SIRT1 (sirtuin 1) is a mammalian homolog of the longevity regulator Sir2p in yeast which catalyzes the removal of acetyl groups from protein substrates. Mammalian SIRT1 acts as an energy and stress sensor contributing to the beneficial effects of calorie restriction by regulating the acetylation status of different intracellular protein targets. Selective over-expressing SIRT1 in adipose tissue of mice prevents ageing induced insulin insensitivity and enhances energy homeostasis by inhibiting ageing related biotin accumulation and reduces the level of biotinylated protein including acetyl-CoA carboxylase (ACC) which is a major reservoir of biotin in adipose tissues. On the other hand, chronic biotin supplementation can facilitate the accumulation of ACC and abolish adipose SIRT1-mediated beneficial effects on insulin sensitivity and lipid metabolism. However, the role of biotin in regulating adipose SIRT1-mediated beneficial effects is still elusive and needs further investigations. The present study shows that overexpression of SIRT1 in adipose tissue downregulates not only the total protein expression of ACC, but also the acetylation and biotinylation of this enzyme. After chronic biotin treatment, both acetylation and biotinylation of ACC are increased, accompanied by elevated total protein expression of this enzyme. Further study using both synthetic peptide and BCCP mutant suggests that the presence of biotin and the biotinylation status of ACC could both influence to the capacity of SIRT1 to regulate the stability of this enzyme. Then the reciprocal causal relationship between ACC, biotin and SIRT1 in both 3T3-L1 adipocytes and mice adipose tissues is established. The reduced ACC can significantly attenuate the accumulation of biotin and enhance the SIRT1 activity in both 3T3-L1 cells and mice adipose tissues suggesting ACC acts as a central regulator of biotin homeostasis in cells and adipose tissues. The in vivo study demonstrates that overexpression of adipose SIRT1 significantly reduces the acetylation but not biotinylation level of histone. The biotin supplementation increases the both biotinylation and acetylation level of histone in adipose tissues. The synthetic peptide study further confirms that direct biotinylation of histone inhibits SIRT1 mediated deacetylation. Biotin also regulates the expression and acetylation of two non-biotinylated SIRT1 substrates P53 and LKB1 differentially. Finally, overexpression dominant negative deacetylase mutant SIRT1 in adipose tissues of mice accelerates ageing induced deterioration of insulin sensitivity and lipid metabolic dysfunction which is restored by fed with biotin deficient diet. Taken in conjunction, the above findings reveal that biotin antagonizes the beneficial effects of SIRT1 by modulating its deacetylation of diversified substrates which provides a potential therapeutic target for the treatment of ageing related metabolic disorders. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Sirtuins, a class of enzymes known as nicotinamide adenine dinucleotide-dependent deacetylases, have been shown to regulate a variety of biological processes, including aging, transcription, and metabolism. Severn human Sirtuins members (Sirt1-7) are involved in various kinds of severe diseases like aging, cancer development, autoimmune diseases and therefore are considered as potential drug targets for treatment. Among them, Sirt4-7 have very weak traditional deacetylation function in contrast to the others. So, investigation on the real functions of these sirtuins is a prerequisite for specific modulator (inhibitor or activator) design. Crystallography is a robust way to study the molecular basis of the catalytic function of these sirtuins. Here we show that the real function of Sirt6 is the de-long-chain-fatty acylase activity from lysine, such as the demyristoylase activity. The crystal structure of Sirt6 complex shows a large hydrophobic pocket accommodating the myristoyl group. Together with the biochemical and physiological data from our collaborators, we confirm that Sirt6 promotes the TNFα secretion via hydrolysis the myristoyl group on K19 and K20. Fatty acylation on lysine occurs in mammalian cells and had been found for years, however, the regulatory mechanism is still unclear. Our results provide the opportunities to understand the regulatory of the long chain fatty acyl modification on lysine via Sirt6, which has been little studied until now. More work will be focused on the structural based development of inhibitors to cure the Sirt6 regulated diseases in the near future. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Meredith L Sosulski

