Breast cancer is the most frequent malignancy in women. Recent studies have proposed that sirtuin 1 (SIRT1) may play a certain role in the tumorgenesis and disease progression of cancer. Therefore, in this study, we demonstrate the localization of SIRT1 in the breast cancer cells by immunohistochemistry method and try to correlate the expression level of SIRT1 with various clinical-pathological parameters as well as the survival time of breast cancer patients. One hundred and eighteen breast cancer cases, arrayed as dual‐cores, were studied in the tissue microarray blocks for their SIRT1 nuclear and cytoplasmic stain. The expression of SIRT1 is found in over 95% of the tumor samples. Although the active functioning site of SIRT1 is known to be mainly the nucleus, both nuclear and cytoplasmic localization score are assessed separately for SIRT1 expression for more accurate statistically analysis. By bi‐variate Pearson correlation analysis, high nuclear localization of SIRT1 is significantly correlated with low tumor grade (p=0.006) and ER (p=0.001) and PR positive status (p=0.044). Moreover, the cytoplasmic localization score of SIRT1 shows positive correlation with tumor grade (p=0.010). The relationship of SIRT1 expression and survival time of breast cancer patient was studied by Kaplan‐Meier analysis. Despite a marginal fail in obtaining a statistically significant result, the trend in survival curve clearly indicated that nuclear localization of SIRT1 is associated with a poorer overall survival (p=0.052). Although the pathway of how SIRT1 affects the survival of breast cancer patient is still unknown, many studies suggested that it is largely due to the deacetylated inactivation of p53 tumor suppressor protein by SIRT1. In conclusion, we propose that nuclear localization of SIRT1 can be a potential prognostic factor of breast cancer patients. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Understanding the structural basis of specificity and promiscuity of paralogous enzymes is important for deciphering molecular mechanisms and is a necessary step towards designing enzyme-specific modulators. The main objective of this thesis is to provide structural insights that relate protein local sequences to their observed binding and activity profiles through the study of two human protein families – cytosolic sulfotransferases (SULTs) and sirtuins (SIRTs). This was achieved by comparing the family-wide ligand binding fingerprints of these two enzyme families with the structural details of their corresponding enzyme-ligand co-crystal structures. The hSULT enzyme family was profiled against a focused library through binding and activity assays. This suggested a number of novel compounds that bind to the less well-characterized SULT members (SULT1C3 and SULT4A1), and revealed additional broad-spectrum hSULT inhibitors. Based on the profiling data, three enzyme/co-factor/ligand complex structures were solved using X-ray crystallography. The structure of SULT1C2•PAP(3'-phosphoadenosine 5'-phosphate)•pentacholorphenol(PCP) provided a rationale for a novel SULTs inhibition mechanism that depends on substrate acidity. The SULT1B1•PAP•resveratrol structure suggested that the hydrogen-bonding coordination of the 5-OH group on resveratrol is the structural determinant for the observed substrate preference towards resveratrol. SULT2A1•PAP•lithocholic acid(LCA) ternary complex structure confirms that the specificity of SULT2A1 for lithocholic acid derives from its high hydrophobicity in the substrate binding pocket. The same approach was used to interrogate the interaction of the sirtuins with their peptide substrates. The binding and enzymatic assays for human sirtuins have suggested that SIRT1 and SIRT2 are generally less discriminate against substrates while class IV sirtuins - SIRT6 and SIRT7 might be highly specific enzymes. Three different biochemical and kinetic assays showed that SIRT6-dependent histone deacetylation is about 1,000 times slower than for other highly active sirtuins. To understand the molecular basis for the specificity and low activity of SIRT6, I determined the first set of crystal structures for SIRT6 in complex with ADPr (ADP ribose) and the non-hydrolyzable analog of OAADPr (2’-O-acetyl-ADP ribose) – NAADPr (2’-N-acetyl-ADP ribose). The structures revealed human SIRT6 has unique structural features including a splayed zinc-binding domain, lacks a helix bundle and the conserved, highly flexible, NAD(+)-binding loop, which contribute to its observed biochemical behavior.

