Non-canonical ERAD as a Regulator of Cardiac Hypertrophy - 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.
