Identification and structural characterization of the function of isoforms ASCc and ASCd in inflammasome regulation

亚型 ASCc 和 ASCd 在炎症小体调节中的功能鉴定和结构表征

基本信息

批准号：
10062397
负责人：
Eva de Alba Bastarrechea
金额：
$ 13.71万
依托单位：
UNIVERSITY OF CALIFORNIA, MERCED
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-24 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062397
关键词：
Adaptor Signaling Protein Address Adult Respiratory Distress Syndrome Affinity Amino Acid Sequence Amino Acids Atherosclerosis Autoimmune Diseases Behavior Binding Cardiovascular Diseases Cells Characteristics Chemicals Chronic Complex Computing Methodologies Cytokine Activation Data Reporting Death Domain Disease Dissociation Future Glues Grant Immune Infection Inflammasome Inflammation Inflammatory Inflammatory Response Injury Innate Immune Response Innate Immune System Kinetics Knowledge Length Life Measures Mediating Mental Depression Mission Molecular Molecular Conformation Molecular Structure Multiprotein Complexes Natural Immunity Parents Peptides Protein Isoforms Proteins Regulation Research Resolution Rheumatoid Arthritis Role Signal Transduction Site Structure Study Subject Techniques Testing Tissues Titrations Transmission Electron Microscopy United States National Institutes of Health base cancer type cytokine design experimental study fighting human disease inhibitor/antagonist innovation nervous system disorder pathogen protein complex protein function recruit response sensor structured data therapeutic target three dimensional structure tissue injury

项目摘要

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. ASC exists in two inflammasome activating isoforms; canonical ASC and ASC_short (both subject of study of the parent R15), and has two more isoforms, one that inhibits inflammasome formation (ASCc) and one isoform with unknown function (ASCd). The presence of protein isoforms is a well- known, natural mechanism for the regulation of protein function. However, little is known on the factors controlling the formation of the inflammasome at the level of the adapter and its isoforms, or how these isoforms impact inflammation. In the parent R15 grant we proposed to study the regulation of the inflammasome via its two activating isoforms (canonical ASC and ASC_short). Both bimodular proteins have two Death Domains connected by a linker, and their amino acid sequences differ solely in the linker length. Our recent findings resulting from the parent R15 indicate that the two isoforms have significantly different kinetics of oligomerization, which in turn correlate with their different dynamic behavior. Our results provide the first explanation at the molecular level of why these isoforms activate the inflammasome to different extent based on their respective oligomerizing capabilities. This Diversity Supplement proposal aims at addressing a knowledge gap in the role of the other two ASC isoforms (ASCc and ASCd) on inflammasome formation and thus in the inflammatory response. The objective of this proposal is innovative because it will decipher the unknown molecular bases for inflammasome regulation mediated by ASCc and ASCd. Our hypotheses are that; 1) ASCc, with an intact Death Domain, inhibits inflammasome formation by competing with ASC self-association, which can account for the observed decreased in the inflammatory response, 2) Based on our preliminary studies, ASCd is an intrinsically disordered protein (IDP), thus we propose that ASCd can fold upon binding to ASC, a typical mechanism of IDPs, resulting in a more moderate inhibition. To test these hypotheses we propose to; 1) study at the atomic level the structural characteristic of ASCc and ASCd with NMR, and 2) quantitatively determine their potential interactions with ASC (identifying the interacting regions and measuring the affinity of the complexes) and 3) discern with Transmission Electron Microscopy the potentially different characteristics of the macrostructures formed by canonical ASC in the presence of ASCc and ASCd.

项目摘要/摘要。炎症是我们先天免疫系统的主要反应对抗感染并自我保护免受伤害。然而，炎症调节功能失调会导致疾病，包括某些类型的癌症、自身免疫性疾病、心血管和神经系统疾病、类风湿病关节炎，甚至抑郁症。炎症的发生取决于多蛋白复合物的组装称为炎症小体。炎症小体组装的主要参与者是： - 反应的传感器蛋白来自病原体或受损组织的危险信号； - 激活炎症细胞因子的 procaspase-1 由于炎性小体组装的结果； - 适配器 ASC 通过连接发挥类似分子胶的作用传感器和 procaspase-1 分子。 ASC 存在两种炎性体激活亚型；规范 ASC 和 ASC_short（都是母体 R15 的研究对象），并且还有两种同工型，一种抑制炎症小体形成（ASCc）和一种功能未知的亚型（ASCd）。蛋白质亚型的存在是一个很好的调节蛋白质功能的已知天然机制。然而，人们对控制因素知之甚少接头及其同工型水平上炎症小体的形成，或这些同工型如何影响炎。在父 R15 资助中，我们建议通过其两个途径来研究炎症小体的调节激活亚型（规范 ASC 和 ASC_short）。两种双模块蛋白都有两个死亡域通过接头连接，并且它们的氨基酸序列仅在接头长度上不同。我们最近的发现来自母体 R15 的结果表明，两种异构体具有显着不同的寡聚动力学，这又与它们不同的动态行为相关。我们的结果提供了第一个解释分子水平上解释了为什么这些亚型根据各自的不同程度不同程度地激活炎症小体低聚能力。该多样性补充提案旨在解决角色中的知识差距其他两种 ASC 亚型（ASCc 和 ASCd）对炎症小体形成的影响，从而影响炎症反应回复。该提案的目标是创新的，因为它将破译未知的分子基础由 ASCc 和 ASCd 介导的炎症小体调节。我们的假设是： 1) ASCc，具有完整的死亡结构域，通过与 ASC 自缔合竞争来抑制炎症小体形成，这可以解释观察到炎症反应减少，2) 根据我们的初步研究，ASCd 本质上是一种无序蛋白（IDP），因此我们提出 ASCd 可以在与 ASC 结合后折叠，这是 ASCd 的典型机制 IDPs，产生更温和的抑制。为了检验这些假设，我们建议： 1）原子研究通过 NMR 水平确定 ASCc 和 ASCd 的结构特征，2) 定量确定它们的潜力与 ASC 的相互作用（识别相互作用区域并测量复合物的亲和力）和 3) 使用透射电子显微镜辨别宏观结构的潜在不同特征由规范 ASC 在 ASCc 和 ASCd 存在的情况下形成。