Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action.

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

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.

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.

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.

Spinal sirtuin 2 attenuates bone cancer pain by deacetylating FoxO3a.

Bone cancer pain (BCP) is refractory to currently used analgesics. Recently, sirtuin 2 (SIRT2) was reported to play a vital role in neuropathic pain but its role in BCP remains unknown. It was hypothesized that spinal SIRT2 attenuates BCP by deacetylating FoxO3a and suppressing oxidative stress. The mouse model of BCP established by injecting tumor cells into the intramedullary space of the femur demonstrated that spinal SIRT2 and FoxO3a were downregulated in BCP development. Intrathecal administration of LV-SIRT2 reduced pain hypersensitivity (mechanical and thermal nociception) in BCP mice. Spinal SIRT2 overexpression upregulated FoxO3a and antioxidant genes (SOD2 and catalase) and inhibited FoxO3a acetylation, phosphorylation, and ubiquitination. Moreover, intrathecal administration of SIRT2 shRNA induced pain hypersensitivity in normal mice. Spinal SIRT2 knockdown downregulated FoxO3a and antioxidant genes and increased FoxO3a acetylation, phosphorylation, and ubiquitination. In summary, spinal SIRT2 increases FoxO3a expression in BCP mice and inhibits oxidative stress by deacetylating FoxO3a and further reducing FoxO3a phosphorylation, ubiquitination, and degradation, leading to BCP relief.

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 promotes base excision repair by transcriptionally activating OGG1 in an ATM/ATR-dependent manner.

Sirtuin 2 (SIRT2) regulates the maintenance of genome integrity by targeting pathways of DNA damage response and homologous recombination repair. However, whether and how SIRT2 promotes base excision repair (BER) remain to be determined. Here, we found that independent of its catalytic activity SIRT2 interacted with the critical glycosylase OGG1 to promote OGG1 recruitment to its own promoter upon oxidative stress, thereby enhancing OGG1 promoter activity and increasing BER efficiency. Further studies revealed that SIRT2 was phosphorylated on S46 and S53 by ATM/ATR upon oxidative stress, and SIRT2 phosphorylation enhanced the SIRT2-OGG1 interaction and mediated the stimulatory effect of SIRT2 on OGG1 promoter activity. We also characterized 37 cancer-derived SIRT2 mutants and found that 5 exhibited the loss of the stimulatory effects on OGG1 transcription. Together, our data reveal that SIRT2 acts as a tumor suppressor by promoting OGG1 transcription and increasing BER efficiency in an ATM/ATR-dependent manner.

Silent information regulator 2 deficiency exacerbates chronic cold exposure-induced colonic injury and p65 activation in mice.

Cold is a common stressor that threatens colonic health by affecting internal homeostasis. From the literature, Silent information regulator 2 (SIRT2) may have important roles during cold stress, but this conjecture requires investigation. To address this knowledge gap, we investigated the effects of SIRT2 on colonic injury in chronically cold-exposure mice. In a previous study, we showed that SIRT2 regulated p65 activation after cold exposure. In the current study, mice were exposed to 4 °C for 3 h/day for 3 weeks to simulate a chronic cold exposure environment. Chronic cold exposure shortened colon length, disrupted tight junctions in colonic epithelial tissue, and disordered colonic flora. Chronic cold exposure also increased p65 acetylation levels, promoted nuclear factor (NF)-κB activation, and increased the expression of its downstream pro-inflammatory factors, while SIRT2 knockdown aggravated the consequences of tissue structure disruption and increased inflammatory factors brought about by chronic cold exposure to some extent, but could alleviate the downregulation of colonic tight junction-related proteins to some extent. We also observed direct SIRT2 regulatory effects toward p65, and in Caco-2 cells treated with lipopolysaccharide (LPS), SIRT2 knockdown increased p65 acetylation levels and pro-inflammatory factor expression, while SIRT2 overexpression reversed these phenomena. Therefore, SIRT2 deletion exacerbated chronic cold exposure-induced colonic injury and p65 activation in mice. Mechanistically, p65 modification by SIRT2 via deacetylation may affect NF-κB signaling. These findings suggest that SIRT2 is a key target of colonic health maintenance under chronic cold exposure conditions.

Sirtuin2 suppresses the polarization of regulatory T cells toward T helper 17 cells through repressing the expression of signal transducer and activator of transcription 3 in a mouse colitis model.