Understanding the structural basis of specificity and promiscuity of paralogous enzymes is important for deciphering molecular mechanisms and is a necessary step towards designing enzyme-specific modulators. The main objective of this thesis is to provide structural insights that relate protein local sequences to their observed binding and activity profiles through the study of two human protein families – cytosolic sulfotransferases (SULTs) and sirtuins (SIRTs). This was achieved by comparing the family-wide ligand binding fingerprints of these two enzyme families with the structural details of their corresponding enzyme-ligand co-crystal structures. The hSULT enzyme family was profiled against a focused library through binding and activity assays. This suggested a number of novel compounds that bind to the less well-characterized SULT members (SULT1C3 and SULT4A1), and revealed additional broad-spectrum hSULT inhibitors. Based on the profiling data, three enzyme/co-factor/ligand complex structures were solved using X-ray crystallography. The structure of SULT1C2•PAP(3'-phosphoadenosine 5'-phosphate)•pentacholorphenol(PCP) provided a rationale for a novel SULTs inhibition mechanism that depends on substrate acidity. The SULT1B1•PAP•resveratrol structure suggested that the hydrogen-bonding coordination of the 5-OH group on resveratrol is the structural determinant for the observed substrate preference towards resveratrol. SULT2A1•PAP•lithocholic acid(LCA) ternary complex structure confirms that the specificity of SULT2A1 for lithocholic acid derives from its high hydrophobicity in the substrate binding pocket. The same approach was used to interrogate the interaction of the sirtuins with their peptide substrates. The binding and enzymatic assays for human sirtuins have suggested that SIRT1 and SIRT2 are generally less discriminate against substrates while class IV sirtuins - SIRT6 and SIRT7 might be highly specific enzymes. Three different biochemical and kinetic assays showed that SIRT6-dependent histone deacetylation is about 1,000 times slower than for other highly active sirtuins. To understand the molecular basis for the specificity and low activity of SIRT6, I determined the first set of crystal structures for SIRT6 in complex with ADPr (ADP ribose) and the non-hydrolyzable analog of OAADPr (2’-O-acetyl-ADP ribose) – NAADPr (2’-N-acetyl-ADP ribose). The structures revealed human SIRT6 has unique structural features including a splayed zinc-binding domain, lacks a helix bundle and the conserved, highly flexible, NAD(+)-binding loop, which contribute to its observed biochemical behavior.

Chen, Juan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Global climate change imposes physiological constraints on marine ecosystems that can alter the distribution of intertidal organisms. In one such instance, the native cold-adapted mussel Mytilus trossulus is being replaced along its southern range by the invasive warm-adapted Mytilus galloprovincialis. These blue mussels occur throughout rocky intertidal zones where they are subjected to greatly varying environmental conditions known to induce oxidative stress. We hypothesize that while under acute stress, related Mytilus congeners undergo a shift in redox potential from NADH-fueled respiratory pathways to pathways producing NADPH as a way to decrease the production of reactive oxygen species (ROS) and provide reducing equivalents to detoxify ROS. Additionally, we hypothesize that sirtuins (SIRT; a family of NAD-dependent deacetylases) might be involved in the regulation of this metabolic transition. To test the latter, a discovery approach will be used to analyze the proteomic response of M. galloprovincialis and M. trossulus to the pro-oxidant menadione, and sirtuin-inhibitors nicotinamide and suramin. Menadione can induce oxidative stress by increasing endogenous peroxide and superoxide radicals, while suramin and nicotinamde both inhibit sirtuin activity. Organisms were exposed to these compounds in filtered seawater for 8 h, followed by a 24.5 h recovery period under constant aeration. A multivariate analysis utilizing 2D-gel electrophoresis and protein identification via mass spectrometry showed that 18% and 17% of all identified protein spots detected demonstrated changes in abundance in M. galloprovincialis and M. trossulus, respectively. Using matrix-assisted laser desorption ionization (MALDI) tandem time-of-light mass spectrometry, we were able to identify 32-41% of proteins, depending on the species. The two Mytilus congeners showed the greatest differences in changes of protein abundance for oxidative stress proteins (including NADP-dependent isocitrate dehydrogenase). Both congeners showed similar effects in response to simultaneous sirtuin inhibition and MIOS for proteins involved in protein degradation (proteasome), cytoskeletal modifications (actin and tubulin), proteins regulating actin filament growth (F-actin capping protein), amino acid metabolism and stress signaling (G-proteins, small G-proteins and MAPK). Results indicate that protein acetylation plays an important role in the oxidative stress response of M. galloprovincialis. More specifically this suggests that sirtuins play an important role in regulating the general stress response in M. galloprovincialis and thus contribute to the greater stress resistance of this species. Furthermore, changes in the abundance of several molecular chaperones suggest a greater effect of sirtuins in regulating the cellular response to heat stress, which could in part explain why this species is more heat-tolerant than the native M. trossulus.

SABP2-interacting protein 428(SIP428) is a SIR2-type deacetylase, also called sirtuins. The SIP428 proteins belong to a family of NAD+-dependent deacetylase enzyme that was identified in tobacco. SABP2 is an important methyl esterase enzyme that catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) during the pathogenic challenge. Accumulation of SA induces systemic acquired resistance (SAR), a broad-spectrum defense mechanism in other uninfected distal parts of the plant. Sirtuins play diverse roles in DNA repair, apoptosis, and stress responses. Cellular proteins are known to undergo posttranslational modifications such as methylation, phosphorylation, and ubiquitination. A more recent addition to the list is acetylation. Protein acetylation is a reversible modification that plays role in regulating transcription, activation, and deactivation of certain pathways by transferring acetyl group to lysine residues. This change neutralizes the positive charge of the amino group thereby affecting the biological function of the affected proteins. Preliminary research has shown that SIP428 is a non-histone deacetylase. To understand better about the role of SIP-428 in plant physiology and how it plays a vital role in SABP2 signaling pathway we will be using transgenic tobacco plant in which the expression of SIP 428 has been silenced/knocked down.

