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: 10024454
• 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/10024454
• 关键词: Alzheimer' s Disease; Apoptosis; Autoimmune Process; 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.

抽象的 该提案的目标是研究细胞焦亡依赖性和新型细胞焦亡的机制- 由半胱天冬酶、分泌性自噬蛋白和分泌性自噬蛋白介导的促炎细胞因子的独立输出 Gasdermin D (GSDMD)，一种最近发现的焦亡效应分子。炎症小体至关重要 先天免疫信号平台涉及针对感染和自身免疫的免疫防御/ 自身炎症性疾病，例如炎症性肠病、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，例如 IL-1β 和 IL-18，以及某些细胞类型中称为细胞焦亡的炎症形式的程序性细胞死亡。 GSDMD 在炎症性 caspase-1、4、5 和 11 以及 caspase-8 裂解后组装膜孔。 在巨噬细胞中，这种孔允许在细胞溶解时释放成熟的细胞因子。有趣的是，焦亡和 GSDMD 在炎症小体激活后分泌 IL-1β 过程中具有独立于膜孔的功能 已在中性粒细胞、上皮细胞和 T 细胞中得到证实（项目 1、2 和 4），其中招募了 GSDMD 通过分泌性自噬蛋白促进细胞因子释放的新型非裂解形式。具体认定 因此，由半胱天冬酶或自噬蛋白引起的 GSDMD 是 GSDMD 在溶解或非溶解细胞因子释放中发挥作用的基础。 我们假设炎性半胱天冬酶在炎症过程中通过不同的蛋白质-蛋白质界面识别 GSDMD 裂解性细胞因子释放。相比之下，GSDMD 被自噬机制招募以促进新型非裂解性 细胞因子的分泌与膜孔的形成无关。我们将着力实现以下具体目标： 协作计划项目来检验上述假设。目标 1. 定义蛋白酶的机制 GSDMD 依赖于细胞因子裂解释放的激活。我们建议阐明其机制 与项目 1、2 和 4 合作，通过半胱天冬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半胱天冬酶抑制剂的研究 界面，可能针对焦亡、细胞凋亡和其他涉及的炎症信号通路 半胱天冬酶。目标 2. 定义 GSDMD 引导的细胞因子非裂解性分泌的机制。我们建议 描述了分子伴侣和自噬蛋白对 IL-1β 和 GSDMD 的招募。这些角色的作用 将与项目 2 和 4 合作探讨非裂解性细胞因子释放中的相互作用。该项目的成功 该项目将揭示促炎细胞因子溶解或非溶解分泌的分子机制 由 GSDMD 介导。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。我们研究的结构见解将为实验设计和数据提供信息 对其他项目的解释。因此，这四个项目的成果是互惠互利的 相互依存和综合的计划。