Role of SIRT6 in the regulation of mTOR signalling and global protein synthesis

Abstract

Cells are constantly engaged in the process of making and breaking proteins in a highly organized manner. However, protein synthesis is an energetically expensive process and often competes with other cellular repair and maintenance processes. The process is thus tightly regulated and any deregulation adversely impacts the overall health of the cell and the organism. Recent studies implicate protein synthesis to be an important determinant of aging. However, the molecular mechanisms regulating protein synthesis is not completely understood. The present work focusses on developing a simple method for studying protein synthesis under in vivo conditions and understanding the role of the anti-aging molecule Sirtuin 6 (SIRT6) in the regulation of protein synthesis. *Development and validation of a non-radioactive assay to measure protein synthesis in in vitro primary cultures and in vivo conditions* Traditional methods employed to study changes in protein synthesis levels are often cumbersome and involve the use of radiolabelled tracers which carry substantial risk. Recently, a nonradioactive assay known as the Surface Sensing of Translation (SUnSET) was described for measuring protein synthesis under in vitro conditions. In this method, the newly synthesized nascent peptides are labelled using the small molecule puromycin, which are then detected immunologically using a specific antibody. We adapted this method, standardized and validated its use for measuring protein synthesis in in vitro primary cultures and in vivo conditions. The results suggest that the SUnSET assay can reliably measure the changes in protein synthesis with high specificity and sensitivity under in vitro as well as in vivo conditions. Further, we successfully tracked the changes in cardiac protein synthesis during different stages of the development of agonist-induced hypertrophy. Overall, we find that SUnSET assay is a simple, reliable and robust method to measure protein synthesis under in vivo conditions...