# Structural Biochemistry/Sirtuins
## Introduction

Sirtuin, a silent information regulator, increases the life span of model organisms. SIR2 has seven family members, SIRT1-SIRT7. SIRT1 is the closest to SIR2. Even though SIRT1 is the closest, the other family members show links to metabolism and aging. SIRT1- SIRT3 and SIRT5- SIRT7 conduct two enzymatic activities in vitro. The two enzymatic activities are NAD+ - dependent protein deacetylase activity and ADP-ribosyltransferase activity. SIRT4's only enzymatic activity is ADP-ribosyltransferase activity. The regulated metabolism and the survival of the cell depend on mammalian sirtuins.

### Founding Member: SIR2

The functions of SIR2, a founding member of the protein family of sirtuins, may provide the link between aging and chromatin regulation. Chromatin is silenced at sub-telomeric DNA, silent mating-type loci and ribosomal DNA by SIR2. SIR2 is effected by its NAD+-dependent histone deacetylase activity. H4 lysine 16 and H3 lysine 56 are the lysine residues where deacetylation happens. This play a key role in SIR2’s silencing effect. The regulation of lifespan in budding yeast is done by SIR2. This is done through two chromatin-silencing activities. The first activity involves suppressing the recombination between repeats of rDNA and thus promoting genomic stability (by preventing senescence-inducing extrachromosomal rDNA circles from being cut out and accumulated). The second activity involves decreasing the Sir2 protein levels by an increase in H$_4$K$_{16}$ acetylation levels in telomeres. In response to nutrient deprivation or mutation, SIR2 can also block lifespan extension in model organisms.

#### SIR2 in Yeast and Other Organisms

In yeast cells, SIR2 has the same function as SIRT6 in human cells. It also segregates damaged proteins which leads to the cell aging due to toxic cell aggregates. In response to lack of nutrients and other cell mutations, SIR2 blocks lifespan extension in yeast cells. Furthermore, in model organisms such as Caenorhabditis elegans and Drosophila melanogaster, SIR2 acts to promote longevity through different pathways. Another action promoted by SIR2 is extension of cell lifespan by inducing dietary restriction adaptions.

## Location of Sirtuins

Nucleus: SIRT1, SIRT6, and SIRT7
Cytosol: SIRT2
Mitochondria: SIRT3, SIRT4, and SIRT5

## Sirtuin in the Nucleus
### SIRT1

SIRT1 is one of the seven Sir2p homologues of yeast called sirtuins. SIRT1 along with SIRT6 and SIRT7 are found in the nucleus. SIRT1 requires nicotinamide adenine dinucleotide (NAD+). SIRT1 is the closest homologue to SIR2. Both control replicative senescence. SIRT1 can block oncogene-induced senescence if over expressed. While SIR2 exclusively deacetylates histones, SIRT1 deacetylates more than 40 different substrates. SIRT1 effects the structure of chromatin directly by deacetylating chromatin-regulating enzymes such as TIP6o and SUV$_{39}$H$_1$. Among other things, SIRT1 also helps regulate many other physiological processes such as apoptosis, metabolism, and stress resistance. SIRT1 is the most studied of the seven SIR2 family members.

### SIRT6

For many years after its initial discovery, SIRT6 was thought to not have any deacetylase activity and it wasn't until later that SIRT6 was discovered to be a histone deacetylase that is very substrate-specific. It is in charge of regulating chromatin function, promoting its proper function in telomere and genome stabilization, gene expression, and DNA repair. The function of SIRT6 in humans parallels the function of SIR2 in yeast. Experimentation with SIRT6-deficient mice revealed that these mice are born completely normal but begin to have phenotypic abnormalities at around two weeks. They develop spinal curvature abnormalities, osteoporosis, and other systemic problems that result in death at around one month of age. At the cellular level, lack of this protein results in genomic instability and hypersensitivity to Ionizing Radiation (IR), methylmethanesulfonate (MMS), and hydrogen peroxide (H$_2$O$_2$). Furthermore, problems with base excision repair have been noticed. This revealed the important role SIRT6 plays in maintaining homeostasis, metabolism, and the life span of the organism.

## Roles of SIRT6
#### Telomeric Chromatin Regulation

One of the most important roles of SIRT6 in relation to telomeres, is its job in maintaining telomeric chromatin integrity. SIRT6 deacetylates H$_3$K$_9$ and H$_3$K$_{56}$. In SIRT6 deficient cell, H$_3$K$_9$ and H$_3$K$_{56}$ are hyperacetylated which leads to stochastic replication-associated telomere sequence loss, accumulation of telomeric DNA damage, and genomic instability with chromosomal end-to-end fusions. With these problems, cell senescence is brought on prematurely in the cell. This discovery has implications in future cancer research because chromosomal instability is corollated with cancer and the healthy function of telomeres plays a large role in maintaining genomic stability in chromosomes.

