NLRP1 and CARD8 Inflammasomes: Assembly, Regulation and Stress Sensing

摘要 含有核苷酸结合域 (NBD) 样和富含亮氨酸重复序列 (LRR) 的蛋白质 (NLR) 在细胞生物学中执行多种功能，介导广泛的基本生物学 从免疫信号传导到胚胎发育的途径。在免疫中，多个 NLR 形成 超分子蛋白信号复合物称为炎症小体。炎症小体激活 caspase-1，一种处理细胞因子白介素-1β (IL-1β) 和 成孔蛋白gasdermin D 可增强焦亡细胞死亡。一种这样的炎症小体—— 形成蛋白 NLRP1 直接激活以响应细胞内病原体和 抑制 DPP9（一种内源性肽酶），同时充当模式识别受体 和信号复合体。虽然大多数 NLR 共享一个公共域架构， NLRP1 的多功能性和调节需要额外的结构组件。 除了特征性的 NBD 和 LRR 结构域外，人类 NLRP1 还包含一个 N- 末端热蛋白结构域 (PYD) 和稀有功能查找结构域 (FIIND)，后跟半胱天冬酶 其 C 末端有激活和招募结构域 (CARD)。唯一具有以下功能的其他蛋白质 FIIND 是 CARD8，它也被证明可以形成炎症小体，从而导致细胞因子 分泌和细胞死亡。从机制上讲，NLRP1 和 CARD8 FIIND 结构域组成 催化自蛋白水解裂解，导致形成两个非共价相关的 肽：一个自抑制性 N 端片段和一个炎性 C 端片段。 此外，二肽基肽酶 8 或 9 (DPP8/9) 结合并抑制 NLRP1 和 CARD8，并且 DPP8/9 的小分子抑制剂通过一些不良作用诱导 NLRP1 和 CARD8 激活 了解途径。在此应用中，我们建议阐明 NLRP1 和 CARD8 使用结构引导方法。我们将确定NLRP1的结构 或 CARD8 与 DPP8 或 DPP9 的复合物，包括 WT 和突变体。我们将分析结构 并进行额外的生化和细胞生物学实验来检验我们的假设。 在一个中心目标中，我们提出 NLRP1 和 CARD8 是内源性压力传感器 细胞失调并在不良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.