NAD+ precursors promote the restoration of spermatogenesis in busulfan-treated mice through inhibiting Sirt2-regulated ferroptosis.

Rationale: In recent years, nicotinamide adenine dinucleotide (NAD+) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and animal models. However, whether and how Npre plays a role in the male reproductive disorder has not been fully clarified. Methods: In the present study, a busulfan-induced non-obstructive azoospermic mouse model was used, and Npre was administered for five weeks following the drug injection, with the objective of reinstating spermatogenesis and fertility. Initially, we assessed the NAD+ level, germ cell types, semen parameters and sperm fertilization capability. Subsequently, testis tissues were examined through RNA sequencing analysis, ELISA, H&E, immunofluorescence, quantitative real-time PCR, and Western blotting techniques. Results: The results indicated that Npre restored normal level of NAD+ in blood and significantly alleviated the deleterious effects of busulfan (BU) on spermatogenesis, thereby partially reestablishing fertilization capacity. Transcriptome analysis, along with recovery of testicular Fe2+, GSH, NADPH, and MDA levels, impaired by BU, and the fact that Fer-1, an inhibitor of ferroptosis, restored spermatogenesis and semen parameters close to CTRL values, supported such possibility. Interestingly, the reduction in SIRT2 protein level by the specific inhibitor AGK2 attenuated the beneficial effects of Npre on spermatogenesis and ferroptosis by affecting PGC-1α and ACLY protein levels, thus suggesting how these compounds might confer spermatogenesis protection. Conclusion: Collectively, these findings indicate that NAD+ protects spermatogenesis against ferroptosis, probably through SIRT2 dependent mechanisms. This underscores the considerable potential of Npre supplementation as a feasible strategy for preserving or restoring spermatogenesis in specific conditions of male infertility and as adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

Biophysical insights into the dimer formation of human Sirtuin 2.

Sirtuin 2 (SIRT2) is a class III histone deacetylase that is highly conserved from bacteria to mammals. We prepared and characterized the wild-type (WT) and mutant forms of the histone deacetylase (HDAC) domain of human SIRT2 (hSIRT2) using various biophysical methods and evaluated their deacetylation activity. We found that WT hSIRT2 HDAC (residues 52-357) forms a homodimer in a concentration-dependent manner with a dimer-monomer dissociation constant of 8.3 ± 0.5 µM, which was determined by mass spectrometry. The dimer was disrupted into two monomers by binding to the HDAC inhibitors SirReal1 and SirReal2. We also confirmed dimer formation of hSIRT2 HDAC in living cells using a NanoLuc complementation reporter system. Examination of the relationship between dimer formation and deacetylation activity using several mutants of hSIRT2 HDAC revealed that some non-dimerizing mutants exhibited deacetylation activity for the N-terminal peptide of histone H3, similar to the wild type. The hSIRT2 HDAC mutant Δ292-306, which lacks a SIRT2-specific disordered loop region, was identified to exist as a monomer with slightly reduced deacetylation activity; the X-ray structure of the mutant Δ292-306 was almost identical to that of the WT hSIRT2 HDAC bound to an inhibitor. These results indicate that hSIRT2 HDAC forms a dimer, but this is independent of deacetylation activity. Herein, we discuss insights into the dimer formation of hSIRT2 based on our biophysical experimental results.

Gene expression patterns of Sirtuin family members (SIRT1 TO SIRT7): Insights into pathogenesis and prognostic of Myelodysplastic neoplasm.

