The Deubiquitinase CYLD Controls Multiple Cell Death Pathways in the Heart

项目总结/摘要 由凋亡和/或坏死引起的心脏细胞死亡是心脏缺血性损伤、病理性重塑 和晚期心力衰竭与凋亡相反，坏死在心脏病发病机制中的作用 一直没有得到充分的研究新出现的证据已经确定了几种形式的"程序性坏死"， 如死亡受体介导的坏死（称为"坏死性凋亡"）、细胞凋亡介导的坏死和氧化性坏死。 应激性坏死程序性坏死在心脏中是如何调节的仍然很不清楚， 防止坏死仍然是一个重要的挑战。此外，目前没有分子策略可用于 同时靶向心脏病中的多个细胞死亡过程。在这里，我们的初步研究发现， CYLD（圆柱瘤病），一种赖氨酸63（K63）特异性去泛素酶，作为一种关键的调节因子， 心肌细胞中的多种细胞死亡途径，包括凋亡、坏死性凋亡和氧化应激诱导的 坏死有趣的是，缺血性损伤后，CYLD的表达在心脏中显著上调。使用 Cyld敲除和转基因小鼠模型，我们的初步数据进一步表明，消除CYLD 减轻，而过度CYLD加剧，心脏缺血性损伤。重要的是，消融 CYLD可抑制心肌细胞凋亡、坏死性凋亡和坏死，而CYLD过表达可抑制心肌细胞凋亡和坏死。 显示出相反的效果。从机制上讲，我们的数据揭示了K63-连接的多聚泛素化（K63-Ub）依赖性 细胞死亡信号传导机制，CYLD控制泛素化状态和三种细胞死亡的活性 调节因子：TRAF2、TAK1和AKT/PKB。因此，我们假设去泛素化酶CYLD是一个关键的调节因子， 多细胞死亡途径和一个有前途的治疗目标，心脏缺血性损伤和重塑。 使用遗传功能丧失和获得策略，我们将解决两个具体目标：目标1）调查 CYLD作为心肌细胞死亡、缺血性损伤和重构的关键调节因子的新作用 敲除和转基因模型以及AAV9-shCYLD载体。目的2）确定CYLD介导的K63-Ub 依赖性细胞死亡信号网络调节心肌细胞的凋亡、坏死性凋亡和坏死。这 该项目研究了一种新的CYLD介导的心脏细胞死亡信号网络及其功能相关性 心脏缺血性损伤和重塑。此外，拟议的研究将定义K63-Ub依赖性 调节细胞凋亡、坏死性凋亡和坏死的机制，这构成了细胞死亡的新范式 调控这些研究还通过提供新的抗细胞死亡的方法， 鉴于我们的初步结果揭示了CYLD作为多个细胞死亡过程的分子靶点，因此，我们可以使用一些新的策略。

Project Summary/Abstract Cardiac cell death by apoptosis and/or necrosis is a hallmark of cardiac ischemic injury, pathological remodeling, and end-stage heart failure. In contrast to apoptosis, the role of necrosis in the pathogenesis of heart disease has been largely understudied. Emerging evidence has identified several forms of “programmed necrosis”, such as death receptor-mediated necrosis (termed “necroptosis”), mitochondria-mediated necrosis, and oxidative stress-induced necrosis. How programmed necrosis is regulated in the heart remains largely unknown, and preventing necrosis is still an important challenge. Moreover, currently no molecular strategies are available to simultaneously target multiple cell death processes in heart disease. Here, our preliminary studies identified an unexpected role for CYLD (cylindromatosis), a lysine 63 (K63)-specific deubiquitinase, as a key regulator of multiple cell death pathways in cardiomyocytes, including apoptosis, necroptosis, and oxidative stress-induced necrosis. Intriguingly, CYLD expression was markedly upregulated in the heart following ischemic injury. Using Cyld knockout and transgenic mouse models, our preliminary data further show that ablation of CYLD attenuated, whereas overexpression of CYLD exacerbated, cardiac ischemic injury. Importantly, ablation of CYLD inhibited apoptosis, necroptosis, and necrosis in cardiomyocytes, whereas overexpression of CYLD showed the opposite effect. Mechanistically, our data reveal a K63-linked polyubiquitination (K63-Ub) dependent cell death signaling mechanism whereby CYLD controls the ubiquitination status and activity of three cell death regulators: TRAF2, TAK1 and AKT/PKB. Therefore, we hypothesize the deubiquitinase CYLD is a key regulator of multiple cell death pathways and a promising therapeutic target for cardiac ischemic injury and remodeling. Using genetic loss- and gain-of-function strategies, we will address two specific aims: Aim 1) To investigate the novel role of CYLD as a key regulator of myocardial cell death, ischemic injury, and remodeling using Cyld knockout and transgenic models and AAV9-shCYLD vectors. Aim 2) To define a CYLD-mediated, K63-Ub dependent cell death signaling network regulating apoptosis, necroptosis, and necrosis in cardiomyocytes. This project investigates a novel CYLD-mediated cell death signaling network in the heart and its functional relevance in cardiac ischemic injury and remodeling. Moreover, the proposed studies will define a K63-Ub dependent mechanism regulating apoptosis, necroptosis, and necrosis, which constitutes a new paradigm of cell death regulation. These studies also have important translational implications by providing new anti-cell death strategies, given our preliminary results revealing CYLD as a molecular target for multiple cell death processes.