#### DNA Repair

SIRT6 has been linked to involvement in DNA repair in humans by allowing efficient DNA DSB repair (DNA double-strand break repair). It was discovered that SIRT6 reacts with proteins(DNA-PKcs and Ku70/80) that are involved in the pathways called non-homologous end-joining pathways (NHEJ). SIRT6's association with chromatin increases drastically in response to DSB in order to decrease the levels of H$_3$K$_9$Ac. The SIRT6 structures were found to be useful in flanking chromatins near the breaks and stabilizing the DNA-PKcs required to perform DSB. When SIRT6 is deficient in the cell, DSB in cells is impaired, leading to instability in the cell.

#### Gene Expression Regulation

Studies have shown a relationship between SIRT6 and the transcription factor nuclear factor-kappa B (NF-κB)which is in charge of gene expressions related to aging, proliferation,and inflammation. A lack of SIRT6 promotes hyperactivation of this transcription factor leading to over-expression of these genes. This is further seen in experiments on SIRT6-deficient mice where these mice were noticed to have metabolic and degenerative defects. In addition to NF-κB regulation, SIRT6 also plays a role in the transcription factor, HIF1α, which is important in glucose regulation that has been connected with lifespan regulation and even cancer.

## Mitochondrial Sirtuin

Sirtuin and Fatty Acid Oxidation During fasting, SIRT3 protein expression is increased as well as its levels and enzymatic activity. The phenotype overlap of SIRT3, AceCS2, and Acadl shows that SIRT3 regulate LCAD and AceCS2 acetylation.
Sirtuin and the Electron Transport Chain Deacetylates Complex I subunits and Succinate Dehydrogenase (Complex II) interacts with SIRT3. Mitochondrial translation is regulated by ATP synthase binding with SIRT3 in proteomic analysis There are less information about the roles of SIRT4 and SIRT5 in the electron transport. A substrate for ATP synthase is created when SIRT4 binds with adenine nucleotide translocator (ANT) which than transports ATP into the cytosol and ADP to the mitochondrial matrix. SIRT5 interacts with cytochrome c. The biological significance of SIRT4 and SIRT5 are unknown
Sirtuin and the Kerb Cycle Mitochondrial matrix is the location of kerb cycle enzymes. The compartmentalization of the mitochondrial matrix provides the cell to utilize metabolites from carbohydrates, fats, and proteins. Several kerb cycle enzymes interact with SIRT3 including succinate dehydrogenase (SDH) and isocitrate dehydrogenase 2 (ICDH2). With deacetylation and activation of AceCS2 and glutamate dehydrogenase (GDH), SIRT3 influence the kerb cycle indirectly. The carbon entry into the kerb cycle are increased by increasing acetyle-CoA and amino acid utilization. SIRT3 activity may provide the general mechanism of these increases. SIRT4 inhibitates GDH via ADP-ribosylation, and SIRT4 via GDH interacts with the kerb cycle.
A group of proteins called sirtuins can help postpone the death time of certain model organisms (non-human organisms that are studied to better understand biological life). To be more specific, sirtuins are the (Sir)2 (silent information regulator) and its orthologs, which are homologs with the same function from different species.
Seven sirtuins, SIRT1-7, are found in mammals and they change a variety of pathways dealing with metabolism and responding to stress. The sirtuin domain has the devices used to bind a co-substrate involved in metabolism, NAD+. In a controlled environment, all sirtuins perform two important enzymatic processes: NAD+-dependent protein deactylase and ADP-ribosyltransferase. However, SIRT4 cannot recognize specific substrates for acetylation, but it can identify ADP-ribosyltransferase. Because the enzymes rely on NAD+, they can perform their functions with the organism’s excited state and are possibly involved with recognizing metabolism. Furthermore, extensive scientific research on mammals’ sirtuins, specifically SIRT1, has shown that they control metabolic processes and the lifespan of cells. To do these, sirtuins specifically focus on different acetylated protein substrates and are put in separate locations. For example, SIRT1, 6, 7 are located in the nucleus.
Three sirtuins, SIRT3-5, are found in the mitochondria and they help by being an important location for metabolism involving oxidation. Compared to SIRT1, SIRT3-5 are smaller in size. Though intense research has been performed on sirtuins, sirtuins of mitochondria have not been studied to the extent of others such as SIRT1. However, reports and information regarding mass spectrometry have been speculating that SIRT3-5 may play an important role in controlling a wide range of activity in the mitochondria, such as making energy, intracellular signaling, and partaking in apoptosis.

