Structural studies of gasdermin E and its recognition by caspase-3.

摘要 经典和非经典炎性体信号传导途径可诱导细胞凋亡和分泌炎性细胞因子。 炎性细胞因子，其在微生物杀伤中作为重要的免疫防御机制起作用， 间隙细胞凋亡是一种由gasdermin家族成员介导的调节性溶解性细胞死亡 其在被蛋白酶切割时组装膜孔。Gasdermin E（GSDME）可以通过以下方式裂解： 凋亡的半胱天冬酶3和7，然后触发质膜孔形成和细胞凋亡，而不是 凋亡GSDME介导的细胞凋亡和细胞因子释放在宿主防御病毒或 细菌感染另一方面，肠上皮细胞中的GSDME与粘膜上皮细胞中的GSDME有关。 克罗恩病的炎症和发病机制，GSDME介导的焦亡有助于肾 纤维化和肾损伤。尽管我们最近在了解招募和承认 Gasdermin D（GSDMD）通过炎症性半胱天冬酶，GSDME识别的分子机制， 细胞凋亡半胱天冬酶的裂解还不清楚。该提案旨在解决我们在以下方面的关键差距： 使用互补结构-功能方法的知识。我们假设不活跃的GSDME是 通过分子内氨基末端结构域（NTD）-羧基-维持自抑制构象 末端结构域（CTD）相互作用，由在NTD-CTD处裂解的凋亡caspase-3识别 连接子区域以释放自身抑制。本研究将探讨GSDME的结构机制 自抑制及其被半胱天冬酶-3识别以检验上述假设。具体目标如下： 我们过去和正在进行的关于gasdermins和pyroptosis的研究目标1将表征分子 GSDME自抑制机制。我们的初步数据表明，GSDME蛋白可以结晶， 晶体很好地衍射X射线。我们将确定GSDME在其自抑制状态下的结构， 晶体学方法，并通过界面的诱变研究验证NTD-CTD界面 使用LDH释放、PI摄取和脂质体渗漏测定法测定残留物。目标2将定义 通过caspase-3识别GSDME。我们将探讨GSDME和caspase-3之间的相互作用， 生物化学和细胞生物学技术，并确定GSDME与caspase-3复合物的结构 使用X射线晶体学。这一提案的成功不仅将揭示对GSDME的重要见解， 凋亡半胱天冬酶的自身抑制和识别，使该领域向前发展，但也促进了细胞凋亡。 基于不同的GSDME-caspase-3界面开发凋亡caspase-3的特异性抑制剂， 这可能是研究细胞凋亡和细胞凋亡信号通路的有价值的试剂。

Abstract The canonical and noncanonical inflammasome signaling pathways can induce pyroptosis and secretion of inflammatory cytokines, which function as crucial immune defense mechanisms in microbial killing and clearance. Pyroptosis is a type of regulated lytic cell death that is mediated by members of the gasdermin family that assemble membrane pores upon cleavage by proteases. Gasdermin E (GSDME) can be cleaved by apoptotic caspases-3 and 7, which then triggers plasma membrane pore formation and pyroptosis instead of apoptosis. GSDME-mediated pyroptosis and cytokine release play crucial roles in host defense against viral or bacterial infections. On the other hand, GSDME in intestinal epithelial cells has been implicated in mucosal inflammation and pathogenesis of Crohn’s disease, and GSDME-mediated pyroptosis contributes to renal fibrosis and kidney injury. Despite recent progress in our understanding of the recruitment and recognition of gasdermin D (GSDMD) by inflammatory caspases, the molecular mechanisms of GSDME recognition and cleavage by apoptotic caspases have been poorly defined. This proposal aims to address the critical gaps in our knowledge using complementary structure-function approaches. We hypothesize that inactive GSDME is maintained in an autoinhibited conformation through intramolecular amino-terminal domain (NTD)-carboxy- terminal domain (CTD) interactions, which is recognized by apoptotic caspase-3 that cleaves at the NTD-CTD linker region to release the autoinhibition. This study will explore the structural mechanisms of GSDME autoinhibition and its recognition by caspase-3 to test the above hypothesis. The following specific aims are built on our past and ongoing studies on gasdermins and pyroptosis. Aim 1 will characterize the molecular mechanisms of GSDME autoinhibition. Our preliminary data show that GSDME protein can be crystallized and the crystals diffracted X-ray well. We will determine the structure of GSDME at its autoinhibitory state using crystallographic approaches, and validate the NTD-CTD interface through mutagenesis studies of the interface residues using LDH release, PI uptake, and liposome leakage assays. Aim 2 will define the mechanisms of GSDME recognition by caspase-3. We will probe the interaction between GSDME and caspase-3 through biochemical and cell biology techniques, and determine the structure of GSDME in complex with caspase-3 using X-ray crystallography. The success of this proposal will not only reveal important insights into GSDME autoinhibition and recognition by apoptotic caspases that move the field forward, but also facilitate the development of specific inhibitors for apoptotic caspase-3 based on the distinct GSDME-caspase-3 interface, which may be valuable reagents in the study of both pyroptosis and apoptosis signaling pathways.