A K63-Ubiquitination Dependent Necroptosis Signaling Network in the Heart

项目摘要/摘要 心肌细胞因凋亡和/或坏死而丧失是心肌缺血损伤的病理标志 重塑和终末期心力衰竭。虽然传统上认为坏死是一种被动的和 不受监管的过程，新出现的证据已经改变了这一范式，并确定了几种形式的 “坏死”，包括死亡受体介导的坏死(称为“坏死性下垂”)、线粒体介导的坏死、 以及其他受监管的坏死过程。尽管最近取得了进展，但坏死性下垂是如何在心脏中调节的 在很大程度上仍不清楚，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连锁的多泛素化(K63-Ub)依赖的坏死性下垂信号网络 对心脏缺血损伤和病理重塑具有重要的调节作用。耐人寻味的是，K63脱泛素酶 CyLD(圆柱瘤病)显著上调，而E3泛素连接酶TRAF2(肿瘤坏死因子受体)表达显著上调 相关因子2)在心脏缺血再灌注损伤后表达下调。我们的初步结果 进一步确定CyLD和TRAF2分别是心肌坏死性下垂的激活物和抑制物。 重要的是，CyLD的心肌细胞特异性消融通过以下途径减轻缺血损伤和不良重构 抑制坏死性下垂。TRAF2在成人心脏中的急性缺失通过促进 而TRAF2基因转移具有心脏保护作用。在机械上，我们提出了一种新的K63- UB依赖的坏死链调节机制，CyLD去泛素化TRAF2和TAK1，破坏 TAK1-RIP1相互作用，并促进坏死复合体的形成。作为一种相反的机制，TRAF2充当 E3连接酶为TAK1，以抑制坏死性下垂信号。因此，可逆的K63-Ub(主要是非蛋白水解性) 构成了具有重要生物学意义的坏死性下垂信号的新范例。我们假设 脱泛素酶CyLD与E3连接酶TRAF2一起，对心肌坏死性下垂起关键调节作用， 缺血性损伤和重塑，因此代表了有希望的治疗靶点。目标1将调查 K63-Ub修饰酶CyLD和TRAF2在调节心肌坏死性下垂中的相反作用 体内缺血性损伤。AIM 2将定义CyLD/TRAF2介导的依赖K63-Ub的坏死性下垂信号 心肌细胞中的网络。使用遗传和分子策略，我们将研究相互对立的作用 CyLD和TRAF2在基础状态和缺血状态下对心肌坏死性下垂的调节作用 受伤。拟议的研究将为分子调控和功能意义提供新的见解 心脏中的坏死性下垂，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CyLD/TRAF2是关键的坏死调节因子，并有望成为治疗缺血性心脏病的靶点。

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.