Heme allocation and disruptions in asthma and the failing heart

哮喘和心脏衰竭中的血红素分配和干扰

• 批准号: 10542447
• 负责人: Elizabeth A. Sweeny
• 金额: $ 24.9万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542447
• 关键词: Actins; Affect; Asthma; Atrial Fibrillation; Biochemical; Biological Assay; Blood Vessels; Calcium; Calcium Signaling; Cardiac Myocytes; Cardiovascular Diseases; Cardiovascular system; Cause of Death; Cell model; Cell physiology; Cells; Cessation of life; Cytoskeleton; Data; Diabetes Mellitus; Disease; Disease Progression; Enzymes; Family; Gene Expression; Heart; Heart failure; Heme; Hemeproteins; Homeostasis; Immunity; Link; Malignant Neoplasms; Membrane; Metabolism; Mitochondria; Molecular; NADPH Oxidase; Oxidation-Reduction; Pathologic; Patients; Phase; Physiological; RNA; Regulation; Role; Signal Transduction; Sinus; Stimulus; Stroke; Superoxides; System; Testing; Tissues; United States; Work; biological adaptation to stress; cardiovascular health; drug discovery; human disease; induced pluripotent stem cell derived cardiomyocytes; knock-down; member; novel; overexpression; prevent; protein expression; protein protein interaction; reconstitution; release of sequestered calcium ion into cytoplasm; response; structural biology

The heme protein NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme which produces superoxide in response to elevated intracellular calcium levels and is emerging as an exciting player in immunity and the cardiovascular system. NOX5 is crucial for proper vascular contraction and appears to be a nexus between cellular redox and calcium signaling, As the most recently discovered member of the NOX family of enzymes, details of NOX5 regulation and its role in the cell remain poorly resolved. It has, however, been implicated in numerous human diseases including cancers, diabetes and cardiovascular disorders. Elucidating details of NOX5 regulation and its role in cardiovascular health and disease is crucial to our understanding of normal cellular functions and how these become disrupted in disease. Based on preliminary data from the K99 phase, the R00 phase will focus on investigating the role of NOX5 in cardiomyocyte function and its contribution to the initiation and progression of atrial fibrillation (AF) (Aim 1) and to probe the significance of novel protein:protein interactions identified in the K99 phase which link NOX5 and the actin cytoskeleton, mitochondria, RNA regulation and stress response systems (Aim 2). Aim 1 will focus on understanding how NOX5 knockdown and overexpression affect gene and protein expression, calcium flux, and cellular metabolism in induced pluripotent stem cell (iPSC) derived cardiomyocytes, and then using patient tissue from hearts in AF or sinus rhythm to test hypothesizes generated from the iPSC derived cardiomyocyte system. Aim 2 will use a model cell system (HEK293 cells and HEK293 cells overexpressing NOX5) as well as iPSC derived cardiomyocytes to probe the interactome of NOX5 in response to stimuli and to understand how these interactions affect NOX5 activity and localization, the actin cytoskeleton, calcium flux, cellular metabolism, gene expression and the stress response system. This project will uncover crucial details about the role of NOX5 in the heart and in the broader context of cellular homeostasis. It will also lay important groundwork for identifying molecular factors responsible for the switch between physiological and pathological responses and identify interactions and interaction networks ideal for further study using purified components for use in reconstitution assays, structural biology projects, mechanistic studies using biochemical approaches and drug discovery projects.

血红素蛋白NADPH氧化酶5（NOX5）是一种跨膜信号传导酶，产生 超氧化物响应升高的细胞内钙水平，并成为令人兴奋的参与者 免疫力和心血管系统。 NOX5对于适当的血管收缩至关重要，似乎是 细胞氧化还原与钙信号传导之间的联系，作为最近发现的成员 NOX酶家族，NOX5调节的细节及其在细胞中的作用仍然很差。它有， 但是，与包括癌症，糖尿病和心血管在内的许多人类疾病有关 疾病。阐明NOX5调节及其在心血管健康和疾病中的作用至关重要的细节至关重要 我们了解正常细胞功能以及它们如何在疾病中破坏。基于 从K99阶段进行初步数据，R00阶段将着重研究NOX5在 心肌细胞功能及其对房颤的起始和进展的贡献（AF）（AIM 1） 并探测新蛋白质的重要​​性：在K99相中鉴定的蛋白质相互作用，该蛋白相互作用 NOX5和肌动蛋白细胞骨架，线粒体，RNA调节和应力反应系统（AIM 2）。目的 1将集中于了解NOX5敲低和过表达如何影响基因和蛋白质 衍生的诱导多能干细胞（IPSC）中的表达，钙通量和细胞代谢 心肌细胞，然后使用AF或窦性节奏中心的患者组织测试假设 由IPSC衍生的心肌细胞系统产生。 AIM 2将使用模型电池系统（HEK293单元格 和过表达NOX5的HEK293细胞以及IPSC衍生的心肌细胞以探测相互作用的组 NOX5响应刺激，并了解这些相互作用如何影响NOX5活动和 定位，肌动蛋白细胞骨架，钙通量，细胞代谢，基因表达和应激 响应系统。该项目将发现有关NOX5在心脏和中的作用的关键细节 细胞稳态的更广泛背景。它还将为识别分子奠定重要的基础 负责生理和病理反应之间转换的因素并确定 相互作用和交互网络非常适合使用纯化的组件进行进一步研究 重建测定，结构生物学项目，使用生化方法的机械研究和 药物发现项目。