INTRODUCTION: Regulatory T cells (Tregs) play an important role in inflammatory bowel diseases (IBDs) through modulating intestinal inflammation. However, the factors affecting Treg function and plasticity during IBD progression are not thoroughly disclosed. The current study aims to reveal new molecular mechanisms affecting Treg plasticity. METHODS: A mouse strain, in which tdTomato and enhanced green fluorescent protein were under the control of the Foxp3 promoter and Il17a promoter, was established and subjected to colitis induction with dextran sulfate sodium. The existence of Tregs and IL-17-expressing Tregs (i.e., Treg/T helper 17 [Th17] cells) were observed and sorted from the spleen, mesenteric lymph nodes, and lamina propria by flow cytometry, followed by measuring Sirtuin2 (Sirt2) expression using quantitative reverse transcription polymerase chain reaction and Immunoblotting. Lentivirus-induced Sirt2 silencing was applied to determine the impact of Sirt2 on Treg polarization to Treg/Th17 cells and even Th17 cells. The effect of Sirt2 on Stat3 was analyzed by flow cytometry and immunoblotting. RESULTS: Sirt2 was highly expressed in lamina propria Tregs and it moderately suppressed Foxp3 expression as well as the immunosuppressive function of Tregs. Surprisingly, lentivirus-mediated Sirt2 silencing promoted the generation of Treg/Th17 cells out of Tregs. Sirt2 silencing also enhanced the generation of Th17 cells out of Tregs under the Th17 induction condition. Furthermore, Sirt2 inhibited Th17 induction by suppressing the protein level of the signal transducer and activator of transcription 3. CONCLUSION: Sirt2 suppresses Treg function but also inhibits Treg polarization toward Treg/Th17 cells and Th17 cells. The ultimate effect of Sirt2 on colitis might depend on the balance among Tregs, Treg/Th17 cells, and Th17 cells.

Bioinformatic identification and experiment validation reveal 6 hub genes, promising diagnostic and therapeutic targets for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease that can cause dementia. We aim to screen out the hub genes involved in AD based on microarray datasets. METHODS: Gene expression profiles GSE5281 and GSE28146 were retrieved from Gene Expression Omnibus database to acquire differentially expressed genes (DEGs). Gene Ontology and pathway enrichment were conducted using DAVID online tool. The STRING database and Cytoscape tools were employed to analyze protein-protein interactions and identify hub genes. The predictive value of hub genes was assessed by principal component analysis and receiver operating characteristic curves. AD mice model was constructed, and histology was then observed by hematoxylin-eosin staining. Gene expression levels were finally determined by real-time quantitative PCR. RESULTS: We obtained 197 overlapping DEGs from GSE5281 and GSE28146 datasets. After constructing protein-protein interaction network, three highly interconnected clusters were identified and 6 hub genes (RBL1, BUB1, HDAC7, KAT5, SIRT2, and ITGB1) were selected. The hub genes could be used as basis to predict AD. Histological abnormalities of brain were observed, suggesting successful AD model was constructed. Compared with the control group, the mRNA expression levels of RBL1, BUB1, HDAC7, KAT5 and SIRT2 were significantly increased, while the mRNA expression level of ITGB1 was significantly decreased in AD groups. CONCLUSION: RBL1, BUB1, HDAC7, KAT5, SIRT2 and ITGB1 are promising gene signatures for diagnosis and therapy of AD.

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.

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.

Glucocorticoids promote steroid-induced osteonecrosis of the femoral head by down-regulating serum alpha-2-macroglobulin to induce oxidative stress and facilitate SIRT2-mediated BMP2 deacetylation.

Our study investigated the possible molecular mechanism of glucocorticoid in steroid-induced osteonecrosis of the femoral head (SINFH) through regulating serum alpha-2-macroglobulin and SIRT2-mediated BMP2 deacetylation. Essential genes involved in glucocorticoid-induced SINFH were screened by transcriptome sequencing and analyzed by bioinformatics, followed by identifying downstream regulatory targets. Rat bone marrow mesenchymal stem cells were isolated and treated with methylprednisolone (MP) for in vitro cell experiments. Besides, a glucocorticoid-induced rat ONFH was established using the treatment of MP and LPS. ChIP-PCR detected the enrichment of SIRT2 in the promoter region of BMP2, and the deacetylation modification of SIRT2 on BMP2 was determined. Bioinformatics analysis revealed that glucocorticoids may induce ONFH through the SIRT2/BMP2 axis. In vitro cell experiments showed that glucocorticoids up-regulated SIRT2 expression in BMSCs by inducing oxidative stress, thereby promoting cell apoptosis. The up-regulation of SIRT2 expression may be due to the decreased ability of α2 macroglobulin to inhibit oxidative stress, and the addition of NOX protein inhibitor DPI could significantly inhibit SIRT2 expression. SIRT2 could promote histone deacetylation of the BMP2 promoter and inhibit its expression. In vitro cell experiments further indicated that knocking down SIRT2 could protect BMSC from oxidative stress and cell apoptosis induced by glucocorticoids by promoting BMP2 expression. In addition, animal experiments conducted also demonstrated that the knockdown of SIRT2 could improve glucocorticoid-induced ONFH through up-regulating BMP2 expression. Glucocorticoids could induce oxidative stress by down-regulating serum α2M to promote SIRT2-mediated BMP2 deacetylation, leading to ONFH.

