Inflammatory Proteases, Ubiquitin Proteasome System, and Myocyte Death

描述（由申请人提供）：心脏缺血再灌注（IR）损伤可导致心功能障碍，进而发展为心力衰竭。这一进展中最早的事件之一被认为涉及炎症细胞及其蛋白酶。虽然在心肌损伤后的早期阶段是有益的，但过度的炎症反应导致肌细胞死亡、瘢痕形成和心肌功能障碍。我们已经表明，中性粒细胞来源的丝氨酸蛋白酶，组织蛋白酶G（CG），诱导心肌细胞死亡的失巢凋亡，涉及下调蛋白质，维持肌节的完整性和收缩功能，包括粘着斑（FA）和细胞骨架蛋白。关于维持FA蛋白的完整性并决定其在心肌细胞中的稳定性的具体机制知之甚少。监测和降解蛋白质的主要效应器之一是泛素蛋白酶体系统（UPS）。原癌基因Casitas b系淋巴瘤（Cbl）是一种具有内在E3泛素连接酶活性的衔接蛋白，其靶向受体酪氨酸激酶信号传导，导致其泛素化和蛋白酶体降解。我们的初步研究表明，增加Cbl激活响应心肌IR损伤，这是与增加泛素化的几种蛋白质，包括FA蛋白。此外，我们发现，CG诱导Cbl激活新生大鼠心肌细胞在体外与FAK的相互作用。我们还发现FAK在其响应于CG而失活和降解之前变得严重丝氨酸磷酸化，这支持蛋白质的丝氨酸磷酸化与其靶向蛋白酶体之间的关系。这些数据支持的假设，丝氨酸磷酸化的FAK响应CG启动其与Cbl的协会，从而导致其泛素化和降解的UPS。这些步骤最终导致FA信号改变和失巢凋亡引起的心肌细胞死亡。目的1：探讨丝氨酸磷酸化在FAK转换和心肌细胞失巢凋亡中的作用。目的2：探讨泛素连接酶Cbl在CG诱导的FAK下调和心肌失巢凋亡中的作用。目的3将确定在CG诱导的心肌细胞失巢凋亡过程中FAK下游导致起始caspase激活的信号通路。最后，目的4将确定CG和Cbl是否参与IR损伤期间体内诱导的FAK泛素化/降解和肌细胞损失。该提议试图将炎症区域中的FAK丝氨酸过度磷酸化与UPS对其的降解联系起来。这项研究的基础新知识应确定新的目标，可以减少心肌缺血再灌注损伤后的心肌细胞死亡和功能障碍性重塑。公共卫生相关性：本申请中提出的研究将描述泛素连接酶在由炎性蛋白酶引起的粘着斑信号传导改变中的作用，并将确定丝氨酸蛋白酶和泛素连接酶Cbl是否可以成为减少心肌IR损伤后肌细胞死亡和功能障碍性重塑的治疗的新靶点。

DESCRIPTION (provided by applicant): Cardiac ischemia reperfusion (IR) injury can cause cardiac dysfunction that progresses to heart failure. One of the earliest events in this progression is thought to involve inflammatory cells and their proteases. Although beneficial at early stages after myocardial injury, excessive inflammatory reaction leads to myocyte death, scar formation, and myocardial dysfunction. We have shown that the neutrophil derived serine protease, cathepsin G (CG), induces myocyte death by anoikis that involves downregulation of proteins that maintain sarcomere integrity and contractile function including focal adhesion (FA) and cytoskeletal proteins. Little is known about specific mechanisms that maintain the integrity of FA proteins and determine their stability in cardiac myocytes. One of the primary effectors of monitoring and degrading proteins is the Ubiquitin Proteasome System (UPS). The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor tyrosine kinase signaling, resulting in their ubiquitylation and proteasomal degradation. Our preliminary studies showed increased Cbl activation in response to myocardial IR injury that was associated with an increase in ubiquitylation of several proteins including FA proteins. Furthermore, we found that CG induced Cbl activation in neonatal rat cardiomyocytes in-vitro that was associated with its interaction with FAK. We also found that FAK becomes heavily serine phosphorylated prior to its inactivation and degradation in response to CG, supportive of a relationship between serine phosphorylation of proteins and their targeting to the proteasome. These data support the hypothesis that serine phosphorylation of FAK in response to CG initiates its association with Cbl, thus leading to its ubiquitylation and degradation by the UPS. These steps ultimately lead to FA signaling alteration and myocyte death by anoikis. Aim 1 will identify the implication of serine phosphorylation in FAK turnover and myocyte anoikis. Aim 2 will identify whether the ubiquitin ligase Cbl play a role in CG-induced FAK downregulation and myocyte anoikis. Aim 3 will identify the signaling pathways downstream from FAK that lead to the activation of initiator caspases during CG-induced myocyte anoikis. Finally, aim 4 will determine if CG and Cbl are involved in FAK ubiquitylation/degradation and myocyte loss induced in-vivo during IR injury. This proposal seeks to link FAK serine hyper-phosphorylation in areas of inflammation to its degradation by the UPS. The fundamental new knowledge resulting from this study should identify novel targets that could diminish myocyte death and dysfunctional remodeling after myocardial IR injury. PUBLIC HEALTH RELEVANCE: The research proposed in this application will delineate the role of ubiquitin ligases in focal adhesion signaling alterations caused by inflammatory proteases and will determine if serine proteases and the ubiquitin ligase Cbl could be novel targets for therapies that diminish myocyte death and dysfunctional remodeling after myocardial IR injury.