To assess and validate the gene expression profile of SIRTs (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) in relation to the pathogenesis and prognostic progression of Myelodysplastic neoplasm (MDS). Eighty bone marrow samples of patients with de novo MDS were diagnosed according to WHO 2022 and IPSS-R criteria. Ten bone marrow samples were obtained from elderly healthy volunteers and used as control samples. Gene expression levels of all SIRTs were assessed using RT-qPCR assays. Downregulation of SIRT2 (p = 0.009), SIRT3 (p = 0.048), SIRT4 (p = 0.049), SIRT5 (p = 0.046), SIRT6 (p = 0.043), and SIRT7 (p = 0.047) was identified in MDS patients compared to control individuals. Also, we identified that while SIRT2-7 genes are typically down-regulated in MDS patients compared to normal controls, there are relative expression variations among MDS patient subgroups. Specifically, SIRT4 (p = 0.029) showed increased expression in patients aged 60 or above, and both SIRT2 (p = 0.016) and SIRT3 (p = 0.036) were upregulated in patients with hemoglobin levels below 8 g/dL. SIRT2 (p = 0.045) and SIRT3 (p = 0.033) were highly expressed in patients with chromosomal abnormalities. Different SIRTs exhibited altered expression patterns concerning specific MDS clinical and prognostic characteristics. The downregulation in SIRTs genes (e.g., SIRT2 to SIRT7) expression in Brazilian MDS patients highlights their role in the disease's development. The upregulation of SIRT2 and SIRT3 in severe anemia patients suggests a potential link to manage iron overload-related complications in transfusion-dependent patients. Moreover, the association of SIRT2/SIRT3 with genomic instability and their role in MDS progression signify promising areas for future research and therapeutic targets. These findings underscore the importance of SIRT family in understanding and addressing MDS, offering novel clinical, prognostic, and therapeutic insights for patients with this condition.

Sirt2 inhibition improves gut epithelial barrier integrity and protects mice from colitis.

Sirt2 is a nicotinamide adenine dinucleotide (NAD+)-dependent protein lysine deacylase that can remove both acetyl group and long-chain fatty acyl groups from lysine residues of many proteins. It was reported to affect inflammatory bowel disease (IBD) symptoms in a mouse model. However, conflicting roles were reported, with genetic knockout aggravating while pharmacological inhibition alleviating IBD symptoms. These seemingly conflicting reports cause confusion and deter further efforts in developing Sirt2 inhibitors as a potential treatment strategy for IBD. We investigated these conflicting reports and elucidated the role of Sirt2 in the mouse model of IBD. We essentially replicated these conflicting results and confirmed that Sirt2 inhibitors' protective effect is not through off-targets as two very different Sirt2 inhibitors (TM and AGK2) showed similar protection in the IBD mouse model. We believe that the differential effects of inhibitors and knockout are due to the fact that the Sirt2 inhibitors only inhibit some but not all the activities of Sirt2. This hypothesis is confirmed by the observation that a PROTAC degrader of Sirt2 did not protect mice in the IBD model, similar to Sirt2 knockout. Our study provides an interesting example where genetic knockout and pharmacological inhibition do not align and emphasizes the importance of developing substrate-dependent inhibitors. Importantly, we showed that the effect of Sirt2 inhibition in IBD is through regulating the gut epithelium barrier by inhibiting Arf6-mediated endocytosis of E-cadherin, a protein important for the intestinal epithelial integrity. This mechanistic understanding further supports Sirt2 as a promising therapeutic target for treating IBD.

Insights into the modulation of bacterial NADase activity by phage proteins.

The Silent Information Regulator 2 (SIR2) protein is widely implicated in antiviral response by depleting the cellular metabolite NAD+. The defense-associated sirtuin 2 (DSR2) effector, a SIR2 domain-containing protein, protects bacteria from phage infection by depleting NAD+, while an anti-DSR2 protein (DSR anti-defense 1, DSAD1) is employed by some phages to evade this host defense. The NADase activity of DSR2 is unleashed by recognizing the phage tail tube protein (TTP). However, the activation and inhibition mechanisms of DSR2 are unclear. Here, we determine the cryo-EM structures of DSR2 in multiple states. DSR2 is arranged as a dimer of dimers, which is facilitated by the tetramerization of SIR2 domains. Moreover, the DSR2 assembly is essential for activating the NADase function. The activator TTP binding would trigger the opening of the catalytic pocket and the decoupling of the N-terminal SIR2 domain from the C-terminal domain (CTD) of DSR2. Importantly, we further show that the activation mechanism is conserved among other SIR2-dependent anti-phage systems. Interestingly, the inhibitor DSAD1 mimics TTP to trap DSR2, thus occupying the TTP-binding pocket and inhibiting the NADase function. Together, our results provide molecular insights into the regulatory mechanism of SIR2-dependent NAD+ depletion in antiviral immunity.

