A K63-Ubiquitination Dependent Necroptosis Signaling Network in the Heart

项目概要/摘要 通过凋亡和/或坏死导致的心肌细胞损失是心脏缺血性损伤、病理性损伤和/或心肌损伤的标志。 重塑和终末期心力衰竭虽然坏死传统上被认为是一种被动的， 然而，新出现的证据已经改变了这一范式，并确定了几种形式的“程序化”。 坏死”，包括死亡受体介导的坏死（称为“坏死性凋亡”），凋亡介导的坏死， 和其他受调节的坏死过程。尽管最近的进展，如何坏死性凋亡是调节在心脏 仍然在很大程度上是未知的，预防坏死性心肌细胞死亡仍然是一个重要的挑战。我们的飞行员 研究发现了一种新的K63连接的多聚泛素化（K63-Ub）依赖性坏死性凋亡信号网络， 关键调节心脏缺血性损伤和病理性重塑。有趣的是，K63-去泛素化酶 CYLD（圆柱瘤病）显著上调，而E3泛素连接酶TRAF 2（TNF受体） 相关因子-2）在缺血再灌注损伤后的心脏中下调。我们的初步结果 进一步鉴定CYLD和TRAF 2分别作为心肌坏死性凋亡的激活剂和抑制剂。 重要的是，心肌细胞特异性消融CYLD通过降低心肌细胞的存活率来减轻缺血性损伤和不良重塑。 抑制坏死性凋亡。成人心脏中TRAF 2的急性缺失通过促进 坏死性凋亡，而TRAF 2基因转移引起心脏保护。从机械学上讲，我们提出了一种新的K63- Ub依赖性坏死性凋亡调节机制，CYLD使TRAF 2和TAK 1去泛素化， TAK 1-RIP 1相互作用，并促进坏死性凋亡复合物。作为一种相反的机制，TRAF 2作为一种 用于TAK 1的E3连接酶以抑制坏死性凋亡信号传导。因此，可逆的K63-Ub（主要是非蛋白水解的） 构成了具有重要生物学意义的坏死性凋亡信号传导的新范例。我们假设 去泛素化酶CYLD与E3连接酶TRAF 2一起，关键性地调节心肌坏死性凋亡， 缺血性损伤和重塑，因此代表了有前途的治疗靶点。目标1将调查 K63-Ub修饰酶CYLD和TRAF 2在调节心肌坏死性凋亡中的相反作用， 体内缺血性损伤。目的2将定义CYLD/TRAF 2介导的K63-Ub依赖性坏死性凋亡信号转导 心肌细胞中的网络。使用遗传和分子策略，我们将调查的对立作用， CYLD和TRAF 2在基础条件下和缺血性心肌坏死中的调节作用 损伤这些研究将为深入了解其分子调控和功能意义提供新的思路 心脏坏死性凋亡，这具有重要的翻译意义，特别是考虑到我们的初步结果， 揭示了CYLD/TRAF 2作为关键的坏死性凋亡调节因子和缺血性心脏病的有希望的靶点。

Project Summary/Abstract Loss of cardiomyocytes by apoptosis and/or necrosis is a hallmark of cardiac ischemic injury, pathological remodeling, and end-stage heart failure. Although necrosis was traditionally regarded as a passive and unregulated process, emerging evidence has shifted this paradigm and identified several forms of “programmed necrosis”, including death receptor-mediated necrosis (termed “necroptosis”), mitochondria-mediated necrosis, and other regulated necrotic processes. Despite recent progress, how necroptosis is regulated in the heart remain largely unknown and preventing necroptotic cardiomyocyte death is still an important challenge. Our pilot studies identified a new K63-linked polyubiquitination (K63-Ub) dependent necroptosis signaling network that critically regulates cardiac ischemic injury and pathological remodeling. Intriguingly, the K63-deubiquitinase CYLD (cylindromatosis) was markedly up-regulated, whereas the E3 ubiquitin ligase TRAF2 (TNF receptor associated factor-2) was down-regulated, in the heart after ischemia-reperfusion injury. Our preliminary results further identified CYLD and TRAF2 as an activator and a suppressor of myocardial necroptosis, respectively. Importantly, cardiomyocyte-specific ablation of CYLD attenuated ischemic injury and adverse remodeling by inhibiting necroptosis. Acute deletion of TRAF2 in the adult heart caused dilated cardiomyopathy by promoting necroptosis, whereas TRAF2 gene transfer elicited cardioprotection. Mechanistically, we propose a new K63- Ub dependent necroptotic regulatory mechanism whereby CYLD deubiquitinates TRAF2 and TAK1, disrupts TAK1-RIP1 interaction, and promotes the necroptotic complex. As an opposing mechanism, TRAF2 acts as an E3 ligase for TAK1 to inhibit necroptosis signaling. Thus, the reversible K63-Ub (mainly non-proteolytic) constitutes a new paradigm of necroptosis signaling with important biological implications. We hypothesize that the deubiquitinase CYLD, in conjunction with the E3 ligase TRAF2, critically regulate myocardial necroptosis, ischemic injury, and remodeling, thus representing promising therapeutic targets. Aim 1 will investigate the opposing roles of K63-Ub modifying enzymes CYLD and TRAF2 in regulating myocardial necroptosis and ischemic injury in vivo. Aim 2 will define a CYLD/TRAF2 mediated, K63-Ub dependent necroptosis signaling network in cardiomyocytes. Using genetic and molecular strategies, we will investigate the opposing roles of CYLD and TRAF2 in regulating myocardial necroptosis under basal conditions and in the setting of ischemic injury. The proposed studies will provide new insights into the molecular regulation and functional significance of necroptosis in the heart, which has important translational implications, especially given our preliminary results revealing CYLD/TRAF2 as key necroptotic regulators and promising targets for ischemic heart disease.