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.

SIRT2 overexpression decreases remifentanil-stimulated post-surgical hyperalgesia via microglia.

Remifentanil (Remi)-induced hyperalgesia is a serious but common postoperative clinical problem. Sirtuin 2 (SIRT2) is essential in the pathogenetic mechanisms of several neurological disorders. However, whether SIRT2 contributes to the modulation of Remi-induced postsurgical hyperalgesia (POH) is unknown. Here, we investigated the regulatory potential of SIRT2 in Remi-stimulated POH. A rat Remi-stimulated POH model was built by infusing Remi in the surgical incision. Mechanical allodynia and thermal hyperalgesia were separately assessed by paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) measurements. SIRT2 and binding adaptor molecule 1 (Iba1) protein expressions and localization in spinal cord samples were detected by western blot and immunofluorescence. The results revealed SIRT2 downregulation in the spinal cord of rats with Remi-stimulated POH. Intrathecal administration of the overexpression plasmid harboring SIRT2 remarkably relieved mechanical allodynia, along with thermal hyperalgesia in the model animals. Iba1 amounts were increased upon intraoperative incision or Remi infusion, and this effect was more pronounced upon combining both treatments. Furthermore, SIRT2 overexpression suppressed microglia activation in the spinal cord of model animals, and starkly relieved incision- and/or Remi-associated pronociceptive processes as well as spinal microglia activation. SIRT2 elevation relieved Remi-associated POH, likely by suppressing spinal microglia activation. Thus, SIRT2 could be a potent target for treating neuropathic pain.

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.

Sirtuin 2 promotes human cytomegalovirus replication by regulating cell cycle progression.

IMPORTANCE: This study expands the growing understanding that protein acetylation is a highly regulated molecular toggle of protein function in both host anti-viral defense and viral replication. We describe a pro-viral role for the human enzyme SIRT2, showing that its deacetylase activity supports HCMV replication. By integrating quantitative proteomics, flow cytometry cell cycle assays, microscopy, and functional virology assays, we investigate the temporality of SIRT2 functions and substrates. We identify a pro-viral role for the SIRT2 deacetylase activity via regulation of CDK2 K6 acetylation and the G1-S cell cycle transition. These findings highlight a link between viral infection, protein acetylation, and cell cycle progression.

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.

SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells.

Transforming growth factor-ß (TGF-ß) is the primary factor that drives fibrosis in most, if not all, forms of chronic kidney disease. In kidneys that are obstructed, specific deletion of Sirt2 in renal tubule epithelial cells (TEC) has been shown to aggravate renal fibrosis, while renal tubule specific overexpression of Sirt2 has been shown to ameliorate renal fibrosis. Similarly, specific deletion of Sirt2 in hepatocyte aggravated CCl4-induced hepatic fibrosis. In addition, we have demonstrated that SIRT2 overexpression and knockdown restrain and enhance TGF-ß-induced fibrotic gene expression, respectively, in TEC. Mechanistically, SIRT2 reduced the phosphorylation, acetylation, and nuclear localization levels of SMAD2 and SMAD3, leading to inhibition of the TGF-ß signaling pathway. Further studies have revealed that that SIRT2 was able to directly interact with and deacetylate SMAD2 at lysine 451, promoting its ubiquitination and degradation. Notably, loss of SMAD specific E3 ubiquitin protein ligase 2 abolishes the ubiquitination and degradation of SMAD2 induced by SIRT2 in SMAD2. Regarding SMAD3, we have found that SIRT2 interact with and deacetylates SMAD3 at lysine 341 and 378 only in the presence of TGF-ß, thereby reducing its activation. This study provides initial indication of the anti-fibrotic role of SIRT2 in renal tubules and hepatocytes, suggesting its therapeutic potential for fibrosis.