The role of palmitoylation in cardiac signal transduction and disease pathogenesis

摘要/项目摘要 心脏病仍然是美国发病率和死亡率的主要原因 而治疗选择仍然有限，而且基本上无效。因此，更好地理解 心肌细胞中的细胞内信号传导是心脏病发病机制的基础， 开发用于治疗心脏病的新疗法和药物靶点。棕榈酰化是一种 可逆的脂质修饰是由最近发现的组氨酸-组氨酸-组氨酸催化， 棕榈酰转移酶的半胱氨酸（zDHHC）家族，并且对蛋白质功能具有多种影响， 包括在细胞信号传导中扮演重要角色。在这里，我们将研究棕榈酰的作用， 转移酶，Godz/Zdhhc 3，在心脏中。我们通过酵母双杂交筛选发现Rho GDP 解离抑制剂（RhoGDI）直接与Godz相互作用，并发现Godz在细胞中的过表达， 心肌细胞增强RhoGDI棕榈酰化，表明RhoGDI是一种新的Godz底物。 RhoGDI作为RhoGTP酶的主要稳态调节剂发挥作用，RhoGTP酶是具有以下功能的信号分子： 在心脏病发病机制中的关键作用。我们培育了心脏特异性 过量表达Godz以研究Godz在体内的作用。转基因Godz小鼠出现充血性 心脏衰竭之前，心脏中RhoGDI棕榈酰化增强， 所有心脏表达的RhoGTP酶的丰度和活性，其由RhoGDI调节。我们 假设Godz通过诱导心肌细胞中的致病性信号传导， 通过RhoGDI的棕榈酰化的RhoGTT水平和活性。这项提案将考验我们的 假设有以下目的：1。为了确定Godz介导的棕榈酰化作用在 心脏信号传导和衰竭的调节，以及2.为了确定RhoGDI棕榈酰化在 RhoGT 3信号传导和心脏病发病机制。该提案的最初部分将是 在著名的分子心脏病学研究员杰弗里·莫肯汀博士的实验室里进行， 我将研究Godz如何调节心肌细胞中的信号传导，并确定 心脏特异性Godz缺失对心脏信号转导、功能和疾病倾向的影响 (Aim 1）。在我的独立实验室里，我将利用基因敲入的小鼠来扩展这些研究。 RhoGDI的棕榈酰化位点的突变（RhoGDIC 79 S），以破译 RhoGDI棕榈酰化在心脏RhoGTT信号传导调节中的作用（Aim 2）。我将进一步 Godz介导棕榈酰化和棕榈酰化依赖性信号转导在心肌细胞中的研究 内稳态和病理生理学。这项工作将揭示 由此动态棕榈酰化调节心脏中的分子信号，这将使我处于 这一新兴领域的前沿，并推动我的研究计划在未来几年。

Abstract/Project Summary Heart disease continues to be the leading cause of morbidity and mortality in the United States while treatment options remain limited and largely ineffective. Therefore, a greater understanding of intracellular signaling in cardiomyocytes that underlies cardiac disease pathogenesis will aid in the development of novel therapies and drug targets for the treatment of heart disease. Palmitoylation is a reversible lipid modification that is catalyzed by the recently discovered aspartate-histidine-histidine- cysteine (zDHHC) family of palmitoyl transferases and has diverse effects on protein function, including playing critical roles in cellular signaling. Here, we will investigate the role of the palmitoyl transferase, Godz/Zdhhc3, in the heart. We discovered by yeast two-hybrid screening that Rho GDP dissociation inhibitor (RhoGDI) directly interacts with Godz and found that overexpression of Godz in cardiomyocytes enhances RhoGDI palmitoylation, indicating that RhoGDI is a novel Godz substrate. RhoGDI functions as the master homeostatic regulator of RhoGTPases, signaling molecules with pivotal roles in cardiac disease pathogenesis. We generated transgenic mice with cardiac-specific overexpression of Godz to study the role of Godz in vivo. Transgenic Godz mice develop congestive heart failure that is preceded by enhanced palmitoylation of RhoGDI in the heart and increased abundance and activity of all cardiac-expressed RhoGTPases, which are regulated by RhoGDI. We hypothesize that Godz plays instrumental roles in pathogenic signaling in cardiomyocytes by inducing RhoGTPase levels and activity through palmitoylation of RhoGDI. This proposal will test our hypothesis with the following aims: 1. To determine the role of Godz-mediated palmitoylation in the regulation of cardiac signaling and failure and 2. To determine the role of RhoGDI palmitoylation in RhoGTPase signaling and cardiac disease pathogenesis. The initial portion of this proposal will be carried out in the laboratory of the renowned molecular cardiology researcher, Dr. Jeffery Molkentin, where I will investigate how Godz regulates signaling in cardiomyocytes and determine the effects of cardiac-specific deletion of Godz on cardiac signal transduction, function, and propensity to disease (Aim 1). In my independent laboratory, I will extend these studies by utilizing mice with a knock-in mutation for the palmitoylation site of RhoGDI (RhoGDIC79S) to decipher the physiological role of RhoGDI palmitoylation in the regulation of cardiac RhoGTPase signaling (Aim 2). I will further investigate Godz-mediated palmitoylation and palmitoylation-dependent signaling in cardiac homeostasis and pathophysiology in my independent laboratory. This work will uncover mechanisms whereby dynamic palmitoylation regulates molecular signaling in the heart, which will place me at the forefront of this emerging field and drive my research program for years to come.