SIRT2-mediated deacetylation of ACLY promotes the progression of oesophageal squamous cell carcinoma.

ATP citrate lyase (ACLY), as a key enzyme in lipid metabolism, plays an important role in energy metabolism and lipid biosynthesis of a variety of tumours. Many studies have shown that ACLY is highly expressed in various tumours, and its pharmacological or gene inhibition significantly inhibits tumour growth and progression. However, the roles of ACLY in oesophageal squamous cell carcinoma (ESCC) remain unclear. Here, our data showed that ACLY inhibitor significantly attenuated cell proliferation, migration, invasion and lipid synthesis in different ESCC cell lines, whereas the proliferation, migration, invasion and lipid synthesis of ESCC cells were enhanced after ACLY overexpression. Furthermore, ACLY inhibitor dramatically suppressed tumour growth and lipid metabolism in ESCC cells xenografted tumour model, whereas ACLY overexpression displayed the opposite effect. Mechanistically, ACLY protein harboured acetylated modification and interacted with SIRT2 protein in ESCC cells. The SIRT2 inhibitor AGK2 significantly increased the acetylation level of ACLY protein and inhibited the proliferation and migration of ESCC cells, while overexpression of ACLY partially reversed the inhibitory effect of AGK2 on ESCC cells. Overall, these results suggest that targeting the SIRT2/ACLY signalling axis may be a potential therapeutic strategy for ESCC patients.

SIRT2 regulates apoptosis by inducing mitophagy in sheep cumulus cells.

Cumulus cells surrounding oocytes furnish nutritional support crucial for oocyte maturation in vitro, and thereby enhance oocyte quality significantly. Our previous studies affirmed the role of SIRT2 in regulation of mitochondrial function in sheep granulosa cells. However, the effect of SIRT2 action on mitophagy in these cells remain unclear. Here, RNA-seq was used to scrutinize pathways where differentially expressed genes (DEGs) are enriched following SIRT2 knockdown in cumulus cells. Prior to SIRT2 knock down, cumulus cells were treated with the mitophagy inhibitor Mdivi-1. Potential mechanisms by which SIRT2 affects apoptosis via mitophagy were explored. Results indicated that DEGs after SIRT2 knockdown were enriched in various pathways including mitochondria, mitophagy, and apoptosis. The expression levels of CASP3/CASP9 were significantly increased after mitophagy activation (P < 0.01), whereas inhibition of mitophagy had no effect on apoptosis (P > 0.05). Pretreatment of cumulus cells with Mdivi-1 prior to SIRT2 knockdown significantly reduced the expression of mitophagy-related genes, the number of autolysosomes, the expression of CASP3/CASP9, and the levels of Ca2+ and cytochrome C (P < 0.05). In addition, an improvement in mitochondrial morphology and increases in ATP levels and mitochondrial DNA (mtDNA) copy numbers were observed. Interestingly, double knockdown of SIRT2 and MAPK15 was found to reverse increased mitophagy and apoptosis activity caused by SIRT2 knockdown. Our findings indicate that SIRT2 modulate apoptosis in cumulus cells by regulating mitophagy, with MAPK15 likely playing a pivotal role in this process.

KLF8 Promotes the Survival of Lung Adenocarcinoma During Nutrient Deprivation by Regulating the Pentose Phosphate Pathway through SIRT2.

BACKGROUND: The pentose phosphate pathway (PPP) is a critical metabolic pathway that generates NADPH and ribose-5-phosphate for nucleotide biosynthesis and redox homeostasis. In this study, we investigated a potential regulatory role for Krüppel-like factor 8 (KLF8) in the control of PPP in lung adenocarcinoma (LUAD) cells. METHODS: Based on a comprehensive set of experimental approaches, including cell culture, molecular techniques, and functional assays, we revealed a novel mechanism by which KLF8 promotes the activation of glucose-6-phosphate dehydrogenase (G6PD), a component enzyme in the PPP. RESULTS: Our findings demonstrate that KLF8 inhibits the acetylation of G6PD, leading to its increased enzymatic activity. Additionally, we observed that KLF8 activates the transcription of SIRT2, which has been implicated in regulating G6PD acetylation. These results highlight the interplay between KLF8, G6PD, and protein acetylation in the regulation of PPP in LUAD. CONCLUSIONS: Understanding the intricate molecular mechanisms underlying the metabolic reprogramming driven by KLF8 in lung cancer provides valuable insights into potential therapeutic strategies targeting the PPP. This study emphasizes the significance of KLF8 as a key modulator of metabolic pathways and indicates the potential of targeting the KLF8-G6PD axis for lung cancer treatment.

