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与损害抗击感染和自我保护。但是，炎症功能失调导致疾病，包括某些类型的癌症，自身免疫性，心血管和神经系统疾病，类风湿病关节炎，甚至抑郁症。炎症的发作取决于多蛋白复合物的组装被称为炎性体。炎症大会的主要参与者是； - 反应的传感器蛋白来自病原体或组织受损的危险信号； - 激活炎症细胞因子的procaspase-1 由于炎症体组装的结果； - 通过连接的适配器ASC的作用像分子胶传感器和procaspase-1分子。 ASC存在于两个炎症体激活同工型中；典型的ASC和 ASC_SHORT（均为母体R15的研究主题），还有另外两种同工型，一种抑制炎症体形成（ASCC）和一个具有未知函数（ASCD）的同工型。蛋白质同工型的存在是一种很好的已知的自然机制，用于调节蛋白质功能。但是，在控制因素上鲜为人知在适配器及其同工型的水平上形成炎症体，或这些同工型如何影响炎。在父母R15赠款中，我们提议通过其两个研究炎症体的调节激活同工型（Canonical ASC和ASC_SHORT）。两种双相蛋白都有两个死亡结构域由接头连接，它们的氨基酸序列仅在接头长度上有所不同。我们最近的发现由母R15产生的这反过来与他们的不同动态行为相关。我们的结果提供了第一个解释分子水平的原因是这些同工型为何根据其各自的程度激活炎症体寡聚功能。这种多样性补充提案旨在解决有关角色的知识差距在炎性体形成上的其他两个ASC同工型（ASCC和ASCD）中，因此在炎症中回复。该提案的目的是创新的，因为它将破译未知的分子碱基 ASCC和ASCD介导的炎性体调节。我们的假设是； 1）ASCC，完整的死亡领域，通过与ASC自我关联竞争，抑制炎症体的形成，这可以解释观察到炎症反应下降，2）根据我们的初步研究，ASCD本质上是一种无序蛋白（IDP），因此我们建议ASCD在与ASC结合时可以折叠，ASC是一种典型的机制 IDP，导致更中等的抑制作用。为了检验这些假设； 1）在原子上学习将ASCC和ASCD的结构特征与NMR保持水平，2）定量确定其潜力与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）用透射电子显微镜辨别宏观结构的潜在不同特征由ASCC和ASCD存在的规范ASC形成。