Molecular Bases of Inflammasome Regulation Mediated by ASC Isoforms

ASC 异构体​​介导的炎症小体调节的分子基础

• 批准号: 9810959
• 负责人: Eva de Alba Bastarrechea
• 金额: $ 44.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810959
• 关键词: Adaptor Signaling Protein; Address; Adopted; Adult Respiratory Distress Syndrome; Affect; Amino Acid Sequence; Amino Acids; Atherosclerosis; Autoimmune Diseases; Behavior; Binding; CASP1 gene; Cardiovascular Diseases; Cells; Characteristics; Chemicals; Chronic; Complement; Complex; Cone; Cytokine Activation; Data; Death Domain; Disease; Engineering; Enzyme-Linked Immunosorbent Assay; Fluorescence Anisotropy; Fluorescence Spectroscopy; Glues; Goals; Grant; Homo; Immune; In Vitro; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Injury; Innate Immune Response; Innate Immune System; Interleukin-18; Interleukins; Kinetics; Knowledge; Length; Life; Macromolecular Complexes; Mediating; Mental Depression; Mission; Modeling; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Molecular Structure; Monitor; Motion; Movement; Multiprotein Complexes; Natural Immunity; Nuclear Magnetic Resonance; Nuclear Structure; Peptides; Play; Property; Protein Isoforms; Proteins; Publishing; Regulation; Relaxation; Relaxation Techniques; Resolution; Rheumatoid Arthritis; Role; Signal Transduction; Site; Spectrometry, Mass, Electrospray Ionization; Structure; Testing; Time; Tissues; Transmission Electron Microscopy; United States National Institutes of Health; Vertebral column; base; cancer type; cytokine; design; experience; experimental study; fighting; flexibility; human disease; innovation; nervous system disorder; organizational structure; pathogen; protein complex; protein function; recruit; self assembly; sensor; stoichiometry; therapeutic development; therapeutic target

PROJECT SUMMARY/ABSTRACT. Inflammation is our primary response from the innate immune system to fight infection and self-protect from damage. However, dysfunctional regulation of inflammation results in disease, including certain types of cancer, autoimmune, cardiovascular and neurological disorders, rheumatoid arthritis, and even depression. The onset of inflammation depends on the assembly of a multiprotein complex known as the inflammasome. The main players in inflammasome assembly are; - sensor proteins that react upon danger signals derived from pathogens or damaged tissue; - procaspase-1 that activates inflammatory cytokines as a result of inflammasome assembly; - the adapter ASC that functions like a molecular glue by connecting sensor and procaspase-1 molecules. Canonical ASC has an isoform, which shows different self-assembly capabilities and modulates the intensity of inflammation in the cellular context. The presence of protein isoforms is a well-known, natural mechanism for the regulation of protein function. Both proteins are bimodular with two Death Domains connected by a linker, and their amino acid sequences differ solely in the linker length. Canonical ASC has a 23 amino acid-long linker, whereas its isoform has a shorter linker of 3 amino acids (ASC_short). We demonstrated that ASC linker plays a key role in defining the orientation of its domains, and show in this proposal that both domains actively participate in ASC self-assembly to form filamentous macrostructures. Evidence indicate that inflammasomes assemble into different supramolecular structures. However, little is known on the factors controlling these structural arrangements, or how the different assemblies impact inflammation. This proposal aims at addressing a knowledge gap in the role of ASC isoforms on inflammasome structural organization and inflammatory response. The long-term goal of this proposal is to gain in-depth knowledge on the interplay between the inflammasome components to understand its function and regulation, which is of great significance to set the grounds for the development of therapeutics to control inflammation. The objective of this grant is innovative because it will decipher the unknown molecular bases for inflammasome regulation mediated by ASC isoforms. Our hypothesis is that; 1) the linker length has important implications in the interaction properties of ASC and ASC_short at the molecular level, which can account for the observed alteration of the inflammatory response, 2) the linker length leads to differences in the interdomain dynamics of the two isoforms and in the resulting macrostructures. To test this hypothesis we propose to; 1) determine the inflammatory activity, precise self-association and interacting capabilities of ASC isoforms, including ASC, ASC_short and an artificial form of ASC engineered with a long linker (3 times the canonical linker length: ASC_long) as a reference for independent domains, 2) determine the interdomain dynamics of the three isoforms using Nuclear Magnetic Resonance; 3) discern with Transmission Electron Microscopy the potentially different characteristics of the macrostructures resulting from ASC isoforms self-association, and their implication in inflammatory activity.

项目总结/摘要。炎症是我们先天免疫系统的主要反应， 抵抗感染和自我保护免受伤害。然而，炎症调节功能失调导致 疾病，包括某些类型的癌症，自身免疫性，心血管和神经系统疾病，类风湿性关节炎 关节炎甚至抑郁症炎症的发生依赖于多蛋白复合物的组装 称为炎性小体。炎性小体组装中的主要参与者是： 来自病原体或受损组织的危险信号; -激活炎性细胞因子的半胱天冬酶原-1 作为炎性小体组装的结果; -适配器ASC，其通过连接 传感器和半胱氨酸天冬氨酸蛋白酶原-1分子。典型的ASC有一个异构体，它显示出不同的自组装 在细胞环境中调节炎症的强度。蛋白质异构体的存在 是一种众所周知的调节蛋白质功能的天然机制。两种蛋白质都是双模的， 死亡结构域通过接头连接，并且它们的氨基酸序列仅在接头长度上不同。Canonical ASC具有23个氨基酸长的接头，而其同种型具有3个氨基酸的较短接头（ASC_short）。我们 证明了ASC接头在定义其结构域的方向方面起着关键作用，并在此建议中显示 这两个结构域都积极参与ASC自组装形成丝状宏观结构。证据 表明炎性小体组装成不同超分子结构。然而，人们对这一问题知之甚少。 控制这些结构排列的因素，或者不同的组件如何影响炎症。这 该提案旨在解决ASC亚型对炎性小体结构的作用方面的知识空白， 组织和炎症反应。本提案的长期目标是深入了解 炎性小体组分之间的相互作用，以了解其功能和调节，这是非常重要的 这对于为控制炎症的治疗方法的发展奠定基础具有重要意义。的目的 格兰特是创新的，因为它将破译未知的分子基础，炎症体调节介导 通过ASC同种型。我们的假设是：1）接头长度对相互作用有重要影响 ASC和ASC_short在分子水平上的性质，这可以解释观察到的 2）接头长度导致两种亚型的结构域间动力学差异 以及由此产生的宏观结构。为了验证这一假设，我们建议：1）确定炎症 活性、精确的自缔合和ASC亚型的相互作用能力，包括ASC、ASC_short和ASC_sort。 用长接头（3倍规范接头长度：ASC_long）工程化的ASC的人工形式作为参照 对于独立畴，2）使用核磁共振确定三种异构体的畴间动力学 共振; 3）用透射电子显微镜辨别共振的潜在不同特征。 由ASC同种型自缔合产生的宏观结构，以及它们在炎症活性中的意义。