SIRT1 (sirtuin 1), a mammalian ortholog of the longevity regulator yeast Sir2p, elicits diversified functions by mediating NAD+ -dependent deacetylation of protein targets. SIRT1 contributes to the beneficial effects of calorie restriction, the non-genetic intervention capable of promoting longevity and reducing the incidence of age-related disorders. In mammals, SIRT1 acts as a metabolic regulator in response to environmental stress signals. Activation of SIRT1 protects mice against diet-induced obesity and insulin resistance. However, the tissue specific metabolic functions of SIRT1 remain to be defined. The present study shows that over-expression of human SIRT1 selectively in adipose tissue decreases circulating lipid levels, reduces whole body fat mass, and elevates systemic insulin sensitivity. By contrast, over-expression of a dominant-negative human SIRT1 mutant H363Y in adipose tissue accelerates the development of aging-associated insulin resistance. Activation or down-regulation of adipose SIRT1 promotes lipid mobilization towards different metabolic organs and alters biotin homeostasis in opposite manners. Adipose SIRT1 positively regulates lipid metabolism, genes expression and adipokines secretion which are negatively influenced by SIRT1 H363Y mutant. Biotin is a water soluble vitamin and plays an important role in energy metabolism. The present study shows that biotin and its metabolites are the endogenous inhibitors of SIRT1 enzymatic activity. Chronic biotin supplementation abolishes adipose SIRT1-mediated beneficial effects on lipid metabolism. These effects are partly explained by the regulation of acetyl-CoA carboxylase (ACC), a key regulator of lipid metabolism and biotin homeostasis. SIRT1 selectively deacetylates and negatively regulate the protein stability of ACC. Over-expression of SIRT1 in fat cells persistently down-regulates ACC expression. The direct interaction between SIRT1 and ACC are subjected to rapid regulation by nutrient status which can explain the beneficial effect of SIRT1 in energy homeostasis. In mice subjected to chronic treatment with biotin, the interactions between SIRT1 and ACC were significantly inhibited. Taken in conjunction, the above findings reveal that SIRT1 in adipose tissue functions to regulate systemic energy metabolism and insulin sensitivity. In particular, it plays a critical role in modulating ACC protein levels and biotin homeostasis in adipose tissue, which in turn facilitate lipid storage and utilization in response to nutrient level changes. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Endothelial senescence represents one of the major characteristics of vascular aging contributing to the development of cardiovascular diseases. SIRT1 is a NAD+-dependent enzyme catalyzing the deacetylation reaction of various signaling molecules and exerts beneficial effects against aging-associated pathologies. SIRT1 is a potent regulator antagonizing endothelial senescence. Both expression and activity of SIRT1 are down-regulated in senescent endothelial cells. However, the molecular mechanisms underlying the loss-of-SIRT1 function during the occurrence of endothelial senescence remain unknown. The present study reveals that phosphorylation at serine 47(S47) contributes to the loss-of-SIRT1 function during endothelial senescence. In both replicative and premature senescent endothelial cells, increased phosphorylation of SIRT1 at S47 was closely associated with the severity of cellular senescence. Replacing serine 47 residue with a phospho-mimicking aspartic acid residue impaired the anti-senescence activity of this protein. In addition, phosphorylation of SIRT1 at serine 47 inhibited its nuclear-cytoplasmic shuttling and protein-protein interactions with LKB1, a senescence-promoting kinase and telomeric repeat-binding factor 2–interacting protein 1, a telomere and inflammation regulator. As a result, the anti-inflammatory function of SIRT1 was also abolished by phosphorylation at serine 47. Cyclin dependent kinase 5 (CDK5) was identified as an upstream kinase responsible for phosphorylation of SIRT1 at serine 47. During the endothelial senescence, the activity of this kinase was up-regulated which was attributed to the augmented P25, a regulatory subunit of CDK5. Inhibition of this kinase by roscovitine, a CDK5 inhibitor, decreased the phosphorylation of SIRT1 at serine 47, reduced cellular senescence, promoted the cytoplasmic translocation of SIRT1 and attenuated the inflammation in endothelial cells triggered by tumor necrosis factor α. Moreover, the kinase activity of CDK5 was significantly elevated in aorta tissues of apolipoprotein E–deficient mice. Chronic administration of roscovitine alleviated endothelial senescence, vascular inflammation and the development of arterial atherosclerosis. These results collectively suggest that CDK 5 is responsible for the phosphorylation of SIRT1 at serine 47, which impairs the anti-senescence activity of enzyme and contributes to loss-of-SIRT1 function during vascular aging. By inhibiting this kinase, SIRT1 function can be improved, in turn preventing the development of endothelial senescence and slowing down the process of vascular aging. In addition to phosphorylation, I have also performed a preliminary study on the ubiquitination of SIRT1. The results demonstrated that SIRT1 ubiquitination was mediated by a Cullin-1-RING E3 ligase complex. Knocking down of cullin-1 enhanced SIRT1 protein expression, promoted proliferation and inhibited senescence in endothelial cells. This discovery may provide novel insights on the anti-vascular aging therapeutic development based on SIRT1 modification. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

