Biological and catalytic functions of sirtuin 6 as targets for small-molecule modulators

SIRT6 is localized to the nucleus, where it mediates DNA repair, regulates the expression of metabolic genes, and maintains genomic stability (Fig. 2). Through these functions, SIRT6 has been described as a tumor suppressor and positive regulator of longevity. Evidence indicates that SIRT6 regulates these processes primarily via deacetylation of histone and nonhistone proteins, although long-chain deacylation, ADP-ribosylation, and recruitment of proteins to chromatin are other biochemical functions ascribed to some SIRT6-dependent phenotypes. This review will focus on processes in which direct SIRT6 substrates or protein-binding partners have been identified.

Some of the earliest phenotypic studies on SIRT6 knockout cells demonstrated increased susceptibility to DNA-damaging agents and higher levels of chromosomal aberrations, suggesting a critical role for SIRT6 in maintaining genomic stability ( ). Later reports demonstrated that SIRT6 is recruited to double-strand breaks (DSBs) within seconds and further recruits DNA repair factors to initiate DNA damage response (DDR) ( , ). This fast response is attributed to the ability of SIRT6 to directly bind to open-ended DSBs, where Onn et al. ( ) postulate that each monomer of a SIRT6 dimer binds one loose ssDNA end of a DSB. Onn et al. suggested a potential DNA-binding tunnel along the core of SIRT6 that mediates ssDNA binding independent of the N and C termini. Another study demonstrated that c-Jun N-terminal kinase phosphorylates SIRT6 on Ser¹⁰ in response to oxidative stress, leading to binding of SIRT6 to DSBs and subsequent recruitment and mono-ADP-ribosylation of poly(ADP-ribose) polymerase 1 (PARP1) ( ). The mechanism by which the DNA repair factor, PARP1, is recruited to DSBs following SIRT6 phosphorylation and whether this is mediated by direct SIRT6 interactions are unknown. Serine 10 is located on the N terminus of SIRT6, which was previously shown to be dispensable for DNA binding, suggesting multiple pathways of passive and active recruitment to DSBs ( ).

Following recruitment of SIRT6 to sites of DNA damage, multiple studies have indicated contexts in which SIRT6 either deacetylates nucleosomes or actively recruits repair factors to mediate DDR. SIRT6 knockout mouse embryonic fibroblasts and knockdown human fibroblasts show impaired nonhomologous end joining (NHEJ) and homologous recombination efficiencies, respectively, whereas SIRT6 overexpression improves both processes ( ). Mao et al. ( ) demonstrated that this effect was facilitated by SIRT6-mediated recruitment of repair factors 53BP1 and NBS1 to sites of DNA damage, and loss-of-function mutants suggested that both deacetylase and ADP-ribosyltransferase activities of SIRT6 were required to facilitate NHEJ. The requirement of SIRT6 ADP-ribosyltransferase activity was attributed to the modification of PARP1 on Lys⁵²¹, resulting in its activation; however, the target of the requisite deacetylase activity in this study was not identified. Separately, SIRT6 has been shown to globally deacetylate H3K9 in response to DNA damage and co-localize with DNA-dependent protein kinase, Ku70, and Ku80 at DSBs ( ), although it remains unknown which factor pioneers DSB binding. Hou et al. ( ) showed that SIRT6 can recruit chromodomain helicase DNA-binding protein 4 (CDH4) to sites of DNA damage to facilitate chromatin relaxation and efficient homologous recombination. This effect was lost with the SIRT6 catalytic mutant H133Y, implicating a role for SIRT6 catalysis in this DDR pathway. Toiber et al. ( ) reported that SIRT6 deacetylates H3K56 in response to DNA damage and recruits the SNF2H chromatin remodeler, making chromatin more accessible and allowing for efficient recruitment of DNA repair factors RPA, 53BP1, and BRCA1 ( ). In this study, sites of DNA damage were identified by the presence of γH2AX, a phosphorylated form of the histone subtype H2AX present at sites of DSBs ( , ). A complex containing SIRT6 and SNF2H was later reported to stabilize γH2AX at DSB sites ( ). In contrast to the well-studied roles of SIRT6 in DSB repair, limited data also suggest that SIRT6 mediates base excision repair (BER). Overexpression of SIRT6 exhibited improved BER efficiency in mouse fibroblasts, whereas deletion increased sensitivity to BER-associated DNA-damaging agents ( , ). Separate studies demonstrated that SIRT6 interacts with and stimulates BER factors, including APE1, MYH, and Rad9-Rad1-Hus1, following oxidative damage ( ). A recent study compared SIRT6 proteins from rodents with varying lifespan and found a correlation between SIRT6-mediated DSB repair efficiency and the lifespan of the host organism ( ). The difference between repair efficiencies of orthologous SIRT6 enzymes was largely isolated to five amino acids that were predicted to be surface-exposed based on homology modeling with human SIRT6. Substitution of these five amino acids from a short-lived mouse SIRT6 sequence to those of long-lived beaver SIRT6 sequence was sufficient to increase NHEJ and decrease global acetylation in culture and improve the survivability of human fibroblasts exposed to γ-irradiation ( ).