FBXO31 is upregulated by METTL3 to promote pancreatic cancer progression via regulating SIRT2 ubiquitination and degradation.

FBXO31, a member of F-box family to comprise of SCF complex, contributes to a pivotal role in cancer progression. However, the possible involvements of FBXO31 in PC are unelucidated. Here, we reported that FBXO31 was overexpressed in PC patients, which was negatively associated with survival in PC patients. Furthermore, FBXO31 significantly enhanced growth, migration and invasion of PC cells in vitro. Consistently, FBXO31 overexpression promoted tumor growth in nude mice. Mechanistically, SIRT2 was a target of FBXO31 and interacted with FBXO31. Protein half-life and ubiquitination analysis demonstrated that FBXO31 promoted proteasome-dependent degradation of SIRT2. In addition, FBXO31 binds to sirtuin-type domain of SIRT2. Moreover, SIRT2 is required for the oncogenic role of FBXO31 in PC progression. Impressively, METTL3 induced m6A modification of FBXO31 and up-regulated FBXO31 expression, subsequently leading to SIRT2 down-regulation in PC cells. The results showed that METTL3 enhanced FBXO31 mRNA translation in YTHDF1-dependent manner. Taken together, we suggest that METTL3-FBXO31-SIRT2 axis was involved in PC tumorigenesis, which could identify new targets for PC treatment.

The role of Sirtuin 2 in liver - An extensive and complex biological process.

Liver disease has become one of the main causes of health issue worldwide. Sirtuin (Sirt) 2 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, and is expressed in multiple organs including liver, which plays important and complex roles by interacting with various substrates. Physiologically, Sirt2 can improve metabolic homeostasis. Pathologically, Sirt2 can alleviate inflammation, endoplasmic reticulum (ER) stress, promote liver regeneration, maintain iron homeostasis, aggravate fibrogenesis and regulate oxidative stress in liver. In liver diseases, Sirt2 can mitigate fatty liver disease (FLD) including non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD), but aggravate hepatitis B (HBV) and liver ischemia-reperfusion injury (LIRI). The role of Sirt2 in liver cancer and aging-related liver diseases, however, has not been fully elucidated. In this review, these biological processes regulated by Sirt2 in liver are summarized, which aims to update the function of Sirt2 in liver and to explore the potential role of Sirt2 as a therapeutic target for liver diseases.

Sirtuin 2 inhibition modulates chromatin landscapes genome-wide to induce senescence in ATRX-deficient malignant glioma.

BACKGROUND: Functional inactivation of ATRX characterizes large subgroups of malignant gliomas in adults and children. ATRX deficiency in glioma induces widespread chromatin remodeling, driving transcriptional shifts and oncogenic phenotypes. Effective strategies to therapeutically target these broad epigenomic sequelae remain undeveloped. METHODS: We utilized integrated multiomics and the Broad Institute Connectivity Map (CMAP) to identify drug candidates that could potentially revert ATRX-deficient transcriptional changes. We then employed disease-relevant experimental models to evaluate functional phenotypes, coupling these studies with epigenomic profiling to elucidate molecular mechanism(s). RESULTS: CMAP analysis and transcriptional/epigenomic profiling implicated the Class III HDAC Sirtuin2 (SIRT2) as a central mediator of ATRX-deficient cellular phenotypes and a driver of unfavorable prognosis in ATRX-deficient glioma. SIRT2 inhibitors reverted Atrx-deficient transcriptional signatures in murine neuroepithelial progenitor cells (mNPCs), impaired cell migration in Atrx/ATRX-deficient mNPCs and human glioma stem cells (GSCs), and increased expression of senescence markers in glioma models. Moreover, SIRT2 inhibition impaired growth and increased senescence in ATRX-deficient GSCs in vivo. These effects were accompanied by genome-wide shifts in enhancer-associated H3K27ac and H4K16ac marks, with the latter in particular demonstrating compelling transcriptional links to SIRT2-dependent phenotypic reversals. Motif analysis of these data identified the transcription factor KLF16 as a mediator of phenotype reversal in Atrx-deficient cells upon SIRT2 inhibition. CONCLUSIONS: Our findings indicate that SIRT2 inhibition selectively targets ATRX-deficient gliomas for senescence through global chromatin remodeling, while demonstrating more broadly a viable approach to combat complex epigenetic rewiring in cancer.

