New insight into arginine and tryptophan metabolism in macrophage activation during tuberculosis.

Arginine and tryptophan are pivotal in orchestrating cytokine-driven macrophage polarization and immune activation. Specifically, interferon-gamma (IFN-γ) stimulates inducible nitric oxide synthase (iNOS) expression), leading to the conversion of arginine into citrulline and nitric oxide (NO), while Interleukin-4 (IL4) promotes arginase activation, shifting arginine metabolism toward ornithine. Concomitantly, IFN-γ triggers indoleamine 2,3-dioxygenase 1 (IDO1) and Interleukin-4 induced 1 (IL4i1), resulting in the conversion of tryptophan into kynurenine and indole-3-pyruvic acid. These metabolic pathways are tightly regulated by NAD+-dependent sirtuin proteins, with Sirt2 and Sirt5 playing integral roles. In this review, we present novel insights that augment our understanding of the metabolic pathways of arginine and tryptophan following Mycobacterium tuberculosis infection, particularly their relevance in macrophage responses. Additionally, we discuss arginine methylation and demethylation and the role of Sirt2 and Sirt5 in regulating tryptophan metabolism and arginine metabolism, potentially driving macrophage polarization.

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.

An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity.

296 million people worldwide are predisposed to developing severe end-stage liver diseases due to chronic hepatitis B virus (HBV) infection. HBV forms covalently closed circular DNA (cccDNA) molecules that persist as episomal DNA in the nucleus of infected hepatocytes and drive viral replication. Occasionally, the HBV genome becomes integrated into host chromosomal DNA, a process that is believed to significantly contribute to circulating HBsAg levels and HCC development. Neither cccDNA accumulation nor expression from integrated HBV DNA are directly targeted by current antiviral treatments. In this study, we investigated the antiviral properties of a newly described allosteric modulator, FLS-359, that targets sirtuin 2 (SIRT2), an NAD+-dependent deacylase. Our results demonstrate that SIRT2 modulation by FLS-359 and by other tool compounds inhibits cccDNA synthesis following de novo infection of primary human hepatocytes and HepG2 (C3A)-NTCP cells, and FLS-359 substantially reduces cccDNA recycling in HepAD38 cells. While pre-existing cccDNA is not eradicated by short-term treatment with FLS-359, its transcriptional activity is substantially impaired, likely through inhibition of viral promoter activities. Consistent with the inhibition of viral transcription, HBsAg production by HepG2.2.15 cells, which contain integrated HBV genomes, is also suppressed by FLS-359. Our study provides further insights on SIRT2 regulation of HBV infection and supports the development of potent SIRT2 inhibitors as HBV antivirals.

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.

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.

Structural basis for phage-mediated activation and repression of bacterial DSR2 anti-phage defense system.

Silent information regulator 2 (Sir2) proteins typically catalyze NAD+-dependent protein deacetylation. The recently identified bacterial Sir2 domain-containing protein, defense-associated sirtuin 2 (DSR2), recognizes the phage tail tube and depletes NAD+ to abort phage propagation, which is counteracted by the phage-encoded DSR anti-defense 1 (DSAD1), but their molecular mechanisms remain unclear. Here, we determine cryo-EM structures of inactive DSR2 in its apo form, DSR2-DSAD1 and DSR2-DSAD1-NAD+, as well as active DSR2-tube and DSR2-tube-NAD+ complexes. DSR2 forms a tetramer with its C-terminal sensor domains (CTDs) in two distinct conformations: CTDclosed or CTDopen. Monomeric, rather than oligomeric, tail tube proteins preferentially bind to CTDclosed and activate Sir2 for NAD+ hydrolysis. DSAD1 binding to CTDopen allosterically inhibits tube binding and tube-mediated DSR2 activation. Our findings provide mechanistic insight into DSR2 assembly, tube-mediated DSR2 activation, and DSAD1-mediated inhibition and NAD+ substrate catalysis in bacterial DSR2 anti-phage defense systems.

Ligand-based design and synthesis of new trityl histamine and trityl cysteamine derivatives as SIRT2 inhibitors for cancer therapy.

The relentless pursuit of novel therapeutic agents against cancer has led to the identification of multiple molecular targets, among which Sirtuin 2 (SIRT2) has garnered significant attention. This study presents an extensive SAR study of our reported trityl scaffold-based SIRT2 inhibitors. This study encompasses a range of different medicinal chemistry approaches to improve the activity of the lead compounds TH-3 and STCY1. The rationally designed and synthesized structures were confirmed using NMR and high-resolution mass spectroscopy before performing SIRT2 inhibition assay, NCI60 cytotoxicity test, and cell cycle analysis. Indeed, our strategies afforded hitherto unreported SIRT2 inhibitors with high activity, particularly 2a, 4a, 7c, and 7f. Remarkably, the presence of a lipophilic para substitution on the phenyl group of a freely rotating or a locked trityl moiety enhanced activity SIRT2 inhibition. Concomitantly, the synthesized compounds showed prominent activity against different cancer lines from the NCI60 assay. Of interest, compound 7c stands out as a potent and highly selective antiproliferative agent against leukemia and colon cancer panels. Furthermore, 7c treatment resulted in cell cycle arrest in MCF-7 cells at G2 phase and did not cause in vitro DNA cleavage.

Resveratrol evokes neuroprotective effects and improves foot stance following kainate-induced excitotoxic damage to the mouse spinal cord.

