| dc.description.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. *Role of SIRT6 in the regulation of mTOR signaling and global protein synthesis* Sirtuins are a class of NAD+-dependent deacetylases which are best studied for their role in aging and regulation of healthspan. Of the seven isoforms of Sirtuins (SIRT1-7) described in mammals, the nuclear localized SIRT6 has been shown to regulate several critical cellular functions including metabolism, inflammation, DNA repair, telomere maintenance etc. However, the role of SIRT6 in the regulation of cellular protein synthesis which is an important determinant of aging is not well understood. In the present work, we find that SIRT6 represses cellular protein synthesis through transcriptional regulation of mTOR signaling. In vivo measurement of protein synthesis using the SUnSET, assay revealed significant upregulation of protein synthesis in multiple tissues of SIRT6 deficient mice. Further, multiple lines of in vitro evidence including polysome analysis and reporter assays indicate that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 deficiency leads to increased activation of mTOR signaling, which is the master regulator of protein synthesis in cells. SIRT6 directly binds to the promoters of mTOR signaling genes and represses their transcription by antagonising the Sp1 transcription factor. NMR and ChIP experiments reveal that SIRT6 directly binds to the zinc finger domain of Sp1 and regulates its occupancy at the mTOR gene promoters. In the absence of SIRT6, increased expression and recruitment of mTOR and its upstream activator Rheb to the surface of lysosomes leads to enhanced phosphorylation of mTORC1 complex and activation of its downstream targets. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle specific SIRT6 knockout mice which spontaneously develop cardiac hypertrophy and heart failure. Our findings have thus unravelled a new layer of regulation of protein synthesis and indicates that SIRT6 could be potentially targeted for treating several age-related diseases where protein synthesis is deregulated. | en_US |