Several studies implicated roles for SIRT6 in maintaining genome stability through the repression of telomeric and pericentric regions of chromatin. SIRT6 was shown to deacetylate telomeric chromatin in mammalian cells at H3K9 and H3K56 ( , ). Michishita et al. ( ) reported that SIRT6 deacetylation of H3K9 was required for stable association of WRN protein, and depletion of SIRT6 resulted in telomeric defects that resemble those observed in Werner syndrome, an aging disorder characterized by mutated WRN. Furthermore, SIRT6-deficient HeLa cells displayed telomeric sequence loss, genomic instability, and chromosomal end-to-end fusions ( ). Following oxidative damage, Gao et al. ( ) observed that SIRT6 was required for directional telomere movement, a process that facilitates efficient DNA damage repair at telomeric chromatin. Additionally, SIRT6 overexpression in mice was reported to up-regulate telomerase reverse transcriptase and telomere repeat binding factor, although this study did not explore the mechanism of up-regulation or potential downstream effects on telomere length ( ). Proper mitosis requires condensed and silenced peri-centric heterochromatin at centromeres to protect against genomic instability and cellular senescence during cell division. Tasselli et al. ( ) showed that SIRT6 repressed transcription at these regions through the deacetylation of H3K18 and also noted that knockdown of SIRT6 resulted in hyperacetylation and aberrant transcription of these pericentric chromatin domains. Collectively, SIRT6's ability to deacetylate proteins, ADP-ribosylate PARP1, or mediate protein-protein interactions with repair proteins has been implicated in controlling DNA stability and repair. Clearly, better genetic and biochemical tools are needed to provide a deeper molecular understanding of which biochemical functions of SIRT6 control various aspects of these processes and dictate histone substrate specificity. The development of better tools will require a robust understanding of catalytic mechanism, regulated activity, and structure-function relationships. Therefore, our current knowledge in these areas is presented later in the review. Despite these outstanding questions, the extensive research summarized here depicts a clear role for SIRT6 in facilitating DNA repair and stabilizing heterochromatic regions.

A number of studies have demonstrated the ability of SIRT6 to maintain cellular homeostasis through the regulation of glucose and lipid metabolism. Early studies characterizing SIRT6 knockout mice showed a lethal phenotype at 2–3 weeks of age, likely resulting from hypoglycemia ( , ). This hypoglycemic phenotype was later attributed to misregulation of glycolytic genes in the absence of SIRT6. Specifically, SIRT6 was found to co-repress the transcription factor hypoxia-inducible factor 1α (HIF-1α), in effect down-regulating glycolytic genes, including glucose transporter-1 (GLUT1), lactate dehydrogenase (LDH), phosphofructokinase-1 (PFK1), and pyruvate dehydrogenase kinase-1 (PDK1) ( ). In the same study, SIRT6 was reported to directly inhibit expression of these genes by deacetylating H3K9 at promoters of targeted genes. SIRT6 knockout resulted in the derepression of these glycolytic genes and increased glucose uptake, reminiscent of the “Warburg effect,” a metabolic hallmark often observed in cancer cells ( ). Accordingly, SIRT6 loss-of-function mutation and down-regulation is observed in several naturally arising human cancers. Mouse model studies by Sebastián et al. ( ) and Kugel et al. ( ) demonstrated that loss-of-function mutation in SIRT6 was sufficient to form tumors and was independent of known oncogene activation. A detailed summary of the roles of SIRT6 in cancer are discussed in a following section. Separately, Gertman et al. ( ) generated two SIRT6 mutants with enhanced in vitro rates of k_cat against long-chain myristoyl substrate. Stable transfection of these mutants in mouse embryonic fibroblasts demonstrated an increased ability to inhibit the expression of GLUT1, PDK1, and PFK1 relative to WT SIRT6. Interestingly, a global increase in acetylation of H3K9 and H3K56 ( ) was noted, suggesting that the demyristoylase activity of SIRT6 may regulate glycolysis and may be distinct from H3 deacetylase activity. However, these mutants also bound HIF-1α more tightly in vitro, making it difficult to definitively conclude whether more efficient recruitment to HIF-1α genes or higher demyristoylase activity was responsible for the down-regulation of these glycolytic genes ( ).

