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

项目摘要 在许多不同的器官中，通过坏死导致的细胞损失是与衰老相关的病理学的主要原因 包括心血管系统。然而，我们对细胞通路的理解， 潜在的坏死还远未完全因此，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以及它们如何促进衰老相关的 疾病从历史上看，坏死被认为是一个“偶然的”，不受管制的过程。然而，最近的工作 已经揭示了坏死可以被严格调控。一种形式的调节性坏死，坏死性凋亡，是典型的 由肿瘤坏死因子-α（TNF α）激活。TNF α受体结合激活激酶受体相互作用 蛋白1（RIPK1）。然后RIPK1与激酶RIPK3结合、磷酸化并激活激酶RIPK3，激酶RIPK3又与 并磷酸化假激酶混合谱系激酶结构域样（MLKL）。MLKL然后寡聚化， 转移到质膜，在那里它直接穿透膜。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。虽然 该途径的"核心"组分（RIPK1、RIPK3、MLKL）已被鉴定和表征，其次是 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，坏死性凋亡信号的最后执行步骤，尚未建立。我们有 发现Ca2+结合蛋白EF手结构域蛋白2（EFhd2）作为一种潜在的负调节剂， MLKL。我们已经发现EFhd2可以结合MLKL，并且其过表达减弱坏死性细胞死亡， 而击倒具有相反的效果。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的相互作用来进行信号传导，并且EFhd2的缺失加速了心脏 衰老过程中的病理本申请的目的是定义通过其 EFhd2抑制坏死性凋亡及其对老年人心功能障碍发展的影响 小鼠在目的1中，我们将确定EFhd2抑制MLKL激活的机制，以及Ca2+在MLKL激活中的作用。 在EFhd2抑制坏死性凋亡的能力中起作用。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。提出这项研究的理由是，一旦关键 当鉴定出调节坏死性凋亡的蛋白质时，可以靶向它们以改善坏死性凋亡的临床结果。 许多与衰老有关的疾病，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.