Sodium benzoate induces pancreatic inflammation and ß cell apoptosis partially via benzoylation.

Epigenetics, specifically histone post-translational modification (HPTM) induced by environmental factors, plays a crucial role in the development of diabetes. Sodium benzoate (NAB) is a widely used additive, however, its potential contribution to diabetes has been largely overlooked. In 2018, a novel HPTM called benzoylation (Kbz) induced by NAB was discovered. This modification can be catalyzed by ACSS2 (acyl-CoA synthetase short-chain member 2) and acyltransferase P300/CBP, and can be reversed by erase enzymes SIRT2. Studies have indicated that Kbz may regulate insulin secretion, although the exact molecular mechanism remains unclear. In our study, C57BL/6J mice were divided into two groups: the NC group and the 1g/kg NAB water feeding group. In vivo experiments were conducted using ß-TC-6 cells, with 6 mM NAB or 100 µM benzoyl-CoA as stimuli, and 10 µM A485 (P300 inhibitor), 5 µM ACSS2 inhibitor (inhibiting benzoyl-CoA synthesis), or 5 µM AGK2 (SIRT2 inhibitor) as intervention factors. Our study found that, although the experimental concentration of NAB is below the maximum allowable concentration in food, it still damaged the insulin secretion function of C57BL/6J mice and induced inflammation and apoptosis of islet ß cells. We observed significant differences in serum benzoyl-CoA levels between healthy individuals and patients with type 2 diabetes. Furthermore, NAB concentration-dependently increases benzoyl-CoA and Kbz levels. When Kbz is down-regulated using A485 and ACSS2 inhibitor, we observed a reduction in ß cell inflammation, apoptosis, and insulin secretion damage. Conversely, up-regulating Kbz using AGK2 resulted in increased levels of ß cell inflammation and apoptosis. In conclusion, our data suggest that NAB, despite being within the safe dose range, may be an overlooked environmental risk factor contributing to the pathogenesis of diabetes through its impact on Kbz.

microRNA-143 targets SIRT2 to mediate the histone acetylation of PLAUR and modulates functions of astrocytes in spinal cord injury.

This study aimed to explore effects of microRNA (miR)-143 on the proliferation, apoptosis, and cytokine secretion in astrocytes after spinal cord injury (SCI). After gain- and loss-of-function assays and transforming growth factor (TGF)-ß stimulation in astrocytes, the cell viability, proliferation, and apoptosis were examined. The expression of miR-143, SIRT2, and PLAUR and levels of astrocyte-related glial fibrillary acidic protein (GFAP), Vimentin, chondroitin sulfate proteoglycan (CSPG), and connective tissue growth factor (CTGF) were also measured. The binding relationship between miR-143 and SIRT2 was assessed, as well as the correlation of PLAUR with SIRT2. In established SCI rat models, the locomotion function and astrocyte hyperplasia were detected. The TGF-ß stimulation decreased miR-143 but increased SIRT2 expression in astrocytes. Mechanistically, miR-143 negatively targeted SIRT2 and SIRT2 down-regulation inhibited the H3K27 deacetylation of PLAUR promoter to increase PLAUR expression. miR-143 up-regulation inhibited TGF-ß stimulated-proliferation, promoted cell apoptosis, and reduced GFAP, Vimentin, CSPG, and CTGF expression in astrocytes, which was counterweighed by SIRT2 overexpression. SIRT2 silencing reduced the proliferation and GFAP, Vimentin, CSPG, and CTGF expression while augmenting the apoptosis in TGF-ß stimulated astrocytes, which was abrogated by PLAUR silencing. The injection of miR-143 agomir improved the locomotion function and reduced the astrocyte hyperplasia in SCI rats, which was reversed by silencing PLAUR. miR-143 targeted SIRT2 to affect PLAUR expression via the regulation of histone acetylation, which repressed the astrocyte activation in vivo and in vitro to improve the locomotion function in SCI rats.

