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

项目总结 细胞因坏死而丧失是许多不同器官衰老相关病理的主要原因。 系统，包括心血管系统。然而，我们对细胞通路的理解 下卧位坏死仍远未完全完成。因此，我们的长期目标是识别和描述 驱动/调节坏死性死亡的分子机制及其对衰老的影响 疾病。从历史上看，坏死被认为是一个“偶然的”、不受监管的过程。然而，最近的工作 已经揭示了坏死是可以严格控制的。一种规律性的坏死，坏死性下垂，是典型的 由肿瘤坏死因子α(肿瘤坏死因子α)激活。肿瘤坏死因子α受体结合激活激酶受体相互作用 蛋白1(RIPK1)。然后RIPK1与RIPK3结合、磷酸化并激活，而RIPK3又与RIPK3结合 并磷酸化伪激酶混合谱系激活域(MLKL)。MLKL然后进行齐聚并 转移到质膜，在那里它直接穿透细胞膜。有趣的是，我们发现 氧化应激诱导的坏死也部分依赖于这一途径的激活。然而，虽然 该途径的“核心”成分(RIPK1、RIPK3、MLKL)已被确定和表征， 对调节这些核心蛋白的关键蛋白一无所知。特别是，调节 MLKL激活是坏死信号转导的最后执行步骤，但尚未确定。我们有 发现钙离子结合蛋白EF Hand DOMAIN Protein 2(EFhd2)是一种潜在的负调控因子 MLKL。我们发现EFhd2可以与MLKL结合，其过表达可以减轻坏死性细胞的死亡。 而击倒则有相反的效果。因此，我们的中心假设是EFhd2抑制坏死性下垂 通过钙离子调节与MLKL相互作用的信号，以及EFhd2的缺失加速心脏 衰老过程中的病理变化。本申请的目的是定义 EFhd2抑制坏死性下垂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激活的机制以及钙离子的作用 发挥EFhd2‘S抑制坏死性下垂的能力。在目标2中，我们将检查坏死性下垂和心脏 对衰老野生型和Efhd2-/-小鼠的作用。提出这项研究的理由是，一旦关键 调节坏死性上睑下垂的蛋白质被识别出来，它们可以作为靶点来改善临床结果 许多与衰老相关的疾病，如心肌病。

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.