Innate Immunity Pathways in Autophagy Signaling in Cardiac Myocytes

 描述(由申请人提供)：通过自噬-溶酶体机制有效地去除受损的亚细胞器对于维持心肌细胞在稳态和缺血再灌注(IR)损伤中的活性至关重要。新出现的证据表明，先天免疫蛋白作为细胞内的“损伤感应器”，在协调其自噬清除方面发挥了重要作用。其中一种蛋白质，TRAF2(肿瘤坏死因子受体相关因子-2)，激活两种肿瘤坏死因子受体下游的细胞保护信号，以防止IR诱导的心肌细胞死亡。我们最近发现了TRAF2作为一种E3泛素连接酶在自噬去除心肌细胞受损线粒体中的新作用。线粒体损伤触发了线粒体膜外膜上PINK1的稳定，PINK1是一种丝氨酸-苏氨酸激酶，它招募了PINK1，一种E3泛素连接酶来标记线粒体蛋白质进行降解。PINK1信号是否也招募TRAF2到受损的线粒体；以及TRAF2是否在体内线粒体自噬中相对于Parkin发挥非冗余作用，尚不清楚。在平行研究中，我们还发现了内质网(ER)在内质网应激和心脏IR损伤条件下通透性的证据。内质网损伤激活IRE1RNA酶，这是一种内质网定位的丝氨酸苏氨酸激酶和α酶，与TRAF2相互作用；体外研究表明，线粒体相关膜上的IRE1TRAF2信号协调受损线粒体和内质网的自噬。在这项提案中，我们将验证这样的假设，即TRAF2信号介导内质网和线粒体的选择性自噬，以调节心肌细胞在稳态和应激状态下的存活；在三个特定目标下。在目标1中，我们将确定TRAF2在稳态和应激状态下心肌细胞存活中的作用。在目标2中，我们将研究TRAF2与Parkin在心肌细胞线粒体自噬中的作用。在目标3中，我们将确定IRE1α-TRAF2信号轴在心肌细胞内质网吞噬中的作用。这些研究将阐明以TRAF2为靶点作为先天免疫传感器的细胞学基础，该传感器协调自噬清除线粒体-内质网界面(MAM)处的受损细胞器，以提高心肌梗死心肌细胞的存活率并预防心力衰竭，这是美国国立卫生研究院的一项关键任务。

 DESCRIPTION (provided by applicant): Efficient removal of damaged sub-cellular organelles via the autophagy-lysosome machinery is critical for maintaining cardiac myocyte viability in homeostasis and with ischemia-reperfusion (IR) injury. Emerging evidence implicates an essential role for innate immunity proteins as intracellular `damage sensors' to orchestrate their autophagic removal. One such protein, TRAF2 (Tumor necrosis factor Receptor-Associated Factor-2), activates cytoprotective signaling downstream of both TNF receptors to prevent IR-induced cardiomyocyte death. We have recently discovered a novel role for TRAF2 as an E3 ubiquitin ligase in autophagic removal of damaged mitochondria in cardiac myocytes. Mitochondrial damage triggers stabilization of PINK1, a serine-threonine kinase, on the outer mitochondrial membrane, which recruits PARKIN, an E3 ubiquitin ligase to ubiquitin- tag mitochondrial proteins for degradation. Whether PINK1 signaling also recruits TRAF2 to damaged mitochondria; and whether TRAF2, plays a non-redundant role vis-à-vis PARKIN in mitochondrial autophagy, in vivo, is not known. In parallel studies, we have also uncovered evidence for endoplasmic reticulum (ER) permeabilization under conditions of ER stress and in cardiac IR injury. ER damage activates IRE1α, an ER-localized serine-threonine kinase and RNAase, which interacts with TRAF2; and in-vitro studies indicate that IRE1α-TRAF2 signaling at the mitochondria associated membranes (MAM) orchestrates autophagy of damaged mitochondria and ER. In this proposal, we will test the hypothesis that TRAF2 signaling mediates selective autophagy of endoplasmic reticulum and mitochondria to regulate cardiac myocyte survival in homeostasis and under stress; under three specific aims. In aim 1, we will determine the role of TRAF2 in cardiac myocyte survival in homeostasis and under stress. In aim 2, we will examine the role of TRAF2 vis-à-vis PARKIN in mitochondrial autophagy in cardiac myocytes. In aim 3, we will determine the role of IRE1α-TRAF2 signaling axis in ER-phagy in cardiac myocytes. These studies will elucidate the cellular basis for targeting TRAF2 as an innate immunity sensor that coordinates autophagic removal of damaged organelles at the mitochondria-ER interface (MAM) to enhance cardiomyocyte survival in myocardial infarction and prevent heart failure, a key mission of the NIH.