The sirtuins (SIRT1~7) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases that regulate lifespan in lower organisms. Recently, studies in the mammalian sirtuins have implicated their roles in cellular stress resistance, genomic stability, tumorigenesis and energy metabolism. Seven mammalian homologues were identified in the sirtuin family of proteins. They vary a lot in the localization and function in physiology. Genetic and cellular studies with specific homologue knock out demonstrated different subcellular compartments for each family member. The Sirt-1, Sirt-2 and Sirt-3 are localized in the cytoplasm or nucleus in regulation of cell cycle or metabolism. Sirt-4 and Sirt-5 are specifically expressed in the mitochondria and play important role in the insulin secretion and ammonia detoxification, respectively. The last two members, Sirt-6 and Sirt-7 are localized in the nucleus with functions of DNA repair or RNA transcription.
Sirtuin signaling pathway
Since the complexity of the different kind of sirtuins in the signaling transduction (Table 1), we choose the Sirt-1 protein as an example to elaborate the upstream and downstream signaling of Sirtuins.
Table 1 Substrates and funcitons of the Sirt1~7
As the best-studied mammalian sirtuin, Sirt-1 has been implicated in a variety of physiological conditions, such as calorie restriction, exercise and oxidative stress. Sirt-1 use nicotinamide dinucleotide (NAD+) as its substrate and the level of NAD+ also regulates the deacetylating activity of the Sirt-1. In the physiology conditions, the NAD+ was synthesized from NAD precursor-NAD riboside, and is sequentially synthesized and controlled by a series of proteins in the NAD salvage pathway. It was illustrated this pathway here to show the upstream regulation of Sirt-1 activity through targeting the upstream NAD+ level in the cellular process. (Figure 1)
The NAD riboside, through some unknown mechanisms transferred to the nicotinamide mononucleotide (NMN). NMN is converted to NAD+ by nicotinamide mononucleotide adenylyltransferase (NMNAT). NAD+ is also recycled by the NAD salvage pathway from nicotinamide (NAM) by nicotinamide phosphoribosyltransferase (NAMPT), which converts NAM to nicotinamide mononucleotide (NMN). NAMPT functions as the rate-limiting enzyme in the mammalian NAD+ salvage pathway. Increased NAMPT activity raises the total cellular NAD level and subsequent transcriptional regulatory activity of SIRT1. In addition, NMNAT can regulate the deacetylating activity of SIRT1 at its target gene promoters. Many Sirt-1 regulators are based on the regulation of NAD+ activity, and importantly the target Sirt-1 activity in this pathway, this will be discussed later.
Figure 1 NAD+ salvage pathway in Sirt-1 regulation
As shown in the figure 2, downstream signaling of Sirt-1 is rather complex due to its amount of targets. Interactions between SIRT1 and AMPK (AMP-activated protein kinase) pathways occur in different types of tissues and cells. In skeletal muscle, AMPK enhances SIRT1 activity by increasing cellular NAD+ levels. In liver, while AMPK and SIRT1 may act in an auto-regulatory loop to regulate lipid metabolism, their impacts on gluconeogenesis during fasting conditions appear to diverge. LKB1 (liver kinase B1) is a primary upstream kinase of AMPK, a necessary element in cell metabolism that is required for maintaining energy homeostasis. Sirt-1 may control the insulin sensitivity and metabolism through the phosphorylation of AMPK by regulating the LKB1 activity. Sirt-1 is also involved in the oxidative stress response. Human antigen R (HuR) associated with the 3' untranslated region of the Sirt-1 mRNA, stabilized the SIRT1 mRNA, and increased SIRT1 expression levels. During oxidative stress, HuR is phosphorylated, resulting in the dissociation of the HuR-Sirt-1 mRNA complex and subsequent Sirt-1 mRNA decay. Active regulator of Sirt-1 (AROS) directly regulates the Sirt-1 function and enhances SIRT1-mediated deacetylation of p53 both in vitro and in vivo, and it inhibits p53-mediated transcriptional activity. Hypermethylated in cancer 1 (HIC1) and deleted in breast cancer 1 (DBC1) have been identified as negative regulators of SIRT1. Sirt-1 also regulates the insulin secretion in the pancreatic beta cells though binding directly to the uncoupling protein UCP2 gene UCP2 promoter. The downregulation of UCP2 then increases the insulin secretion and depletion of Sirt-1 blunts the insulin secretion by increasing the UCP2 activity. Sirt-1 deacetylates the peroxisome proliferator-activated receptor Gamma Coactivator-1α (PGC-1α) and which was proposed to partially underlie the Sirt-1’s role in mitochondrial physiology, gluconeogenesis and the lipid homeostasis. Sirt-1 was also an essential regulator of FoXOs family proteins. Sirt-1 catalyzes its deacetylation in an NAD-dependent manner, and thereby increases its transactivation activity. It’s known that activity of FOXO4 is suppressed or enhanced by SIRT1 inhibitor, nicotinamide, or its activator, resveratrol, respectively. Other substrates, such as NF-kB, PTP1B and E2F1, they were involved in the modulation of apoptosis, insulin sensitivity and tumorigenesis.
Figure 2 The Sirt-1 cell signaling pathway[Chong, 2012 #2027;Bonkowski, 2016 #3949]
Sirtuin agonist in cancer therapy
Additional expression of sirtuin family proteins (such as Sirt-1 or Sirt-6), or treated with the sirtuin-activating compounds (STACs) such as resveratrol and SRT2104 or with NAD+ precursors, have improved organ function, physical endurance, disease resistance and longevity. Clinical studies on the primate and humans have indicated that STACs might be safe and effective for the metabolic and inflammatory disorders.
The first generation of STACs were a group of related plant polyphenols that included, among others, quercetin, butein and resveratrol, which is a molecule found in red wine. These molecules lower the binding affinity of Sirt protein for its substrates by 10 folds. Resveratrol rapidly became the molecule of choice to test SIRT-1 activation because it was potent, nontoxic, naturally available and inexpensive. Many researchers have proved that resveratrol have functions in extending the lifespan in yeast, worm and even honey bees. However, the resveratrol is a nonspecific compound that also binds to other proteins. Except for the Sirt-1, it also binds to AMPK, phoshodiesterases, F1-ATPase, complex III of the mitochondrial electrical transport chain and PARP1. Establishing which effect was induced by the activation of Sirt-1 by resveratrol is difficult. Some studies have shown that resveratrol activates Sirt-1 by deacetylating and activating the AMPK kinase LKB1. The AMPK in turn would activate Sirt-1 again. Recent studies suggest that resveratrol may activate both the AMPK and Sirt-1.
By now, more than 14,000 synthetic STACs have been discovered and dozens of them have been tested in animal models of neurodegeneration disease, type-2 diabetes and colitis. These STACs have more than 1,000-fold greater potency in vitro than resveratrol. The SRT2104 is such a STAC that mimics the calorie restriction and enlarges the male mouse lifespan. More meaningfully is that the Phase I and phase II studies have shown the SRT2104 has little side effects (Bonkowski and Sinclair, 2016) . The clinical trials in elderly volunteers and otherwise healthy smokers showed a little decrease of body weight, a 15%~30% improvement in the cholesterol ratio and a 19% decrease in triglyceride levels. However, additional clinical trials are needed to prove its efficacy and pharmaceutical properties in the future.
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