Acetylated adenosine diphosphoribose (OAADPR) is a product of protein deacetylation catalysed by class III of histone deacetylases called sirtuins. Sirtuins deacetylate histones and other proteins by unique mechanism coupled with consumption of stoichiometric amount of NAD+ . Sirtuins and OAADPR are implicated in the regulation of gene transcription, signalling and metabolic pathways and lifespan extension, thus preventing the development of age-related diseases. Even though, sirtuins are well studied, the exact biological role of OAADPR remains mainly unknown. Its further exploration is restricted by OAADPR's proneness to enzymatic hydrolysis. Therefore, non-hydrolysable analogues of OAADPR are needed to establish its biological function. These analogues are also expected to be competitive inhibitors of sirtuins, which may reveal their potential as therapeutic agents. A series of OAADPR analogues in which the acetate moiety was replaced with alkylcarbonate functionality has been synthesized. The studies of alkylcarbonate migration on furanoside scaffold have established the stability of alkylcarbonate vs. acetate under various conditions. Generally, alkylcarbonates are more stable than acetate under acidic or neutral conditions whereas under basic conditions they seem to be less stable....

Background: Cardiovascular disease continues to remain a leading cause of morbidity and mortality in both developing and developed worlds. The sirtuin and urocortin systems are novel hormone systems in humans with an emerging role in cardiovascular physiology and pathophysiology. Through a series of studies, this thesis examines the cardiovascular effects of SRT2104 (a novel small molecule SIRT1 activator) in otherwise healthy cigarette smokers and in patients with type 2 diabetes mellitus, and of urocortins 2 and 3 in healthy volunteers and in patients with heart failure. Methods: Twenty-four otherwise healthy cigarette smokers and 15 subjects with stable type 2 diabetes participated in a randomised, double blind, placebo controlled, crossover trial and received 28 days of oral SRT2104 (2.0 g/day) or matched placebo. Plasma SRT2104 concentrations, serum lipid profile, plasma fibrinolytic factors, markers of platelet and monocyte activation and pulse wave analysis and velocity were measured at baseline and the end of each treatment period together with an assessment of forearm blood flow during intra-arterial bradykinin, acetylcholine and sodium nitroprusside infusions. The pharmacodynamic profile of urocortins 2 and 3 were assessed in 18 healthy male volunteers recruited into a series of randomised, double blind, placebo controlled, crossover studies. Bilateral forearm venous occlusion plethysmography was performed during incremental intra-arterial infusions of urocortin 2 (3.6-120 pmol/min), urocortin 3 (1.2-36 nmol/min) and substance P (2-8 pmol/min) in the presence or absence of inhibitors of cyclooxygenase (aspirin), cytochrome P450 metabolites of arachidonic acid (fluconazole) and nitric oxide synthase (L-NG-monomethyl-arginine (L-NMMA)). Finally, 12 patients with stable heart failure (New York Heart Association (NYHA) II-IV) and 10 age- and sex-matched healthy volunteers were recruited to attend once each. Bilateral forearm arterial blood flow was measured using forearm venous occlusion plethysmography during incremental intra-arterial infusions of urocortin 2 (3.6-36 pmol/min), urocortin 3 (360-3600 pmol/min) and substance P (2-8 pmol/min). Results: SRT2104 was safe and well tolerated in otherwise healthy cigarette smokers and subjects with type 2 diabetes mellitus. There were no significant differences in fibrinolytic or blood flow parameters between placebo and SRT2014. Treatment with SRT2104 was associated with a significant reduction in augmentation pressure (P=0.0273) and a trend towards improvement in the augmentation index (AIx) and corrected augmentation index (0.10 > P > 0.05 for both) without significant changes in pulse wave velocity (PWV) and time to wave reflection (Tr) (P > 0.05). Administration of SRT2104 had a favourable effect on lipid profile in otherwise healthy cigarette smokers in comparison to placebo. Urocortins 2 and 3 evoked arterial vasodilatation (P < 0.0001) without tachyphylaxis but with a slow onset and offset of action. Inhibition of nitric oxide synthase with L-NMMA reduced vasodilatation to substance P and urocortin 2 (P≤0.001 for both) but had little effect on urocortin 3 (P > 0.05). Neither aspirin nor fluconazole affected vasodilatation induced by any of the infusions (P > 0.05 for all). In the presence of all three inhibitors, urocortin 2- and urocortin 3-induced vasodilatation were attenuated (P < 0.001 for all) to a greater extent than with L-NMMA alone (P≤0.005). The vasodilatory effects of urocortins 2 and 3 were preserved in patients with heart failure. Conclusion: Activation of SIRT1 through SRT2104 improved lipid profile but did not produce demonstrable differences in vascular or platelet function with some effect on measures of arterial stiffness. Urocortins 2 and 3 appear to be potent arterial vasodilators whose vasomotor responses remained preserved in patients with heart failure and were at least partly mediated via the endothelium. Both hormone systems hold potential in their role in cardiovascular disease in man but require further studies to help translate findings of this thesis to clinical practice.