In blood vessel of mature animal, endothelial cells remain quiescent for years, before apoptosis and being replaced by newly generated endothelial cells. During aging, this turnover process is accelerated and the fast generated endothelial cells become dysfunctional. Endothelial dysfunction in blood vessel is characterized by the imbalanced production of endothelium-dependent relaxing factors (EDRF) and endothelium-dependent contracting factors (EDCF). The NAD-dependent deacetylase SIRT1 is an anti-aging protein with therapeutic potential for aging related cardiovascular diseases. Endothelium-specific over-expression of human SIRT1 promotes endothelium-dependent vasodilatation and endothelium-selective inhibition of human SIRT1 inhibit it. It is accepted that SIRT1 plays a protective role in endothelium dysfunction. However, the underlying mechanisms remain unclear. In the present study, the endothelial functions of a transgenic mouse model with endothelium-selective over-expression of human SIRT1 (hSIRT1) were evaluated and compared with those of wild type mice. Aging-induced deterioration in endothelium-dependent vasodilatation was observed in wild type but not hSIRT1 mice. Endothelium-specific over-expression of SIRT1 prevented aging-induced reduction of NO bioavailability in aortae, without changing endothelial nitric oxide synthase (eNOS) expression levels. Enhanced phosphorylation of eNOS at serine 1177 was detected in hSIRT1 mice aorta. In the presence of Nω-Nitro-L-arginine methyl ester (L-NAME), the nitric oxide synthase (NOS) inhibitor, EDCF induced contraction to acetylcholine was significantly decreased in carotid arteries of hSIRT1 mice. Cyclooxygenase-2 (COX-2) expression was induced by aging in wild-type mice but not in hSIRT1 mice. Thus, both the augmented NO bioavailability and the reduced production of COX-2-derived EDCF in hSIRT1 mice enhanced their endothelial function. To further explore the eNOS-independent mechanism underlying the vasoprotective role of SIRT1, the eNOS deficient and hSIRT1 endothelium-specific over-expression (eNOS-hSIRT1) mice was generated. Decreased endothelium-dependent contraction to acetylcholine was observed in both the carotid artery and aorta of eNOS-hSIRT1 mice, when compared to the controlled eNOS deficient mice. Besides, ATP induced endothelium-dependent contraction, which was COX-dependent, was also decreased in aortae of eNOS-hSIRT1 mice. Thus, the improved endothelial function induced by the endothelium-selective overexpression of SIRT1 was partly attributed to the reduced COX function, independent of eNOS signaling pathway. In summary, endothelium-selective overexpression of human SIRT1 prevented aging-induced impairment of endothelial function via both eNOS dependent and independent mechanisms. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

No description available.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

No description available.

Sirtuins (Silent Information Regulator Two (SIR2) protein) are NAD-dependent protein deacetylases, originally discovered in yeast. Sirtuins play a critical role in the regulation of different cellular processes involving aging, chromatin silencing and cellular differentiation. SIRT6 is a member of Sirtuins and plays a role in regulation of DNA repair and suppression of genomic instability. Many studies have shown SIRT6 to be associated with diseases of aging, including cancer. The finding by our collaborator that SIRT6 expression was found in chemotherapy-resistant breast cancer cell lines stimulated this study which aims to explore the role of SIRT6 expression as a prognostic marker in breast cancer. One hundred and eighteen breast cancer samples in tissue microarray blocks were examined for SIRT6 expression by immunohistochemistry. As SIRT6 expression is predominantly located in the nucleus but with a small fraction in cytoplasm, the calculation of nuclear or cytoplasmic localization scores were divided by total localization scores to increase accuracy. The nuclear localization scores represent the SIRT6 expression in breast cancer. Statistical analysis was performed using SPSS software. SIRT6 overexpression in the nucleus was significantly associated with poorer overall survivals (p=0.018) while low cytoplasmic expression of SIRT6 was also associated with poorer overall survivals (p=0.014). There was no relationship between SIRT6 expression and disease-specific survivals. By multivariate analysis, SIRT6 expression was an independent predicator of poorer overall survivals. These results suggest that SIRT6 overexpression induces apoptosis in cancer cells through deacetylation of transcription factor p65. SIRT6 interacts with and deacetylates p65 to activate nuclear factor kappa B gene linked to cancer. Also high levels of SIRT6 were associated with resistance to paclitaxel and epirubicin inMCF-7 breast cancer cell lines. This provides evidence that Sirt6 is an important prognostic marker and therapeutic target for breast cancer. / published_or_final_version / Pathology / Master / Master of Medical Sciences