Abstract 15500: The Proteasome is a Target of Protein Kinase G
The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein quality control and degradation in the cell. A characteristic phenotype of most heart diseases is abnormal protein aggregation and increased ubiquitinated proteins, indicative of UPS dysfunction. More importantly, experimental studies have demonstrated that cardiac proteasome functional insufficiency plays a major pathogenic role in heart failure resulting from a large subset of heart disease.
Clinical trials and studies on animal models reveal that cGMP-dependent kinase (PKG) activation by inhibition of phosphodiesterase type 5 (e.g., sildenafil) protects against a variety of cardiac pathological conditions. We have recently reported that PKG activation facilitates proteasome-mediate degradation of misfolded proteins and slows down the progression of cardiac proteinopathy in mice (Ranek MJ et al, Circulation 2013). To investigate further the mechanism by which PKG regulates proteasome function, we performed in vitro tests with purified proteasomes treated with active PKG. We found that 26S proteasomes showed increased chymotrypsin-like peptidase activity relative to untreated proteasomes. “Gel-free” 2D nano-LC MS/MS analysis identified increased phosphorylation at proteasome subunits including 19S regulatory subunit Rpn2 and 20S catalytic subunit β-7. Sites of phosphorylation were within predicted PKG targeted sequences. Neonatal rat ventricular myocytes (NRVMs) were employed for in vivo investigation. NRVMs were treated with sildenafil or vehicle control and proteasomes were immunoprecipitated against α-3 subunit. We found that PKG was co-precipitated with the 26S proteasome and that this interaction was augmented by sildenafil. Additionally, we found that NRVM 26S proteasome chymotrypsin-like activity was significantly increased by sildenafil treatment.
These findings suggest that proteasome activity may be positively regulated by PKG and that the mechanism may be through direct interaction.
- © 2013 by American Heart Association, Inc.