## NAD+ Metabolism and Sirtuin Activity

Sirtuins are absolutely dependent on NAD+, meaning the excess of free NAD+ and its biosynthetic and broken down products in the cells are important to how the activity of the enzyme of the sirtuins work. There are essentially two primary ways to the NAD+ biosynthesis in yeast and mammals. One is the a de novo kynurenine pathway, which is formed from tryptophan. The other one is a known as a salvage pathway that is usues nicotinamide that is created from NAD+ by sirtuins in addition to ADP-ribosyl-transferases and polymerases or exogenous nicotinic acid. Two researches Bieganowski and Brenner recently found a special pathway to NAD+ in yeast and humans. It is initiated from nicotinamide riboside, which is provided from the outside. In addition, aonoter significant discovery is that mammalian cells operate differently at a basic level in terms of their pathways compared to bacteria and yeast. In yeast, nicotinamide is deaminated by the enzyme Pnc1p, which transforms to nicotinic acid. Then the nicotinic acid is changed to NaMN by the nicotinic phosphoribosyltransferase. Nicotinamide in mammalian cells, on the other hand, are changed directly to nicotinamide mononucleotide by the Nampt. The level of expression of Nampt in response to a variety of stresses makes the levels of cellular NAD+ higher. In effect this regulated catalytic activity of Sir2. Recent studies have found that changes in the NAD+ metabolites potentially possess tissue-specific effects. Take NAD+ for example. NAD+ makes the level of nuclear neurons higher which prevents axonal degeneration in a SitT1-dependent way. In addition, mammalian de novo biosynthesis is also organized in a different manner compared to plants and prokaryotes.

## Sirtuins, caloric restriction, and aging

The relationship between Sir2 proteins, caloric restriction, and aging have been studied in detail. Evidence has shown that sirtuns are associated in encouraging longer life, especially longevity dealing with CR regimens, in a few organisms. There are two key early discoveries that support this in that the discovery that excess amounts of sirtuins encourage longevity in C. elegans. The relationship of yeast mother cell longevity with Sir2 interactions and SIr poteins has been shown that there is a correlation. When the yeast cells divide, they divide in an asymmetric way. The mother cells have only the ability to divide a certain amount of times, which isa bout 20-30 times. Mutants that don't have Sir2 have a decreased life span in the respect that they divide less time. Sir2 mutant mother cells that age prematurely were found to build up extrachromasal rDNA circles, which build up because rDNA combinated is not regulated anymore in the Sir2 mutant.

## Possible Link to Cancer

Due to the large role that sirtuins, particularly SIRT6, play in aiding in genome stability and the regulation of the metabolism, problems with SIRT6 function and availability are thought to be linked to oncogenic transformation and tumorigenesis. For example, certain cancers such as myeloid leukemia have breakages at the SIRT6 chromosomal locus. Furthermore, cancer cells exhibit a change from aerobic respiration to glycolysis that is seen in the Warberg effect which causes cancer cells to switch from oxidative phosphorylation to aerobic glycolysis. Other studies have shown that acetylation of H$_3$K$_{56}$ is increased in many cancers such as skin, thyroid, breast, liver, and colon cancers.
SIRT3 and Oral Cancer It has been studied and reported that SIRT3 has a connection to the beginnings of oral squamous cell carcinoma (OSCC) cancer formation, that is, it inhibits cell growth and induces early cell death. When this sirtuin is produced in excess in breast cancer, it modifies how the protein p53 to prevent cell arrest and deterioration in bladder cancer cells as they age. When compared to human oral keratinocytes, SIRT3 levels were higher than what it should have been and this was evidence for overexpression of SIRT3 in OSCC carcinogenesis. From there, the sirtinol and nicotinamide inhibitors were tested, which resulted in a blockage of cell growth and induced cell death in OSCC cells, which furthers evidence for SIRT3 overexpression in these cells. But people have also studied and reported that SIRT3 does the complete opposite, that is, it helps keep cells alive. It decreases stress on the cells and keeps them away from inducing of cell death and apoptosis. For example, Nampt, which regulates the response to stress and diet, requires SIRT3 to keep cells alive when they are exposed to harmful substances that alter the gene. It also works to keep the heart from failing and protects from other cardiac problems.

## References

Verdin, Eric, et al. Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling. Cell Press. 669-675.
Tennen,Ruth I., and Chua, Katrin F. Chromatin regulation and genome maintenance by mammalian SIRT6 Kwon, Hye-Sook and Ott, Melanie. The Ups and Downs of SIRT1
Anthony A. Sauve,1 Cynthia Wolberger,2 Vern L. Schramm,3 and Jef D. Boeke4(2006). Biochemistry of Sirtuins'. "PubMed", p. 7-9.
Alhazzazi, Turki Y., et al. Sirtuin-3 (SIRT3), a Novel Potential Therapeutic Target for Oral Cancer. (2011). "PubMed".