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

Gasdermin E 的结构研究及其被 caspase-3 识别。

• 批准号: 10571048
• 负责人: Tsan Sam Xiao
• 金额: $ 8.05万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-03 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10571048
• 关键词: Address; Adopted; Apoptosis; Apoptotic; Autoimmune Diseases; Bacterial Infections; Binding; Biochemical; Biological Assay; CASP1 gene; CASP3 gene; Caspase; Cell Death; Cell membrane; Cellular biology; Complex; Crohn' s disease; Crystallization; Crystallography; Data; Defense Mechanisms; Development; E protein; Epithelial Cells; Extravasation; Family; Family member; Host Defense; Human; Immune; Inflammasome; Inflammatory; Injury to Kidney; Knowledge; Length; Liposomes; Lytic; Mediating; Membrane; Molecular; Molecular Conformation; Mucositis; Mus; Mutagenesis; Pathogenesis; Peptide Hydrolases; Peptides; Play; Reagent; Reporting; Rest; Role; Signal Pathway; Site; Structure; Techniques; Testing; Virus Diseases; X ray diffraction analysis; X-Ray Crystallography; cytokine; enzyme substrate complex; inhibitor; insight; intestinal epithelium; kidney fibrosis; member; membrane assembly; microbial; prevent; recruit; success; tissue injury; uptake

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.

抽象的 规范和非规范的炎性体信号传导途径可以诱导和分泌 炎性细胞因子，在微生物杀伤和 清除。凋亡是一种由Gasdermin家族成员介导的调节的裂解细胞死亡 蛋白酶裂解后，膜孔组装。 Gasdermin E（GSDME）可以通过 凋亡病例-3和7，然后触发质膜孔的形成和凋亡而不是 凋亡。 GSDME介导的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已隐含在粘膜中 克罗恩氏病的炎症和发病机理以及GSDME介导的凋亡有助于肾脏 纤维化和肾脏损伤。尽管我们对招聘和认可的理解最近取得了进步 Gasdermin d（GSDMD）通过炎症胱天蛋白酶，GSDME识别的分子机制和 凋亡胱天蛋白酶的裂解的定义很差。该建议旨在解决我们的关键差距 使用完整的结构功能方法的知识。我们假设无活动的GSDME是 通过分子内氨基末端结构域（NTD） - 羧基维持在自身抑制的构象中 末端结构域（CTD）相互作用，该相互作用被裂片在NTD-CTD处裂解的凋亡caspase-3识别 链接区域以释放自身抑制。这项研究将探讨GSDME的结构机制 caspase-3自身抑制及其识别以检验上述假设。建立以下特定目标 关于我们过去和正在进行的关于毒气和凋亡的研究。 AIM 1将表征分子 GSDME自身抑制的机制。我们的初步数据表明，GSDME蛋白可以结晶，并且 晶体衍射的X射线很好。我们将使用使用GSDME在其自身抑制状态的结构 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摄取和脂质体泄漏的残留物。 AIM 2将定义 caspase-3的GSDME识别。我们将通过 生化和细胞生物学技术，并确定与caspase-3复合物中GSDME的结构 使用X射线晶体学。该提案的成功不仅会揭示对GSDME的重要见解 凋亡胱天蛋白酶的自身抑制和识别，使田间向前移动，但也有助于 基于独特的GSDME-Caspase-3界面，开发凋亡caspase-3的特异性抑制剂， 这可能是对凋亡和凋亡信号通路的研究中有价值的试剂。