SIRT6 also regulates glucose metabolism by inhibiting gluconeogenesis, a metabolic process that generates glucose from noncarbohydrate sources. Mostoslavsky et al. ( ) found that SIRT6 knockout mice are hypoglycemic and display elevated expression of gluconeogenic genes. Although this observation alone could simply be dismissed as a proper liver response to hypoglycemia, work by Dominy et al. demonstrated active regulation of gluconeogenesis in the liver by SIRT6. Mechanistically, SIRT6 was described to deacetylate general control nonrepressed protein 5 (GCN5), which enhanced its acetyl-transferase activity toward peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) ( ). PGC-1α is the primary regulator of hepatic gluconeogenesis and induces the expression of gluconeogenic genes ( ). Following acetylation by GCN5, PGC-1α was found to relocate away from the promoters of gluconeogenic genes, resulting in decreased expression ( ). Another study demonstrated that SIRT6 overexpression reduced gluconeogenic gene expression in WT, but not Forkhead box O1 (FoxO1) knockout mice ( ). FoxO1 is a transcription factor required for activation of gluconeogenic genes in hepatic cells ( ). Subsequent work by Zhang et al. ( ) mechanistically described the FoxO1/SIRT6 regulatory axis by demonstrating that SIRT6 directly deacetylates FoxO1, leading to its nuclear export, thus resulting in decreased gluconeogenic gene expression.

SIRT6 has also been implicated in the regulation of lipid metabolism with studies reporting roles in both lipolysis and lipogenesis. Early work in mice demonstrated that liver-specific knockout of SIRT6 resulted in fatty liver formation owing to increased triglyceride synthesis and reduced β-oxidation, whereas mice overexpressing SIRT6 were protected from the accumulation of visceral fat, LDL-cholesterol, and triglycerides when fed high-fat diets ( , ). Fat-specific SIRT6 knockout increased obesity-related phenotypes in mice ( , ). Mechanistically, SIRT6 was found to represses the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) and sterol-regulatory element–binding protein 1 and 2 (SREBP1 and SREBP2). This was demonstrated to occur through SIRT6 recruitment to the promoter of Pcsk9, a critical regulator of LDL-cholesterol, via interactions with the transcription factor FoxO3 where SIRT6 deacetylates H3K9 and H3K56 to reduce expression of Pcsk9, resulting in lowered LDL-cholesterol levels ( ). FoxO3 was also shown to recruit SIRT6 to promoters of SREBP genes, positive regulators of lipogenic genes, resulting in inhibition of expression by promoter-specific SIRT6 deacetylation of H3K9 and H3K56 ( , , ). Interestingly, miRNAs expressed from the introns of SREBP, miR-33a and miR-33b, as well as the most common hepatic miRNA, miR-122, were shown by Elhanati et al. ( , ) to down-regulate SIRT6 expression, indicating intricate regulation of SIRT6 toward lipid metabolism. Separately, SIRT6 was noted to promote β-oxidation of fatty acids during fasting through regulation of the PPARα coactivator, NCOA2. In this distinct mechanism, SIRT6 was found to decrease NCOA2 acetylation at Lys⁷⁸⁰, resulting in the activation of PPARα and increased expression of hepatic β-oxidation genes ( ). It remains to be determined whether NCOA2 Lys⁷⁸⁰ is a direct substrate of SIRT6 deacetylation.

