Novel mechanisms in ER regulation in heart

描述（由申请人提供）：ER应激，也称为未折叠蛋白反应（UPR），是一种关键的信号传导机制，与神经元变性、癌症、糖尿病和其他疾病有关。最近的证据表明，在血流动力学超负荷和缺血/再灌注损伤的心肌细胞中也诱导UPR。UPR的激活可以提供细胞保护以对抗来自蛋白质聚集体的细胞毒性，如在淀粉样变性或氧化和缺血性损伤的情况下。然而，长时间刺激UPR也可引发细胞凋亡。因此，UPR的校准调节可能在心脏保护和损伤中具有重要意义。IRE 1（a或B同种型）是具有特异性RNA酶活性的ER膜靶向的ser/thr蛋白激酶，其对于UPR以及ER应激诱导的JNK活化和细胞死亡是关键的，并且对于正常胚胎发育是必需的。IRE 1活性在UPR期间通过二聚化和Ser/Thr转磷酸化被诱导。然而，其脱磷酸化的分子机制尚不清楚。通过基因组挖掘，我们发现了一种新的丝氨酸/苏氨酸蛋白磷酸酶（PP 2Ce），该酶在脑和心脏中高度富集，并且专门靶向ER膜，在体外和体内对IRE 1具有显著的去磷酸化选择性。初步研究表明，PP 2Ce抑制IRE 1磷酸化并负性调节IRE 1介导的ER应激信号。这一令人兴奋的发现导致我们目前的假设，这种新的PP 2C亚型是内源性IRE 1特异性蛋白磷酸酶，并在病理条件下调节心脏中的UPR中发挥重要作用。为了严格检验这一假设，我们建议实现以下三个具体目标：目标1。我们将确定PP 2Ce在培养的心肌细胞中调节UPR的作用。目的2：探讨PP 2Ce调控IRE 1活性的分子机制。目的3：研究PP 2Ce在斑马鱼胚胎发育中的作用。目的4：我们将使用心脏特异性和可诱导的PP 2Ce过表达和PP 2Ce敲除小鼠模型来确定PP 2Ce活性对完整动物心脏功能和缺血再灌注损伤的影响。通过这些研究，我们将建立一个新的ER应激信号组分心脏的功能作用和分子机制，并为心力衰竭的潜在机制提供新的见解。

DESCRIPTION (provided by applicant): ER stress, also known as unfolded protein response (UPR) is a critical signaling mechanism that has been implicated in neuron degeneration, cancer, diabetics and other diseases. Recent evidence suggests that UPR is also induced in cardiac myocytes following hemodynamic overload and ischemia/reperfusion insults. Activation of UPR can provide cellular protection against cytoxicity from protein aggregate as in the cases of amyloidosis, or oxidative and ischemic injury. However, prolonged stimulation of UPR can also trigger apoptosis. Therefore, calibrated regulation of UPR may have significant implication in cardiac protection and injury. IRE1 (a or b isoform) is an ER membrane targeted ser/thr protein kinase with specific RNase activity that is critical to UPR as well as ER stress induced JNK activation and cell death, and is essential for normal embryonic development. IRE1 activity is induced during UPR via dimerization and ser/thr trans-phosphorylation. However, the molecular mechanism involved in its dephosphorylation is unknown. Through genome mining, we found a novel Ser/Thr protein phosphatase (PP2Ce) that is highly enriched in brain and heart, and is exclusively targeted on ER membrane and possesses a remarkable selectivity to dephosphorylate IRE1 in vitro and in vivo. Preliminary studies demonstrate that PP2Ce inhibits IRE1 phosphorylation and negatively modulate IRE1 mediated ER stress signaling. This exciting finding leads to our current hypothesis that this novel PP2C isoform is the endogenous IRE1 specific protein phosphatase and has an important role in regulating UPR in heart under pathological conditions. To rigorously test this hypothesis, we propose to accomplish the following three specific aims: Aim 1. we will determine the role of PP2Ce in regulating UPR in cardiomyocytes in culture. Aim 2, we will explore the molecular mechanisms of PP2Ce mediated regulation of IRE1 activity. Aim 3, we will determine the functional significance of PP2Ce mediated regulation in embryonic development in zebrafish. Aim4, we will use cardiac specific and inducible PP2Ce over-expressor and PP2Ce knockout mouse models to determine the impact of PP2Ce activity on cardiac function and ischemia reperfusion injury in intact animals. From these studies, we will establish the functional role and molecular mechanisms of a novel ER stress signaling component heart and shed new insights to the underlying mechanisms of heart failure.