Novel mechanisms in ER regulation in heart

描述（由申请人提供）：内质网应激，也被称为未折叠蛋白反应（UPR），是一种关键的信号机制，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，在血流动力学超载和缺血/再灌注损伤后，心肌细胞也会诱导UPR。在淀粉样变、氧化和缺血性损伤的情况下，UPR的激活可以提供细胞保护，防止蛋白质聚集的细胞毒性。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。IRE1 （a或b亚型）是一种内质网膜靶向丝氨酸/苏氨酸蛋白激酶，具有特异性RNase活性，对UPR和内质网应激诱导的JNK激活和细胞死亡至关重要，对正常胚胎发育至关重要。IRE1活性在UPR过程中通过二聚化和丝氨酸/苏氨酸反式磷酸化诱导。然而，参与其去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（PP2Ce），它在大脑和心脏中高度富集，并且专门靶向内质网膜，在体外和体内对IRE1脱磷酸化具有显著的选择性。初步研究表明，PP2Ce抑制IRE1磷酸化并负向调节IRE1介导的内质网应激信号。这一令人兴奋的发现导致了我们目前的假设，即这种新的PP2C异构体是内源性IRE1特异性蛋白磷酸酶，在病理条件下调节心脏的UPR中起重要作用。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。我们将在培养的心肌细胞中确定PP2Ce在调节UPR中的作用。目的2，我们将探索PP2Ce介导的IRE1活性调控的分子机制。目的3，我们将确定PP2Ce介导的调控在斑马鱼胚胎发育中的功能意义。在第四阶段，我们将使用心脏特异性和诱导型PP2Ce过表达和PP2Ce敲除小鼠模型来确定PP2Ce活性对完整动物心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内质网应激信号元件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.