Abstract 144: An Intact NEDD8 Pathway Protects Cardiomyocytes against Proteotoxic Stress

Abstract

NEDD8 is a ubiquitin (Ub)-like protein whose modification on cellular proteins is crucial to various cellular processes. Conjugation of NEDD8 to target proteins (neddylation) is mediated by its unique set of E1, E2 and E3, and can be reversed by the actions of deneddylase such as the COP9 signalosome (CSN) and NEDP1, a process termed deneddylation. Canonical neddylation involves NEDD8 E1 (NAE1) and directly attaches NEDD8 to the substrate, whereas atypical neddylation utilizes a Ub activating enzyme (UBE1) to incorporate NEDD8 to the Ub chain of target proteins. The physiological significance of such atypical neddylation remains unknown in any cell types. Here we report a protective role of atypical neddylation to proteotoxic stress in cardiomyocytes. Proteasome inhibitor Bortezomib (BZM) increases high molecular weight species of neddylated proteins in a dose- and time- dependent manner. Immunoprecipitation analysis reveals mixed modification of these proteins by Ub and NEDD8. Silencing of UBE1, but not inhibition of NAE1, diminishes BZM-induced neddylation response and aggravates the BZM-induced cell death. Interestingly, forced expression of deneddylase NEDP1 also reduces BZM-induced neddylation but surprisingly attenuates cell injury. The protective effect of NEDP1 is dependent on its deneddylation activity. We thus propose that atypical neddylation is an adaptive response and that dynamic cycling of neddylation and deneddylation is essential to cardiomyocytes to antagonize proteotoxic stress, possibly through preventing depletion of ubiquitin. Additionally, imposing proteotoxic stress by H2O2, simulated ischemia reperfusion or expression of misfolded proteins all elicits such atypical neddylation in cardiomyocytes. Furthermore, increased neddylated proteins are also evident in mouse hearts suffering from proteotoxic stress, which is caused by either expression of misfolded proteins or deficiency of a 19S proteasome subunit. These findings, combined with our previous reports that the CSN regulates proteasomal and autophagic proteolysis in the heart, strongly suggest that a fine-tuned NEDD8 system is critical to protein quality control and is essential to cardiomyocyte survival and functioning.