Non-canonical ERAD as a Regulator of Cardiac Hypertrophy

项目总结 病理性心肌肥厚时蛋白质合成增加对蛋白质折叠的要求 避免有毒的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的蛋白质，是一个识别和 降解错误折叠的蛋白质，即内质网相关(蛋白质)降解，或ERAD。我们发现了一个不同的， ERAD作为生长激素促进激酶、血清糖皮质激素水平调节的非典范作用 蛋白激酶1(SGK1)。SGK1是一种胞浆激酶，参与其他类型细胞的生长，如癌细胞。 有趣的是，SGK1可以通过ERAD机制传递到内质网，在那里它被泛素化，随后 被胞浆蛋白酶体降解；然而，这一独特的调节机制尚未在 心。我们的初步证据表明，非典型的ERAD可以调节心脏中SGK1的水平， 从而调节病理条件下的心脏生长。我们还发现了一种SGK1结合蛋白， 被称为糖皮质激素诱导的亮氨酸拉链蛋白(GILZ)，可与SGK1结合并保护其免受非正则基因的影响 ERAD介导的降解，我们认为它增加了SGK1介导的心脏在压力下的生长 超载。因此，我们的假设是SGK1是压力超负荷所致心脏疾病的主要诱因。 病理学。在压力超负荷期间，SGK1水平，从而，SGK1介导的心肌肥厚和 随后的病理改变是由于依赖GILZ的SGK1从内质网转移而增加的，内质网减少 非典型ERAD对SGK1的降解。SGK1多肽的异位表达破坏GILZ-SGK1 相互作用，增加SGK1的降解，从而减少SGK1介导的心肌肥厚和随后的 病理学。这一假说将在遭受压力超负荷诱导的心脏病理的小鼠身上得到验证。 在我们的特定目标中，1-将心脏特异的SGK1缺失与编码SGK1-WT的AAV9结合 (激活；内质网靶向)、SGK1-KD(激酶死亡内质网靶向)或SGK1-∆60(激活；非内质网靶向)进行检查 SGK1和ERAD对超负荷心脏病理的影响2-联合AAV9-SGK1-WT或AAV9- 用AAV9介导的GILZ过表达或被敲除来确定GILZ是否转移SGK1-∆- 来自ER的WT并保护其免受心脏ERAD的影响，3-评估潜在的治疗、抗肥厚 一种新的SGK1多肽阻断GILZ-SGK1结合并增加非规范ERAD-1的作用 介导SGK1的降解。这些研究具有重要意义，因为它们将揭示以前未被认识到的 SGK1、GILZ和ERAD在病理性心肌肥厚中的作用我们将使用一种创新的分子策略 GILZ和非典型性ERAD作为SGK1信号和心脏调节因子的机制分析 病理学。基于多肽的破坏SGK1-GILZ相互作用可能是一种高度特异的抑制方法 SGK1选择性降解在心脏中的非适应性病理效应

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.