Structural basis for allosteric, substrate-dependent stimulation of SIRT1 activity by resveratrol.

Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.

Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrol-induced secretome effects.

Obesity is characterized by dysfunctional white adipose tissue (WAT) that ultimately may lead to metabolic diseases. Calorie restriction (CR) reduces the risk for age and obesity-associated complications. The impact of CR on obesity has been examined with human intervention studies, which showed alterations in circulating adipokines. However, a direct effect of CR on the human adipocyte secretome remains elusive. Therefore, the effect of a 96h low glucose CR on the secretion profile of in vitro cultured mature human SGBS adipocytes was investigated by using proteomics technology. Low-glucose CR decreased the adipocyte triglyceride contents and resulted in an altered secretion profile. Changes in the secretome indicated an improved inflammatory phenotype. In addition, several adipocyte-secreted proteins related to insulin resistance showed a reversed expression after low-glucose CR. Furthermore, 6 novel CR-regulated adipocyte-secreted proteins were identified. Since resveratrol (RSV) mimics CR we compared results from this study with data from our previous RSV study on the SGBS adipocyte secretome. The CR and RSV adipocyte secretomes partly differed from each other, although both treatment strategies lead to secretome changes indicating a less inflammatory phenotype. Furthermore, both treatments induced SIRT1 expression and resulted in a reversed expression of detrimental adipokines associated with metabolic complications.

The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop.

Silent information regulator 1 (SIRT1) represents an NAD(+)-dependent deacetylase that inhibits proapoptotic factors including p53. Here we determined whether SIRT1 is downstream of the prototypic c-MYC oncogene, which is activated in the majority of tumors. Elevated expression of c-MYC in human colorectal cancer correlated with increased SIRT1 protein levels. Activation of a conditional c-MYC allele induced increased levels of SIRT1 protein, NAD(+), and nicotinamide-phosphoribosyltransferase (NAMPT) mRNA in several cell types. This increase in SIRT1 required the induction of the NAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of the NAD(+) salvage pathway and enhances SIRT1 activity by increasing the amount of NAD(+). c-MYC also contributed to SIRT1 activation by sequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1) from the SIRT1 protein. In primary human fibroblasts previously immortalized by introduction of c-MYC, down-regulation of SIRT1 induced senescence and apoptosis. In various cell lines inactivation of SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1 enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directly bound to and deacetylated c-MYC. Enforced SIRT1 expression increased and depletion/inhibition of SIRT1 reduced c-MYC stability. Depletion/inhibition of SIRT1 correlated with reduced lysine 63-linked polyubiquitination of c-Myc, which presumably destabilizes c-MYC by supporting degradative lysine 48-linked polyubiquitination. Moreover, SIRT1 enhanced the transcriptional activity of c-MYC. Taken together, these results show that c-MYC activates SIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1 suppressed cellular senescence in cells with deregulated c-MYC expression and also inhibited c-MYC-induced apoptosis. Constitutive activation of this positive feedback loop may contribute to the development and maintenance of tumors in the context of deregulated c-MYC.

Cloning, purification, crystallization and preliminary crystallographic analysis of the human histone deacetylase sirtuin 1.

Human sirtuin 1 is a member of the histone deacetylase family and is involved in cellular aging, tumourigenesis and cellular metabolism. Recombinant sirtuin 1 comprising residues 140-747 was crystallized using the hanging-drop vapour-diffusion method. The crystal diffracted to 3.4 Šresolution and belonged to space group P622, with unit-cell parameters a = b = 203.1, c = 625.3 Å, and is estimated to contain between six and 12 molecules per asymmetric unit.

Activation of PKA/SIRT1 signaling pathway by photobiomodulation therapy reduces Aß levels in Alzheimer's disease models.

A hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-ß (Aß), which correlates significantly with progressive cognitive deficits. Although photobiomodulation therapy (PBMT), as a novel noninvasive physiotherapy strategy, has been proposed to improve neuronal survival, decrease neuron loss, ameliorate dendritic atrophy, and provide overall AD improvement, it remains unknown whether and how this neuroprotective process affects Aß levels. Here, we report that PBMT reduced Aß production and plaque formation by shifting amyloid precursor protein (APP) processing toward the nonamyloidogenic pathway, thereby improving memory and cognitive ability in a mouse model of AD. More importantly, a pivotal protein, SIRT1, was involved in this process by specifically up-regulating ADAM10 and down-regulating BACE1, which is dependent on the cAMP/PKA pathway in APP/PS1 primary neurons and SH-SY5Y cells stably expressing human APP Swedish mutation (APPswe). We further found that the activity of the mitochondrial photoacceptor cytochrome c oxidase (CcO) was responsible for PBMT-induced activation of PKA and SIRT1. Together, our research suggests that PBMT as a viable therapeutic strategy has great potential value in improving cognitive ability and combatting AD.

Capillary electrophoretic enzyme assays.

In the past years, capillary electrophoresis has become a frequently used technique for enzyme assays due to the high separation efficiency and versatility as well as small sample size and low consumption of chemicals. The capillary electrophoresis assays can be divided into two general categories: pre-capillary (or offline) assays and in-capillary (or online) assays. In pre-capillary assays, the incubation is performed offline and substrate(s) and product(s) are subsequently analyzed by capillary electrophoresis. In in-capillary assays enzyme reaction and separation of the analytes are performed inside the same capillary. In such assays the enzyme is either immobilized or in solution. The latter techniques is also referred to as electrophoretically mediated microanalysis (EMMA) indicating that the individual steps of the incubation as well as analysis are performed via electrophoretic phenomena. This chapter describes both techniques using the deacetylation of acetyl-lysine residues in model peptides by sirtuin enzymes as well as the hydrolysis of acetylthiocholine by acetylcholinesterase as examples.