Role of SYVN1 in the control of airway remodeling in asthma protection by promoting SIRT2 ubiquitination and degradation.

BACKGROUND: Asthma is a heterogenous disease that characterized by airway remodeling. SYVN1 (Synoviolin 1) acts as an E3 ligase to mediate the suppression of endoplasmic reticulum (ER) stress through ubiquitination and degradation. However, the role of SYVN1 in the pathogenesis of asthma is unclear. RESULTS: In the present study, an ovalbumin (OVA)-induced murine model was used to evaluate the effect of SYVN1 on asthma. An increase in SYVN1 expression was observed in the lungs of mice after OVA induction. Overexpression of SYVN1 attenuated airway inflammation, goblet cell hyperplasia and collagen deposition induced by OVA. The increased ER stress-related proteins and altered epithelial-mesenchymal transition (EMT) markers were also inhibited by SYVN1 in vivo. Next, TGF-ß1-induced bronchial epithelial cells (BEAS-2B) were used to induce EMT process in vitro. Results showed that TGF-ß1 stimulation downregulated the expression of SYVN1, and SYVN1 overexpression prevented ER stress response and EMT process in TGF-ß1-induced cells. In addition, we identified that SYVN1 bound to SIRT2 and promoted its ubiquitination and degradation. SIRT2 overexpression abrogated the protection of SYVN1 on ER stress and EMT in vitro. CONCLUSIONS: These data suggest that SYVN1 suppresses ER stress through the ubiquitination and degradation of SIRT2 to block EMT process, thereby protecting against airway remodeling in asthma.

Effects of Dimerization on the Deacylase Activities of Human SIRT2.

Human sirtuin isoform 2 (SIRT2) is an NAD+-dependent enzyme that functions as a lysine deacetylase and defatty-acylase. Here, we report that SIRT2 readily dimerizes in solution and in cells and that dimerization affects its ability to remove different acyl modifications from substrates. Dimerization of recombinant SIRT2 was revealed with analytical size exclusion chromatography and chemical cross-linking. Dimerized SIRT2 dissociates into monomers upon binding long fatty acylated substrates (decanoyl-, dodecanoyl-, and myristoyl-lysine). However, we did not observe dissociation of dimeric SIRT2 in the presence of acetyl-lysine. Analysis of X-ray crystal structures led us to discover a SIRT2 double mutant (Q142A/E340A) that is impaired in its ability to dimerize, which was confirmed with chemical cross-linking and in cells with a split-GFP approach. In enzyme assays, the SIRT2(Q142A/E340A) mutant had normal defatty-acylase activity and impaired deacetylase activity compared with the wild-type protein. These results indicate that dimerization is essential for optimal SIRT2 function as a deacetylase. Moreover, we show that SIRT2 dimers can be dissociated by a deacetylase and defatty-acylase inhibitor, ascorbyl palmitate. Our finding that its oligomeric state can affect the acyl substrate selectivity of SIRT2 is a novel mode of activity regulation by the enzyme that can be altered genetically or pharmacologically.

SIRT2 transgenic over-expression does not impact lifespan in mice.

The NAD+ -dependent deacylase family of sirtuin enzymes have been implicated in biological ageing, late-life health and overall lifespan, though of these members, a role for sirtuin-2 (SIRT2) is less clear. Transgenic overexpression of SIRT2 in the BubR1 hypomorph model of progeria can rescue many aspects of health and increase overall lifespan, due to a specific interaction between SIRT2 and BubR1 that improves the stability of this protein. It is less clear whether SIRT2 is relevant to biological ageing outside of a model where BubR1 is under-expressed. Here, we sought to test whether SIRT2 over-expression would impact the overall health and lifespan of mice on a nonprogeroid, wild-type background. While we previously found that SIRT2 transgenic overexpression prolonged female fertility, here, we did not observe any additional impact on health or lifespan, which was measured in both male and female mice on standard chow diets, and in males challenged with a high-fat diet. At the biochemical level, NMR studies revealed an increase in total levels of a number of metabolites in the brain of SIRT2-Tg animals, pointing to a potential impact in cell composition; however, this did not translate into functional differences. Overall, we conclude that strategies to enhance SIRT2 protein levels may not lead to increased longevity.

