Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma.

The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor-intensive. To this end, we present a polyadenylation signal-trap construct for N'-tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c-CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants.

Characterization of physiological defects in adult SIRT6-/- mice.

The NAD+-dependent SIRT6 deacetylase was shown to be a major regulator of lifespan and healthspan. Mice deficient for SIRT6 develop a premature aging phenotype and metabolic defects, and die before four weeks of age. Thus, the effect of SIRT6 deficiency in adult mice is unknown. Here we show that SIRT6-/- mice in mixed 129/SvJ/BALB/c background reach adulthood, allowing examination of SIRT6-related metabolic and developmental phenotypes in adult mice. In this mixed background, at 200 days of age, more than 80% of the female knock-out mice were alive whereas only 10% of male knock-out mice survived. In comparison to their wild-type littermates, SIRT6 deficient mice have reduced body weight, increased glucose uptake and exhibit an age-dependent progressive impairment of retinal function accompanied by thinning of retinal layers. Together, these results demonstrate a role for SIRT6 in metabolism and age-related ocular changes in adult mice and suggest a gender specific regulation of lifespan by SIRT6.

The sirtuin SIRT6 regulates lifespan in male mice.

The significant increase in human lifespan during the past century confronts us with great medical challenges. To meet these challenges, the mechanisms that determine healthy ageing must be understood and controlled. Sirtuins are highly conserved deacetylases that have been shown to regulate lifespan in yeast, nematodes and fruitflies. However, the role of sirtuins in regulating worm and fly lifespan has recently become controversial. Moreover, the role of the seven mammalian sirtuins, SIRT1 to SIRT7 (homologues of the yeast sirtuin Sir2), in regulating lifespan is unclear. Here we show that male, but not female, transgenic mice overexpressing Sirt6 (ref. 4) have a significantly longer lifespan than wild-type mice. Gene expression analysis revealed significant differences between male Sirt6-transgenic mice and male wild-type mice: transgenic males displayed lower serum levels of insulin-like growth factor 1 (IGF1), higher levels of IGF-binding protein 1 and altered phosphorylation levels of major components of IGF1 signalling, a key pathway in the regulation of lifespan. This study shows the regulation of mammalian lifespan by a sirtuin family member and has important therapeutic implications for age-related diseases.

SIRT6 protects against pathological damage caused by diet-induced obesity.

The NAD+-dependent SIRT6 deacetylase is a therapeutic candidate against the emerging metabolic syndrome epidemic. SIRT6, whose deficiency in mice results in premature aging phenotypes and metabolic defects, was implicated in a calorie restriction response that showed an opposite set of phenotypes from the metabolic syndrome. To explore the role of SIRT6 in metabolic stress, wild type and transgenic (TG) mice overexpressing SIRT6 were fed a high fat diet. In comparison to their wild-type littermates, SIRT6 TG mice accumulated significantly less visceral fat, LDL-cholesterol, and triglycerides. TG mice displayed enhanced glucose tolerance along with increased glucose-stimulated insulin secretion. Gene expression analysis of adipose tissue revealed that the positive effect of SIRT6 overexpression is associated with down regulation of a selective set of peroxisome proliferator-activated receptor-responsive genes, and genes associated with lipid storage, such as angiopoietin-like protein 4, adipocyte fatty acid-binding protein, and diacylglycerol acyltransferase 1, which were suggested as potential targets for drugs to control metabolic syndrome. These results demonstrate a protective role for SIRT6 against the metabolic consequences of diet-induced obesity and suggest a potentially beneficial effect of SIRT6 activation on age-related metabolic diseases.

Regulation of SIRT1 protein levels by nutrient availability.

The mammalian NAD+ dependent deacetylase, SIRT1, was shown to be a key protein in regulating glucose homeostasis, and was implicated in the response to calorie restriction. We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions. These results have important implications on the relationship between sirtuins, nutrient availability and aging.

Regulation of SIRT6 protein levels by nutrient availability.

Sirtuins have been shown to regulate life-span in response to nutritional availability. We show here that levels of the mammalian sirtuin, SIRT6, increased upon nutrient deprivation in cultured cells, in mice after fasting, and in rats fed a calorie-restricted diet. The increase in SIRT6 levels is due to stabilization of SIRT6 protein, and not via an increase in SIRT6 transcription. In addition, p53 positively regulates SIRT6 protein levels under standard growth conditions but has no role in the nutrient-dependent regulation of SIRT6. These observations imply that at least two sirtuins are involved in regulation of life-span by nutrient availability.