The yeast homolog and founding member of the sirtuin family, Sir2, is implicated in lifespan extension through repression of tandem rRNA gene arrays (rDNA) ( , ). Subsequent studies have solidified links between aging and the mammalian homolog, SIRT6. SIRT6 overexpression in mice was found to extend the lifespan of male mice by 15% in a mechanism thought to be driven through reduced insulin-like growth factor 1 (IGF1) signaling. Zhang et al. ( ) demonstrated that caloric restriction prevented aging-related renal insufficiency and suggested that this effect was mediated via the up-regulation of SIRT6 in response to caloric restriction, which resulted in the repression of nuclear factor-κB (NF-κB). NF-κB is a nuclear transcription factor associated with inflammatory responses, aging, and apoptosis ( ). SIRT6 was found to represses NF-κB activity both by deacetylating H3K9 at NF-κB promoters and by deacetylating Lys³¹⁰ of the NF-κB p65 subunit (RelA), resulting in its nuclear export ( ). Xu et al. ( ) observed a decrease in SIRT6 levels with age in human fibroblasts. In separate studies, aging-related decreases in SIRT6 levels impaired the reprogramming potential of iPSCs from old mice and decrease DNA repair efficiency ( , ). Another study demonstrated that increased copy number of Sirt6 was sufficient to restore lifespan in Caenorhabditis elegans deficient in Atm, a DNA repair factor ( ). Studies on human populations have discovered various SNPs that correlate both positively and negatively with longevity ( , , ). The mechanistic outcomes of these polymorphisms are yet unknown; however, they present a potentially powerful tool for further probing the link between SIRT6 and aging. Work by Tian et al. ( ), discussed previously, compared long- and short-lived rodents and demonstrated a SIRT6-mediated increase in DNA-repair efficiency among long-lived species corresponding to increased in vitro deacetylase and self-mono-ADP-ribosyltransferase activities. Transgenic Drosophila expressing beaver (long-lived) SIRT6 showed increased lifespan relative to those expressing mouse (short-lived) SIRT6, directly linking SIRT6 activity to increased lifespan.

With dysregulated DNA damage repair and metabolic homeostasis as hallmarks of cancer, SIRT6 has been implicated in oncogenesis, proliferation, and poor prognosis. Down-regulation of SIRT6 in human cancers has been reported in hepatocellular carcinomas, ovarian cancers, and colon cancers ( , , , ). Furthermore, the Cancer Cell Line Encyclopedia reports that the SIRT6 gene is deleted in 35% of all cancer cell lines in their database ( , ). SIRT6 knockout in mouse embryonic fibroblasts led to tumor formation in mouse xenograft studies independent of known oncogene activation ( ). This tumorigenesis was attributed to derepression of glycolytic genes resulting in a Warburg-like phenotype characterized by increased glucose uptake and anaerobic glycolysis. Similarly, SIRT6 point mutations identified in human cancers were shown to be loss-of-function, and xenograft studies by Kugel et al. ( ) demonstrated that these loss-of-function mutations were sufficient to drive tumor formation in mice. Copy loss number of SIRT6 has been observed in 60% of pancreatic cancer cell lines, and studies on low SIRT6-expressing cell lines demonstrated accelerated tumorigenesis in a mechanism independent of enhanced glycolysis ( ). Rather, SIRT6 was shown to deacetylate H3K9 and H3K56 at the promoter of Lin28b, an RNA-binding fetal oncogene that promotes transformation and tumor progression ( , ). It was therefore concluded by Kugel et al. that loss of SIRT6 leads to c-Myc transcription factor–driven overexpression of Lin28b and subsequent tumor growth. Additionally, SIRT6 was shown to deacetylate H3K9 at the promoter of survivin, an oncoprotein up-regulated in most cancers that impairs tumorigenesis ( , ). Positive outcomes for breast cancer patients correlated with SIRT6 levels and inversely correlated with SIRT6 phosphorylation at Ser³³⁸ ( ). This was mechanistically supported through the demonstration that Ser³³⁸ phosphorylation of SIRT6 by the kinase AKT1 induced SIRT6 ubiquitination by MDM2, targeting SIRT6 for protease degradation. In colon cancer cells, the tumor suppressor USP10, a ubiquitin-specific peptidase, was found to be critical for deubiquitinating SIRT6 and stabilizing protein levels to negatively regulate the transcriptional activity of the c-Myc oncogene ( ). In separate studies, SIRT6 SUMOylation was shown to promote interaction with c-Myc, resulting in the deacetylation of H3K56, but not H3K9, and transcriptional repression at the promoters of c-Myc genes ( ). The site of SIRT6 SUMOylation was localized to the C terminus and isolated to either Lys²⁹⁶, Lys³⁰⁰, Lys³¹⁶, or Lys³³². Furthermore, Bhardwaj et al. ( ) demonstrated that SIRT6 interacts with and deacetylates nuclear pyruvate kinase M2 (PKM2), an oncogenic transcriptional coactivator, on Lys⁴³³, which resulted in its nuclear export and a downstream reduction in cell proliferation, migration potential, and invasiveness.

