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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对抵抗感染，自我保护免受损害。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、心血管和神经疾病、类风湿关节炎，甚至抑郁症。炎症的发生取决于多蛋白复合体的组装。被称为炎症症。在炎症体组装中的主要角色是；-感受器蛋白，它与来自病原体或受损组织的危险信号；-激活炎症细胞因子的原天冬氨酸酶-1 作为炎症体组装的结果；-适配器ASC通过连接起到分子胶的作用感应器和proaspase-1分子。Asc存在于两种炎症体激活亚型：典型性Asc和 Asc_Short(都是亲本R15的研究对象)，还有两种异构体，一种抑制炎症体形成(ASCC)和一个功能未知的异构体(ASCD)。蛋白质异构体的存在是一个很好的- 已知的，调节蛋白质功能的自然机制。然而，人们对其控制因素知之甚少。在接头及其异构体水平上炎性小体的形成，或这些异构体如何影响发炎。在母方R15拨款中，我们建议通过其两个分支研究炎症小体的调节激活异构体(规范Asc和Asc_Short)。这两种双峰蛋白都有两个死亡结构域由连接子连接，并且它们的氨基酸序列仅在连接子长度上不同。我们最新的发现来自母体R15的结果表明两种异构体具有显著不同的齐聚动力学，这反过来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这些异构体激活炎症体的不同程度的分子水平取决于它们各自的齐聚能力。这项多样性补充提案旨在解决角色中的知识差距另外两种ASC亚型(ASCC和ASCD)在炎症性小体形成中的作用回应。这项提议的目的是创新的，因为它将破译未知的分子基础 ASCC和ASCD介导的炎性小体调节。我们的假设是：1)ASCC，死亡完好无损结构域，通过与ASC自结合竞争来抑制炎症体的形成，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)，因此我们认为ASCD在与ASC结合时可以折叠，这是ASCD的一种典型机制国内流离失所者，导致了更适度的抑制。为了验证这些假设，我们建议：1)研究原子用核磁共振对ASCC和ASCD的结构特征进行分级，2)定量测定它们的潜力与ASC的相互作用(确定相互作用区域并测量络合物的亲和力)和3) 用透射电子显微镜识别宏观结构的潜在不同特征由规范的ASC在ASCC和ASCD存在下形成的。