而被激活， 抑制DPP 9，一种内源性肽酶，作为模式识别受体 和信号复合物。虽然大多数NLR共享一个通用的域体系结构， NLRP 1的多功能性和调节需要额外的结构组分。 除了特征性的NBD和LRR结构域之外，人NLRP 1还含有一个N- 末端pyrin结构域（PYD）和一个罕见的功能发现结构域（FIIND），随后是一个半胱天冬酶 激活和募集结构域（CARD）。另一种蛋白质 FIIND是CARD 8，其也已被证明形成炎性小体，导致细胞因子 分泌和细胞死亡。从机制上讲，NLRP 1和CARD 8 FIIND结构域组成性地 催化自身蛋白水解裂解，导致形成两个非共价缔合的 肽：自身抑制性N-末端片段和炎性C-末端片段。 另外，二肽基肽酶8或9（DPP 8/9）结合并抑制NLRP 1和CARD 8， DPP 8/9的小分子抑制剂诱导NLRP 1和CARD 8活化，通过一些不良的 理解路径在本申请中，我们建议阐明 NLRP 1和CARD 8使用结构引导的方法。我们将确定NLRP 1的结构 或与DPP 8或DPP 9复合的CARD 8，WT和突变体。我们将分析 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NLRP 1和CARD 8是内源性的应激传感器， 细胞失调，并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.