Despite the vast majority of data supporting the roles of SIRT6 as a tumor suppressor, some evidence suggests that SIRT6 may promote tumor survival in a context-dependent manner. It has been observed that SIRT6 is up-regulated in cases of squamous cell carcinoma, chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia ( , , , ). Three of the aforementioned diseases are hematopoietic cancer lines, which curiously are the only tumor type exhibiting gain of SIRT6 gene copies according to the Cancer Cell Line Encyclopedia ( , ). In the case of multiple myeloma, a disease characterized by a highly unstable genome, Cea et al. ( ) noted increased SIRT6 expression as an adaptive response to combat genomic instability. In acute myeloid leukemia, SIRT6 overexpression was demonstrated to protect tumors from DNA damage through the deacetylation and activation of DNA-dependent protein kinase, a DDR element that facilitates DSB repair ( , ). In the same study, SIRT6 depletion in multiple myeloma cells was found to potentiate genotoxic treatment and increase proliferation rate. Mechanistically, SIRT6 was shown to interact with the transcription factor ELK1 to deacetylate H3K9 and suppress the expression of ERK signaling–related genes and reduce proliferation ( ). Bauer et al. ( ) reported that SIRT6 promotes metastasis through the up-regulation of pro-inflammatory cytokines, including IL-8 and TNF-α. This effect was noted to occur through SIRT6 catalytic activity, resulting in increased intracellular ADP-ribose levels, an activator of the Ca²⁺ channel TRPM2 required for LPS-induced production of IL-8 and TNF-α ( , ). In hepatocellular carcinoma (HCC), Lee et al. ( ) identified SIRT6 as a tumor promoter that prevented DNA damage and cellular senescence. Separate studies of HCC by Ran et al. ( ) identified a separate oncogenic role for SIRT6. Namely, SIRT6 was found to deacetylate H3K9 at the promoter of Bcl-2–associated X protein (Bax), an apoptotic activator, resulting in HCC protection from apoptosis. Contrasting research by Zhang et al. ( ) suggested a tumor-suppressing role of SIRT6 toward HCC cells by blocking the ERK1/2 signaling pathway, although the mechanism of SIRT6/ERK cross-talk is unknown.

Clearly, SIRT6 is an important regulator of metabolism and genome maintenance, and the ability to suppress tumor formation is well-documented. Nevertheless, cell- and context-dependent examples of SIRT6-driven tumor survival have also been reported. These contrasting studies suggest a complex role for SIRT6 in tumor development and survival that is still being evaluated. One interpretation of current data are that within the context of a healthy cell, DNA repair and genomic stability prevents oncogenesis; however, following specific types of transformation, these same functions can protect the aberrant cells from genotoxic therapies or the accumulation of excess deleterious mutations. The context-dependent role of SIRT6 as either a tumor suppressor or tumor driver opens the door to informed disease-specific pharmacological activation or inhibition. Likewise, the development of small-molecule SIRT6 modulators will aid the study of SIRT6 activity in varying disease contexts. To provide context for a subsequent review of small-molecule modulators, we will first discuss the overall structure of SIRT6 and unique features that differentiate this sirtuin from other family members.

Sirtuins 1–7 are an evolutionarily conserved family of enzymes that share a core catalytic domain spanning ∼250 amino acids (Fig. 3). The catalytic core comprises an NAD⁺-binding Rossmann fold domain and a structural zinc-binding domain containing four conserved cysteine residues, which coordinate a zinc ion ( ). Catalysis occurs in a hydrophobic cleft situated between these two domains that varies in size and composition between sirtuin family members to confer specificity toward acyl substrates ( , , ). Additionally, sirtuins contain diverse N and C termini that can direct cellular localization and protein-protein interactions.

