Molecular mechanisms of gasdermin recognition by proteases and autophagy proteins in cytokine release

细胞因子释放中蛋白酶和自噬蛋白识别gasdermin的分子机制

• 批准号: 10654580
• 负责人: Tsan Sam Xiao
• 金额: $ 40.25万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-24 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654580
• 关键词: Alzheimer' s Disease; Apoptosis; Autoimmune; Autophagocytosis; Binding Sites; CASP1 gene; CASP3 gene; CASP8 gene; Caspase; Caspase Inhibitor; Catalytic Domain; Cell Death; Cell membrane; Cells; Collaborations; Complex; Crystallization; Cytolysis; Data; Data Analyses; Disease; Epithelial Cells; Event; Experimental Designs; Goals; Human; IL18 gene; Immune; Immune signaling; Immunity; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interleukin-1 beta; Investigation; Knowledge; Lead; Length; Lipid Binding; Lytic; Macrophage; Mediating; Membrane; Molecular; Molecular Chaperones; Multiple Sclerosis; Mus; Neutrophil Activation; Nonlytic; Outcome; Peptide Hydrolases; Peptide Signal Sequences; Play; Proteins; Proteomics; Regulation; Reporting; Role; Serine Protease; Signal Pathway; Signal Transduction; Site; Structure; T-Lymphocyte; Testing; Tissues; Vesicle; autoinflammatory diseases; cell type; cytokine; enzyme substrate; enzyme substrate complex; inflammatory modulation; insight; mast cell; membrane assembly; neutrophil; novel; pathogen; programs; receptor; recruit; repaired; success; therapeutic development; therapeutic target

Abstract The goal of this proposal is to investigate mechanisms for pyroptosis-dependent and novel pyroptosis- independent export of proinflammatory cytokines mediated by caspases, secretory autophagy proteins, and gasdermin D (GSDMD), a recently identified effector molecule for pyroptosis. The inflammasomes are crucial innate immune signaling platforms implicated in immune defense against infections and autoimmune/ autoinflammatory disorders such as inflammatory bowel disease, multiple sclerosis, and Alzheimer’s disease. Activation of the inflammasome signaling pathways leads to the maturation and secretion of cytokines such as IL-1β and IL-18, and an inflammatory form of programmed cell death in certain cell types called pyroptosis. GSDMD assembles membrane pores upon cleavage by inflammatory caspases-1, 4, 5 and 11 and caspase-8. In macrophages, such pores allow the release of mature cytokines upon cytolysis. Intriguingly, pyroptosis- and membrane pore-independent function of GSDMD during secretion of IL-1β upon inflammasome activation has been demonstrated for neutrophils, epithelial cells and T cells (Projects 1, 2, and 4), in which GSDMD is recruited by secretory autophagy proteins to facilitate a novel non-lytic form of cytokine release. Specific recognition of GSDMD by caspases or autophagy proteins thus underlie GSDMD function in lytic or non-lytic cytokine release. We hypothesize that inflammatory caspases recognize GSDMD through distinct protein-protein interfaces during lytic cytokine release. By contrast, GSDMD is recruited by autophagy machinery to facilitate novel non-lytic cytokine secretion independent of membrane pore formation. We will focus on the following specific aims in a collaborative Program Project to test the above hypothesis. Aim 1. Define the mechanisms of protease- dependent activation of GSDMD in lytic release of cytokines. We propose to elucidate the mechanisms of GSDMD recognition by caspases and serine proteases in collaboration with Projects 1, 2 and 4. Discovery from our proposal may facilitate investigation into specific caspase inhibitors based on distinct enzyme-substrate interfaces, which may target pyroptosis, apoptosis and other inflammatory signaling pathways involving caspases. Aim 2. Define the mechanisms of GSDMD-guided non-lytic secretion of cytokines. We propose to characterize the recruitment of IL-1β and GSDMD by chaperones and autophagy proteins. The roles of these interaction in non-lytic cytokine release will be probed in collaboration with Projects 2 and 4. The success of this project will reveal the molecular mechanisms for the lytic or non-lytic secretion of proinflammatory cytokines mediated by GSDMD. This proposal draws on the strength of the other three Projects to facilitate and validate our structural studies. Structural insights from our studies will in turn inform experimental design and data interpretation for the other Projects. As such, outcomes from the four Projects benefit each other as an interdependent and integrated Program.

摘要 这项建议的目标是研究上睑下垂依赖和新型上睑下垂的机制。 由caspase、分泌性自噬蛋白和 Gasdermin D(GSDMD)，最近发现的一种治疗上睑下垂的效应分子。炎性小体至关重要 天然免疫信号平台参与免疫防御感染和自身免疫/ 自身炎症性疾病，如炎症性肠病、多发性硬化症和阿尔茨海默病。 炎症小体信号通路的激活导致细胞因子的成熟和分泌，如 IL-1β和IL-18，以及某些细胞类型中的一种炎性形式的程序性细胞死亡，称为下垂。 GSDMD在炎症性caspase-1、4、5和11以及caspase-8的作用下组装膜孔。 在巨噬细胞中，这种毛孔允许在细胞溶解时释放成熟的细胞因子。耐人寻味的是，上睑下垂 炎症小体激活时GSDMD在分泌IL-1β过程中的膜孔非依赖性作用 已证实中性粒细胞、上皮细胞和T细胞(方案1、2和4)，其中招募了GSDMD 通过分泌型自噬蛋白促进一种新的非裂解形式的细胞因子的释放。具体认识到 因此，半胱氨酸酶或自噬蛋白导致的GSDMD在裂解性或非裂解性细胞因子的释放中起重要作用。 我们假设炎性半胱氨酸酶通过不同的蛋白质-蛋白质界面识别GSDMD 裂解细胞因子的释放。相比之下，GSDMD通过自噬机制招募，以促进新的非裂解 细胞因子的分泌不依赖于膜孔的形成。我们将重点关注以下具体目标 协作计划项目来验证上述假设。目的1.确定蛋白酶的作用机制-- GSDMD在细胞因子裂解释放中的依赖激活。我们建议阐明以下机制： 与项目1、2和4合作，通过半胱氨酸酶和丝氨酸蛋白酶识别GSDMD。 我们的建议可能有助于基于不同酶-底物的特定caspase抑制剂的研究。 接口，可能针对下垂、细胞凋亡和其他炎性信号通路，涉及 半胱氨酸酶。目的2.明确GSDMD诱导细胞因子非溶血性分泌的机制。我们建议 研究伴侣蛋白和自噬蛋白对IL-1、β和GSDMD的募集作用。这些角色的作用 将与项目2和项目4合作探讨非溶血性细胞因子释放中的相互作用。 该项目将揭示促炎症细胞因子裂解或非裂解分泌的分子机制 由GSDMD调解。该提案借鉴了其他三个项目的优势，以促进和验证 我们的结构研究。我们研究的结构洞察力将反过来为实验设计和数据提供信息 其他项目的解释。因此，四个项目的成果是互惠互利的 相互依存、相互融合的规划。