Reading and erasing of histone crotonyllysine mimics by the AF9 YEATS domain and SIRT2 deacylase.

Lysine acylations on histones and their recognition by chromatin-binding reader domains and removal by histone deacylases function as an important mechanism for eukaryotic gene regulation. Histone lysine crotonylation (Kcr) is an epigenetic mark associated with active transcription, and its installation and removal are dynamically regulated by cellular epigenetic enzymes. Here, we report binding studies and enzyme assays with histone H3K9 peptides bearing simplest Kcr analogs with varying hydrocarbon chain length, bulkiness, rigidity and polarity. We demonstrate that the AF9 YEATS domain displays selectivity for binding of different acylation modifications on histone H3K9 peptides and exhibits preference for bulkier cinnamoylated lysine over crotonylated lysine and its mimics. SIRT2 shows deacylase activity against most of acylated H3K9 peptides bearing different crotonyllysine mimics, however, it displays a poor ability for the removal of cinnamoyl and trifluorocrotonyl groups. These results demonstrate different substrate selectivities of epigenetic proteins acting on crotonyllysine and pave the way for rational design and development of AF9 YEATS and SIRT2 inhibitors for treatment of human diseases, including cancer.

SIRT2 negatively regulates the cGAS-STING pathway by deacetylating G3BP1.

SIRT2, a cytoplasmic member of the Sirtuin family, has important roles in immunity and inflammation. However, its function in regulating the response to DNA virus infection remains elusive. Here, we find that SIRT2 is a unique regulator among the Sirtuin family that negatively modulates the cGAS-STING-signaling pathway. SIRT2 is down-regulated after Herpes simplex virus-1 (HSV-1) infection, and SIRT2 deficiency markedly elevates the expression levels of type I interferon (IFN). SIRT2 inhibits the DNA binding ability and droplet formation of cGAS by interacting with and deacetylating G3BP1 at K257, K276, and K376, leading to the disassembly of the cGAS-G3BP1 complex, which is critical for cGAS activation. Administration of AGK2, a selective SIRT2 inhibitor, protects mice from HSV-1 infection and increases the expression of IFN and IFN-stimulated genes. Our study shows that SIRT2 negatively regulates cGAS activation through G3BP1 deacetylation, suggesting a potential antiviral strategy by modulating SIRT2 activity.

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation.

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

Pyruvate kinase is post-translationally regulated by sirtuin 2 in Aedes aegypti mosquitoes.

We previously demonstrated that Aedes aegypti pyruvate kinase (AaPK) plays a key role in the regulation of both carbon and nitrogen metabolism in mosquitoes. To further elucidate whether AaPK can be post-translationally regulated by Ae. aegypti sirtuin 2 (AaSirt2), an NAD+-dependent deacetylase that catalyzes the removal of acetyl groups from acetylated lysine residues, we conducted a series of analysis in non-starved and starved female mosquitoes. Transcriptional and protein profiles of AaSirt2, analyzed by qPCR and western blots, indicated that the AaSirt2 is differentially modulated in response to sugar or blood feeding in mosquito tissues dissected at different times during the first gonotrophic cycle. We also found that AaSirt2 is localized in both cytosolic and mitochondrial cellular compartments of fat body and thorax. Multiple lysine-acetylated proteins were detected by western blotting in both cellular compartments. Furthermore, western blotting of immunoprecipitated proteins provided evidence that AaPK is lysine-acetylated and bound with AaSirt2 in the cytosolic fractions of fat body and thorax from non-starved and starved females. In correlation with these results, we also discovered that RNAi-mediated knockdown of AaSirt2 in the fat body of starved females significantly decreased AaPK protein abundance. Notably, survivorship of AaSirt2-deficient females maintained under four different nutritional regimens was not significantly affected. Taken together, our data reveal that AaPK is post-translationally regulated by AaSirt2.