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: 10223159
• 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/10223159
• 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; Immune; Immune signaling; Immunity; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Interleukin-1 beta; Interleukin-18; Investigation; Knowledge; Lead; Length; Lipid Binding; Lytic; Masks; 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; base; cell type; cytokine; enzyme substrate; enzyme substrate complex; insight; macrophage; 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 该提案的目标是研究焦亡依赖性和新型焦亡的机制， 由半胱天冬酶、分泌性自噬蛋白介导的促炎细胞因子的独立输出， gasdermin D（GSDMD），最近鉴定的焦亡效应分子。炎性小体是关键 先天性免疫信号平台涉及对感染和自身免疫的免疫防御， 自身炎性疾病，如炎性肠病、多发性硬化和阿尔茨海默病。 炎性体信号传导途径的激活导致细胞因子的成熟和分泌， IL-1β和IL-18，以及在某些细胞类型中称为细胞凋亡的程序性细胞死亡的炎性形式。 GSDMD在被炎性半胱天冬酶-1，4，5和11以及半胱天冬酶-8切割后组装膜孔。 在巨噬细胞中，这种孔允许在细胞溶解时释放成熟细胞因子。有趣的是， GSDMD在炎性小体激活后分泌IL-1β期间的膜孔非依赖性功能， 中性粒细胞、上皮细胞和T细胞（项目1、2和4），其中招募GSDMD 通过分泌性自噬蛋白促进细胞因子释放的新的非溶解形式。特异性识别 因此，通过半胱天冬酶或自噬蛋白的GSDMD是GSDMD在裂解或非裂解细胞因子释放中的功能的基础。 我们假设炎症性半胱天冬酶通过不同的蛋白质-蛋白质界面识别GSDMD， 溶解性细胞因子释放。相比之下，GSDMD被自噬机制招募，以促进新的非溶解性细胞凋亡。 细胞因子分泌独立于膜孔形成。我们将集中在以下具体目标， 我们的合作计划项目，以测试上述假设。目标1.定义蛋白酶的机制- GSDMD在细胞因子的裂解释放中的依赖性活化。我们建议阐明的机制 半胱天冬酶和丝氨酸蛋白酶与项目1、2和4合作识别GSDMD。发现从 我们的建议可能有助于基于不同酶底物的特异性半胱天冬酶抑制剂的研究 界面，其可能靶向焦亡、凋亡和其他炎症信号通路， 半胱天冬酶目标二。明确GSDMD引导的细胞因子非溶解性分泌的机制。我们建议 通过分子伴侣和自噬蛋白表征IL-1β和GSDMD的募集。这些角色 将与项目2和4合作探讨非溶解性细胞因子释放中的相互作用。的成功 该项目将揭示促炎细胞因子溶解性或非溶解性分泌的分子机制 由GSDMD介导。本建议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。从我们的研究中获得的结构见解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，这四个项目的成果作为一个整体， 相互依存的综合方案。