SIRT6 contains a Rossmann fold structural motif responsible for NAD⁺ binding that is common among nucleotide-binding proteins (Fig. 3) ( ). This domain spans residues 27–132 and 195–268 and is structurally represented by a six-stranded parallel β-sheet in between two helices on one side and four helices on the other ( ). The Rossmann fold domain forms a platform for the NAD⁺-binding site, whereas the flanking cofactor binding loop forms additional contacts with NAD⁺ to stabilize the bound form ( ). Structural analysis of bound adenosine diphosphate ribose (ADPr) revealed 18 possible polar contacts between primarily the Rossmann fold domain and ADPr (Fig. 4C). ADPr is an NAD⁺ metabolite lacking the nicotinamide moiety that inhibits SIRT6 catalysis, suggesting that it similarly occupies the NAD⁺-binding pocket ( , ). Substrate binding by SIRT1-3 has been reported to follow an ordered binding mechanism in which acetyl peptide precedes that of NAD⁺ ( , ). SIRT6 is the only known sirtuin to bind NAD⁺ in the absence of acyl substrate ( , ). The ability to bind NAD⁺ in the absence of acyl peptide substrate is structurally explained by the NAD⁺ binding loop of SIRT6 maintaining an ordered helix in the absence of peptide substrate ( ), whereas this loop is disordered in the absence of substrate for SIRT2,3,5 ( , , ). Interestingly, a naturally occurring point mutation (D63Y) in human cancer exists in the NAD⁺-binding loop and has been shown to abolish deacetylase activity ( ). Additionally, a study by Mao et al. ( ) reported that S56Y and R65A variants lacked cellular deacetylase activity. Subsequent studies further demonstrated that Arg⁶⁵ is critical for in vitro activation of deacetylation ( ). These studies suggest that the NAD⁺-binding domain of SIRT6 is a hotspot for loss-of-function mutation.

The Zn²⁺-binding domain of the sirtuins (class III HDACs) is thought to be strictly structural and does not participate in catalysis, in contrast to the active-site zinc ion in the zinc-dependent class I, II, and IV HDACs ( ). The sirtuin zinc domain utilizes four conserved cysteine residues to coordinate Zn²⁺ (SIRT6: Cys¹⁴¹, Cys¹⁴⁴, Cys¹⁶⁷, and Cys¹⁷⁸). Most sirtuins contain the conserved sequence motif Cys-X-X-Cys-X_15-20-Cys-X-X-Cys; however, SIRT6 incorporates a 10-amino acid insert between the third and fourth cysteine, resulting in a unique extended flexible loop (Fig. 4E) ( ). Interestingly, C terminus of Hsp70-interacting protein (CHIP) has been shown to noncanonically ubiquitinate SIRT6 on Lys¹⁷⁰, a residue that resides in the extended flexible loop on the Zn²⁺-binding domain ( ). This ubiquitination prevented proteasome-mediated degradation of SIRT6 and led to increased protein t_1/2. Studies involving SIRT1 and the yeast homolog, Sir2, demonstrated that cysteine S-nitrosation or mutation of these zinc-coordinating cysteines to alanine results in loss of deacetylase function ( , , ). Molecular dynamics simulations suggested that loss of activity was due to repositioning of the Zn²⁺-binding domain away from the catalytic domain following zinc release, resulting in allosteric changes that disrupt NAD⁺ and acyl substrate binding ( ). Trichostatin A (TSA) is well-documented as a Class I and II HDAC inhibitor that functions by chelating the active-site zinc of these enzymes ( ). Curiously, it was demonstrated that TSA specifically inhibits SIRT6 (Ki = 2 μm) over SIRT1–3 and SIRT5 (Ki > 50 μm) against H3K9ac peptide substrate ( ). Subsequent kinetic analyses suggested that TSA competitively inhibited SIRT6 with respect to H3K9ac substrate ( ). A structure of SIRT6 bound to TSA was solved in a separate study revealing binding to a SIRT6 specific region at the distal end of the hydrophobic cleft, explaining its isoform selectivity ( ). TSA is often used in an effort to specifically inhibit class I and II HDACs in cellular experiments at concentrations ranging from 0.1 to 2 μm. Given this expanded role of TSA as a zinc chelation–independent inhibitor of SIRT6 with a Ki of 2 μm, methodological caution should be taken to rule out “nonspecific” inhibition of SIRT6.

