Non-canonical ERAD as a Regulator of Cardiac Hypertrophy

项目摘要 病理性心肌肥大过程中蛋白质合成的增加对蛋白质折叠提出了要求 避免有毒错误折叠蛋白质积累的机制。与内质网有关 (ER)在心肌细胞中合成许多重要蛋白质的系统，是一种识别和 降解错误折叠的蛋白质，即ER相关的（蛋白质）降解，或ERAD。我们发现了一种不同的， ERAD作为促生长激酶、血清糖皮质激素水平调节剂的非典型作用 激酶1（SGK 1）。SGK 1是一种参与其他细胞类型（如癌细胞）生长的胞质激酶。 有趣的是，SGK 1可以运输到ER，在ER中它被ERAD机制泛素化， 被胞质蛋白酶体降解;然而，这种独特的调节机制尚未在 心我们的初步证据表明，非经典ERAD可以调节心脏中的SGK 1水平， 从而调节病理条件下的心脏生长。我们还发现了SGK 1结合蛋白， 称为糖皮质激素诱导的亮氨酸拉链蛋白（GILZ），可以结合并保护SGK 1免受非典型的 ERAD介导的降解，我们认为在压力下增加了SGK 1介导的心脏生长 超载。因此，我们的假设是SGK 1是压力超负荷诱导的心脏损害的主要诱导物。 病理在压力超负荷期间，SGK 1水平，因此，SGK 1介导的心肌肥大和 GILZ依赖性SGK 1从ER转移，从而降低了随后的病理学， 非典型ERAD引起的SGK 1降解。SGK 1肽的异位表达破坏GILZ-SGK 1 相互作用，增加SGK 1降解，从而减少SGK 1介导的心脏肥大和随后的 病理这一假设将在压力超负荷诱导的心脏病理学小鼠中进行检验 在我们的具体目标中，其是将心脏特异性SGK 1缺失与编码SGK 1-WT的AAV 9 1-偶联 （活性; ER靶向）、SGK 1-KD（激酶死亡ER靶向）或SGK 1-KD 60（活性;非ER靶向）进行检查 SGK 1和ERAD对超负荷诱导的心脏病理学的影响，2-联合收割机AV 9-SGK 1-WT或AV 9- 用AAV 9介导的GILZ过表达或敲低SGK 1-SGK 60，以确定GILZ是否转移SGK 1-SGK 60。 WT从ER和保护它从ERAD在心脏，3-评估潜在的治疗，抗肥大 一种新的SGK 1肽的作用，中断GILZ-SGK 1结合，并增加非经典ERAD- 介导的SGK 1降解。这些研究意义重大，因为它们将揭示以前未被重视的 SGK 1、GILZ和ERAD在病理性心脏肥大中的作用。我们将使用创新的分子策略 从机制上剖析GILZ和非经典ERAD作为SGK 1信号传导和心脏信号传导调节因子的作用， 病理基于肽的SGK 1-GILZ相互作用的破坏可能是抑制GILZ表达的高度特异性方法。 通过选择性降解SGK 1，SGK 1在心脏中的适应不良病理效应。

PROJECT SUMMARY Increases in protein synthesis during pathological cardiac hypertrophy places demands on protein-folding machinery to avert the accumulation of toxic misfolded proteins. Associated with the endoplasmic reticulum (ER), where many important proteins are synthesized in cardiac myocytes, is a system that recognizes and degrades misfolded proteins, i.e. ER associated (protein) degradation, or ERAD. We discovered a different, non-canonical role for ERAD as a regulator of the levels of the growth-promoting kinase, serum glucocorticoid kinase 1 (SGK1). SGK1 is a cytosolic kinase involved in growth of other cell types, such as cancer cells. Interestingly, SGK1 can traffic to the ER where it is ubiquitylated by ERAD machinery and subsequently degraded by cytosolic proteasomes; however, this unique regulatory mechanism has not been studied in the heart. Our preliminary evidence shows that non-canonical ERAD could regulate SGK1 levels in the heart and, thereby regulating cardiac growth under pathological conditions. We also found that an SGK1-binding protein, called glucocorticoid-inducible leucine zipper protein (GILZ), can bind to and protect SGK1 from non-canonical ERAD-mediated degradation, which we believe increases SGK1-mediated growth of the heart during pressure overload. Accordingly, our hypothesis is that SGK1 is a major inducer of pressure overload-induced cardiac pathology. During pressure overload, SGK1 levels, and thus, SGK1-mediated cardiac hypertrophy and subsequent pathology, are increased by GILZ-dependent diversion of SGK1 away from the ER, which decreases SGK1 degradation by non-canonical ERAD. Ectopic expression of an SGK1 peptide disrupts the GILZ-SGK1 interaction, increases SGK1 degradation, thus decreasing SGK1-mediated cardiac hypertrophy and subsequent pathology. This hypothesis will be examined in mice subjected to pressure overload-induced cardiac pathology in our specific aims, which are to 1-couple cardiac-specific SGK1 deletion with AAV9 encoding SGK1-WT (active; ER-targeted), SGK1-KD (kinase-dead ER-targeted) or SGK1-∆60 (active; not ER-targeted) to examine the effect of SGK1 and ERAD on overload-induced cardiac pathology, 2-combine AAV9-SGK1-WT or AAV9- SGK1-∆60 with AAV9-mediated GILZ overexpression or knockdown to determine whether GILZ diverts SGK1- WT from the ER and protects it from ERAD in the heart, 3-evaluate the potential therapeutic, antihypertrophic effects of a novel SGK1 peptide that interrupts GILZ-SGK1 binding, and increases non-canonical ERAD- mediated SGK1 degradation. These studies are significant because they will reveal previously unappreciated roles for SGK1, GILZ and ERAD in pathologic cardiac hypertrophy. We will use an innovative molecular strategy to mechanistically dissect roles for GILZ and non-canonical ERAD as regulators of SGK1 signaling and cardiac pathology. Peptide-based disruption of the SGK1-GILZ interaction could be a highly specific method for inhibiting the maladaptive pathological effects of SGK1 in the heart by selective degradation of SGK1.