The overall objective of this thesis was to test the hypothesis that maternal overnutrition has adverse effects on the expression of central metabolic regulators in offspring but could be offset by supplementing green tea extract (GTE) to the dams during gestation and/or lactation. This thesis focuses on two aspects of central metabolic regulation: the leptin signaling that links to appetite regulation and the sirtuin 1(SIRT1)/oxidative stress pathway that links to insulin sensitivity (IS). This study was initiated based on previous findings of this laboratory that via developmental programming energy intake of offspring born to dams given GTE during lactation was suppressed and that IS was improved in offspring of dams supplemented with GTE during gestation and/or lactation. The diets used included low fat (LF), high-fat (HF), and HF diet added with 0.75% or 1%GTE (GT1, GT2). In experiment 1, female rats were given the respective diet 8 weeks prior to mating till the end of lactation. Male offspring were weaned to the HF, GT1 or GT2 diet for 10 weeks forming the LF/HF, HF/HF, GT1/HF, GT2/HF, HF/GT1 and HF/GT2 groups. Maternal and postweaning GTE supplementation increased hypothalamic leptin receptor (OB-Rb) and signal transducer activator of transcription 3 (STAT3) mRNA suggestive of enhanced leptin signaling but pro-opiomelanocortin (POMC) mRNA expression, an appetite inhibitor was only elevated in the HF/GT1 group which was associated with reduction in food intake in this group. Central oxidative status was improved in GT1/HF and GT2/HF offspring through enhanced hypothalamic SIRT1 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) expression compared with the HF/HF group. These improvements coincided with better IS in the HF offspring born of GTE supplemented dams. Experiment 2 was designed to determine the relative importance of gestation and lactation as the critical period for GTE supplementation. Female rats were assigned to LF, HF or GT1 diet 9 weeks prior to mating till the end of pregnancy. During lactation half of the HF and GT1 dams had the diet switched to GT1 and HF, respectively. Male offspring were fed the LF or HF diet until 22 weeks of age forming 10 offspring groups: LF/LF/LF, LF/LF/HF, HF/HF/LF, HF/HF/HF, HF/GT1/LF, HF/GT1/HF, GT1/HF/LF, GT1/HF/HF, GT1/GT1/LF, and GT1/GT1/HF. Consistent with a reduction in energy intake in offspring born to dams receiving GTE supplementation during lactation, there was an increase in melanocortin 4 receptor (MC4R) expression in the hypothalamus (P<0.05). Regardless of postweaning diet, offspring of dams given GTE during gestation and/or lactation had elevated hypothalamic PGC-1α and reduced protein phosphorylation of c-Jun N-terminal kinase-1 when compared with offspring of unsupplemented dams(P<0.05) which was associated with improved IS. Hence, leptin signaling and appetite regulators in the offspring were selectively affected by GTE supplementation during lactation whereas offspring exhibited improved ability to handle oxidative stress if dams received GTE supplementation during gestation and/or lactation. Collectively, these results support the notion that central mechanisms with roles in appetite control and oxidative status are susceptible to the programming phenomenon triggered by maternal nutritional status. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Abiotic stress leads to a change in the water content of plants. Salinity and osmotic stress affect both the morphology and physiology of plants. Plants have therefore responded to these environmental changes by adapting and tolerating them. The SABP2-interacting proteins (SIP) 428-silenced RNAi transgenic tobacco lines were subjected to various abiotic stresses (salinity, osmotic, and drought). The effect of SIP428-silencing on the tobacco plants subjected to these abiotic stresses was monitored. The results from the root growth data show that the sip428-silenced lines exhibit enhanced tolerance to the stressors compared to the wild-type plants. Interestingly, results of the relative chlorophyll content show no significant difference between the wild-type plants and sip428-silenced transgenic plants. In summary, based on the results presented in this study it could be concluded that SIP428 is a negative regulator of salinity, osmotic and drought stresses. Further studies are required to understand the mechanism.