A conserved salt bridge between the Rossmann fold and zinc-binding domains of SIRT5 and many Sir2 homologs is thought to contribute to the stabilized positioning of these domains ( , ). SIRT6 lacks this salt bridge (predicted by homology modeling to occur between Arg¹⁸² and Asp¹⁹⁰), resulting in a structure that is splayed open relative to other sirtuins ( ). This expanded cleft between domains contains an elongated hydrophobic pocket that accommodates long-chain acyl substrates. A structure of SIRT6 bound to H3K9myr revealed nonpolar contacts of the myristoyl chain with Ala¹³, Pro⁶², Phe⁶⁴, Trp⁷¹, Pro⁸⁰, Phe⁸², Phe⁸⁶, Val¹¹⁵, Leu¹³², Met¹⁵⁷, Leu¹⁸⁶, and Ile²¹⁹ (Fig. 4D) ( ). Furthermore, structures containing bound myristoyl peptide are the only ones to date in which the SIRT6 N terminus became structured, revealing that it covers part of this hydrophobic pocket at the interface of the Rossmann fold and zinc-binding domains ( , ). Together, this suggests that acyl substrate occupation of the hydrophobic pocket can lead to an ordered structure and provides one possible explanation for why long-chain deacylation is more efficient than short-chain deacetylation. This hydrophobic pocket has also been implicated as an activator-binding site through both kinetic and structural studies. Kinetically, the activators CL5D and myristic acid displayed competitive inhibition toward myristoyl substrate ( , ). Crystal structures of SIRT6 in the presence of the activators UBCS039, MDL-801, and cyanidin demonstrated their occupation of the distal hydrophobic pocket away from the active site ( , ). Structural alignment of these deacetylase activators with myristoylated peptide demonstrates varying levels of overlap, resulting in differing effects toward competitive inhibition of long-chain deacylation. These kinetics will be discussed further in subsequent sections of this review. Structural analysis of H3K9 peptide binding reveals primarily hydrogen bonds between the main chain of the peptide and the surface of SIRT6 with just Thr¹¹ and Gln⁶ of the peptide substrate participating in side-chain interactions (Fig. 4B). Microarray analysis using ∼6,800 acetyllysine substrates demonstrated a SIRT6 preference for uncharged residues around acetyllysine except for negatively charged residues at –4 and +2 ( ); however, none of the three identified in vivo histone substrates (H3K9/18/56ac) satisfy these reported in vitro preferences. This suggests that selectivity for histone substrate may be dependent on other factors such as protein-protein binding and optimized orientation between SIRT6 and target sites on nucleosomes. The nature of the acyl group modification on lysine might also facilitate SIRT6-substrate interactions. Consistent with this notion, SIRT6 performs more efficient deacylation in vitro against substrates containing acyl groups ranging from 8 to 14 carbons in length ( ); however, identification and validation of long-chain acyl substrates in vivo remains limited ( , ). Directed evolution of SIRT6 conducted using SIRT6 ancestral libraries identified multiple point mutations that resulted in enhanced demyristoylation of peptide substrate ( ). The mutation M157H, located in the hydrophobic pocket, was identified in two separately evolved SIRT6 variants and resulted in improved k_cat of demyristoylation. In silico modeling led the authors to propose improved catalysis through better positioning of NAD⁺ and peptide substrate; however, the precise contributions of this mutation are unknown. Despite the structural and biochemical evidence that SIRT6 preferentially hydrolyzes long-chain acyl groups, compelling cellular evidence suggests that SIRT6 exerts biological regulation primarily through the direct deacetylation of specific sites on histone and nonhistone proteins ( , , ).

In addition to the conserved catalytic core consisting of a Rossmann fold and zinc-binding domain, individual sirtuins harbor unique N- and C-terminal extensions (Fig. 3). Structurally, the 24-amino acid N terminus of SIRT6 is surface-exposed and lacks secondary structural elements. The second half of the N terminus (Asp¹⁴–Phe²⁴) is ordered in crystal structures and is adjacent to the Rossmann fold domain. The first half of the N terminus (Met¹–Ala¹³) has only been observed to be ordered upon H3K9myr peptide binding, which makes contacts with both the Rossmann fold domain and zinc-binding domain to seal the hydrophobic pocket. Given that SIRT6 performs more efficient long-chain deacylation in vitro, it follows that the ordering of this domain may facilitate catalysis. The N terminus of SIRT6 has been shown to be critical for H3K9 and H3K56 deacetylase activity and for chromatin association in cells; however, this region is dispensable for nuclear localization ( ). No SIRT6 structures to date have been solved in the presence of acetylated substrates, so it remains to be seen whether canonical acetyl substrates can induce ordering of the N terminus. The N-terminal domain of SIRT6 is highly conserved among mammalian homologs and can be phosphorylated at Ser¹⁰ to facilitate DNA repair ( , ).

