Characterization of EFhd2 as a novel inhibitor of necroptosis and aging-related cardiovascular pathology

EFhd2 作为坏死性凋亡和衰老相关心血管病理的新型抑制剂的表征

• 批准号: 10263964
• 负责人: Christopher P Baines
• 金额: $ 19.41万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10263964
• 关键词: Affect; Age; Aging; Animals; Attenuated; Binding; Binding Proteins; Biochemical; Cardiac; Cardiac development; Cardiomyopathies; Cardiovascular Diseases; Cardiovascular Pathology; Cardiovascular system; Cell Culture Techniques; Cell Death; Cell membrane; Cells; Cessation of life; Clinical; Core Protein; Development; Disease; EF-Hand Domain; Functional disorder; Goals; Heart; Heart failure; Measures; Membrane; Molecular; Mus; Myocardial dysfunction; Necrosis; Neurodegenerative Disorders; Organ; Outcome; Oxidative Stress; Pathology; Pathway interactions; Patients; Phosphotransferases; Play; Predisposition; Process; Proteins; Proteomics; RIPK1 gene; RIPK3 gene; Reactive Oxygen Species; Research; Role; Signal Transduction; TNF gene; TNFRSF1A gene; Techniques; Tertiary Protein Structure; Testing; Time; Work; aged; body system; genetic approach; heart function; improved; in vivo; indexing; inhibitor/antagonist; insight; knock-down; mutant; novel; overexpression; receptor binding

PROJECT SUMMARY The loss of cells through necrosis is a major cause of aging-related pathologies in many different organ systems, including the cardiovascular system. Nevertheless, our understanding of the cellular pathways that underlie necrosis is still very far from complete. Thus, our long-term goal is to identify and characterize the molecular mechanisms that drive/regulate necrotic death and how they contribute to aging-associated disease. Historically, necrosis was thought of as an “accidental”, unregulated process. However, recent work has revealed that necrosis can be tightly regulated. One form of regulated necrosis, necroptosis, is canonically activated by tumor necrosis factor-α (TNFα). TNFα receptor binding activates the kinase receptor-interacting protein 1 (RIPK1). RIPK1 then binds to, phosphorylates, and activates the kinase RIPK3, which in turn binds and phosphorylates the pseudokinase mixed lineage kinase domain-like (MLKL). MLKL then oligomerizes and translocates to the plasma membrane, where it directly perforates the membrane. Intriguingly, we have found that oxidative stress-induced necrosis is also partially dependent on activation of this pathway. However, while the “core” components of the pathway (RIPK1, RIPK3, MLKL) have been identified and characterized, next to nothing is known of the key proteins that modulate these core proteins. In particular, proteins that regulate MLKL activation, the final execution step in necroptotic signaling, have yet to be established. We have discovered the Ca2+-binding protein EF hand domain protein 2 (EFhd2) as a potential negative regulator of MLKL. We have found that EFhd2 can bind to MLKL, and its overexpression attenuates necroptotic cell death, while knockdown has the opposite effect. Thus, our central hypothesis is that EFhd2 inhibits necroptotic signaling through a Ca2+-regulated interaction with MLKL, and that loss of EFhd2 accelerates cardiac pathology during aging. The objective of the present application is to define the mechanisms by which EFhd2 inhibits necroptosis and the consequences this has for the development of cardiac dysfunction in aged mice. In Aim 1 we will determine the mechanisms by which EFhd2 inhibits MLKL activation, and the role Ca2+ plays in EFhd2's ability to inhibit necroptosis. In Aim 2 we will examine indices of necroptosis and cardiac function in aged wildtype and Efhd2-/- mice. The rationale for the proposed research is that once the key proteins that modulate necroptosis are identified, they can be targeted to improve the clinical outcome of many aging-related diseases such as cardiomyopathy.

项目概要 细胞坏死损失是许多不同器官中与衰老相关的病理的主要原因 系统，包括心血管系统。尽管如此，我们对细胞通路的理解 底层坏死还远未完成。因此，我们的长期目标是识别和表征 驱动/调节坏死性死亡的分子机制及其如何促进衰老相关的 疾病。从历史上看，坏死被认为是一种“意外”、不受监管的过程。然而最近的工作 已经表明坏死可以被严格调控。规范性坏死的一种形式，坏死性凋亡，是规范的 由肿瘤坏死因子-α (TNFα) 激活。 TNFα 受体结合激活激酶受体相互作用 蛋白 1 (RIPK1)。然后 RIPK1 结合、磷酸化并激活激酶 RIPK3，RIPK3 又结合 并磷酸化假激酶混合谱系激酶结构域样 (MLK​​L)。然后 MLKL 寡聚并 易位至质膜，直接刺穿质膜。有趣的是，我们发现 氧化应激诱导的坏死也部分依赖于该途径的激活。然而，虽然 该通路的“核心”成分（RIPK1、RIPK3、MLKL）已被识别和表征，接下来 对于调节这些核心蛋白的关键蛋白一无所知。特别是调节蛋白质 MLKL 激活是坏死性凋亡信号传导的最终执行步骤，尚未确定。我们有 发现 Ca2+ 结合蛋白 EF 手结构域蛋白 2 (EFhd2) 作为潜在的负调节因子 MLKL。我们发现 EFhd2 可以与 MLKL 结合，并且其过度表达可减弱坏死性细胞死亡， 而击倒则具有相反的效果。因此，我们的中心假设是 EFhd2 抑制坏死性凋亡 通过 Ca2+ 调节的与 MLKL 相互作用的信号传导，EFhd2 的缺失会加速心脏 衰老过程中的病理学。本申请的目的是定义机制，通过该机制 EFhd2 抑制坏死性凋亡及其对老年人心功能障碍发展的影响 老鼠。在目标 1 中，我们将确定 EFhd2 抑制 MLKL 激活的机制，以及 Ca2+ 的作用 EFhd2 具有抑制坏死性凋亡的能力。在目标 2 中，我们将检查坏死性凋亡和心脏指数 在老年野生型和 Efhd2-/- 小鼠中的功能。拟议研究的基本原理是，一旦关键 调节坏死性凋亡的蛋白质已被鉴定出来，它们可以有针对性地改善临床结果 许多与衰老相关的疾病，例如心肌病。