The C terminus of SIRT6 (Leu²⁶⁹–Ser³⁵⁵) is significantly larger than the N terminus and is reported to contain a nuclear localization signal ( ). This C terminus is predicted to be an intrinsically disordered region (IDR) and is composed of 24.7% prolines with a pI of 10.1 ( ). To date, only a short region (Leu²⁶⁹–Glu²⁹⁸) of this C terminus has been solved by crystallography, with the rest remaining unstructured (Fig. 3). Given the difficulty in crystallizing this inherently disordered domain, some researchers have opted to remove this region to facilitate crystal formation ( , ). Interestingly, it has recently been suggested that heterochromatin can form through phase separation into liquid droplets, and many of the proteins that form these condensates contain IDRs ( , ). Given the role of SIRT6 in maintaining heterochromatin and telomeres ( , , ), it is an emerging and exciting question whether the C-terminal IDR participates in heterochromatic condensates. In vivo phosphorylation of the C terminus at Thr²⁹⁴, Ser³⁰³, Ser³³⁰, and Ser³³⁸ has been reported ( , , ). The SIRT6 variant S338A disrupted interactions with nuclear pore complexes as determined by immunoprecipitation-MS, suggesting that phosphorylation of the C terminus may modulate protein-protein interactions ( ). However, there are no additional data suggesting the cellular function for this potentially regulated interaction.

Sirtuins remove acyl moieties from the ε-amino group of lysines and, in the process, consume one molecule of NAD⁺ for every one molecule of acyl chain removed ( ). Three products are generated: nicotinamide, deacylated peptide/protein, and acylated ADP-ribose. After both substrates are bound, the glycosidic bond between nicotinamide and ribose is cleaved, and the acyl oxygen attacks the C1 of ribose, resulting in an adduct between the two substrates (Fig. 5). Kinetic isotope effect experiments suggest that the glycoside bond is largely broken before the addition of the acyl oxygen ( ), although the net rate constant for this first chemical step is greatly influenced by the nucleophilicity of the attacking oxygen ( ). The lifetime of the resulting alkylimidate intermediate can be extended by replacing a catalytic histidine (His¹³³ in SIRT6), which would normally facilitate (via general base action) the attack of the ribose 2′-OH on the imidate intermediate (Fig. 5), forming a bicyclic adduct ( , ). During catalysis, the histidine to alanine mutant protein is partially stalled at the imidate intermediate, allowing water or other solvent nucleophiles (methanol/ethanol) to react, essentially turning the enzyme into an acetyl substrate–dependent NAD⁺ hydrolase, with no net deacetylation ( ). These results have implications for the weak ability of sirtuins to mediate protein ADP-ribosylation under some circumstances (discussed further below). During the normal catalytic cycle for deacylation, formation of the bicyclic intermediate (Fig. 5, species iii) is followed by attack of a water molecule to form intermediate iv, followed by cleavage of the C–N bond to liberate the final two products, O-acyl-ADP-ribose and deacetylated lysine. Evidence for these catalytic steps and intermediates has come from biochemical, structural, and theoretical experiments ( , , , , ).

Early reports on the possible reactions catalyzed by sirtuins included both deacetylation and ADP-ribosylation ( , , , , , ). To date, a preponderance of data supports the main catalytic activity as NAD⁺-dependent deacylation, with unique specificities between sirtuins for various acyl groups on the lysine ε-amine. Whereas most studies have ascribed deacylation as the biological function of sirtuins, a few others have suggested that the protein ADP-ribosylation activity of some sirtuins is responsible for certain cellular functions. Such reports suggest that SIRT4 can ADP-ribosylate GDH ( ), that SIRT2 can target BSA ( ), and that SIRT6 can ADP-ribosylate PARP1 ( ). The role of sirtuin-dependent ADP-ribosylation remains enigmatic. From a biochemical perspective, protein ADP-ribosylation can be explained through the intrinsic catalytic properties of sirtuins ( , , ). However, parsing deacylation from ADP-ribosylation in cellular assays remains challenging, largely because both activities occur through the same active site. In vitro, ADP-ribosylation activity pales compared with deacylation, and the ability of SIRT6 to perform multiple rounds of catalytic turnover for ADP-ribosylation has not been reported. Nevertheless, in cellular contexts, it is possible that there are molecular circumstances that greatly promote ADP-ribosylation over deacylation. With SIRT6, the unique kinetic mechanism might allow SIRT6 to facilitate such a proposed switch toward more favorable ADP-ribosylation.