Excitotoxicity, characterized by over-activation of glutamate receptors, is a major contributor to spinal cord injury (SCI) pathophysiology, resulting in neuronal death and loss of locomotor function. In our previous in vitro studies, we showed that excitotoxicity induced by the glutamate analogue kainate (KA) leads to a significant reduction in the number of neurons, providing a model for SCI. Our current objective was to assess the neuroprotective role of resveratrol (RESV), a natural polyphenol, following KA-induced SCI. In vivo excitotoxicity was induced by intraspinal injection of KA immediately followed by RESV administration to Balb/C adult male mice. In neonatal mouse spinal cord preparations, excitotoxicity was transiently induced by bath-applied KA, either with or without RESV. KA administration resulted in a significant deterioration in hindlimb motor coordination and balance during locomotion, which was partially reverted by RESV. Additionally, RESV preserved neurons in both dorsal and ventral regions. Sirtuin 2 (SIRT2) immunoreactive signal was increased by RESV, while the selective SIRT1 inhibitor 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide (EX-527) attenuated RESV neuroprotective effects. These findings suggest that RESV attenuation of excitotoxic-induced neuronal loss and locomotor deficits is mediated, at least in part, through the activation of SIRT1, potentially involving SIRT2 as well. Indeed, our results highlight the potential use of RESV to enhance neuroprotective strategies for SCI.

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.

Oligodendrocyte-derived exosomes-containing SIRT2 ameliorates depressive-like behaviors and restores hippocampal neurogenesis and synaptic plasticity via the AKT/GSK-3ß pathway in depressed mice.

AIMS: To investigate the antidepressant role of oligodendrocyte-derived exosomes (ODEXs)-containing sirtuin 2 (SIRT2) and the underlying mechanism both in vivo and in vitro. METHODS: Oligodendrocyte-derived exosomes isolated from mouse serum were administered to mice with chronic unpredictable mild stress (CUMS)-induced depression via the tail vein. The antidepressant effects of ODEXs were assessed through behavioral tests and quantification of alterations in hippocampal neuroplasticity. The role of SIRT2 was confirmed using the selective inhibitor AK-7. Neural stem/progenitor cells (NSPCs) were used to further validate the impact of overexpressed SIRT2 and ODEXs on neurogenesis and synapse formation in vitro. RESULTS: Oligodendrocyte-derived exosome treatment alleviated depressive-like behaviors and restored neurogenesis and synaptic plasticity in CUMS mice. SIRT2 was enriched in ODEXs, and blocking SIRT2 with AK-7 reversed the antidepressant effects of ODEXs. SIRT2 overexpression was sufficient to enhance neurogenesis and synaptic protein expression. Mechanistically, ODEXs mediated transcellular delivery of SIRT2, targeting AKT deacetylation and AKT/GSK-3ß signaling to regulate neuroplasticity. CONCLUSION: This study establishes how ODEXs improve depressive-like behaviors and hippocampal neuroplasticity and might provide a promising therapeutic approach for depression.

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.

Acetylation of c-Myc at Lysine 148 Protects Neurons After Ischemia.

This study focuses on understanding the role of c-Myc, a cancer-associated transcription factor, in the penumbra following ischemic stroke. While its involvement in cell death and survival is recognized, its post-translational modifications, particularly acetylation, remain understudied in ischemia models. Investigating these modifications could have significant clinical implications for controlling c-Myc activity in the central nervous system. Although previous studies on c-Myc acetylation have been limited to non-neuronal cells, our research examines its expression in perifocal cells during stroke recovery to explore regulatory mechanisms via acetylation. We found that in peri-infarct neurons, c-Myc is upregulated with acetylation at K148 but not K323 during the acute phase of stroke, with SIRT2 deacetylase primarily affecting K148 acetylation. Molecular dynamics simulations suggest that lysine 148 plays a crucial role in stabilizing c-Myc spatial structure. Increased acetylation at K148 reduces c-Myc compaction, potentially limiting its nuclear penetration, promoting calpain-mediated cleavage, and decreasing nuclear localization. Additionally, cytoplasmic acetylation at K148 may alter c-Myc's interaction with unidentified proteins, potentially influencing its pro-apoptotic effects and promoting cytoplasmic accumulation. Targeting SIRT2 with selective inhibitors could be a promising avenue for future stroke therapy strategies.

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.

Isobavachalcone, a natural sirtuin 2 inhibitor, exhibits anti-triple-negative breast cancer efficacy in vitro and in vivo.

Triple-negative breast cancer (TNBC) is the most aggressive and lethal clinical subtype and lacks effective targeted therapies at present. Isobavachalcone (IBC), the main active component of Psoralea corylifolia L., has potential anticancer effects. Herein, we identified IBC as a natural sirtuin 2 (SIRT2) inhibitor and characterized the potential mechanisms underlying the inhibition of TNBC. Molecular dynamics analysis, enzyme activity assay, and cellular thermal shift assay were performed to evaluate the combination of IBC and SIRT2. The therapeutic effects, mechanism, and safety of IBC were analyzed in vitro and in vivo using cellular and xenograft models. IBC effectively inhibited SIRT2 enzyme activity with an IC50 value of 0.84 ± 0.22 µM by forming hydrogen bonds with VAL233 and ALA135 within its catalytic domain. In the cellular environment, IBC bound to and stabilized SIRT2, consequently inhibiting cellular proliferation and migration, and inducing apoptosis and cell cycle arrest by disrupting the SIRT2/α-tubulin interaction and inhibiting the downstream Snail/MMP and STAT3/c-Myc pathways. In the in vivo model, 30 mg/kg IBC markedly inhibited tumor growth by targeting the SIRT2/α-tubulin interaction. Furthermore, IBC exerted its effects by inducing apoptosis in tumor tissues and was well-tolerated. IBC alleviated TNBC by targeting SIRT2 and triggering the reactive oxygen species ROS/ß-catenin/CDK2 axis. It is a promising natural lead compound for future development of SIRT2-targeting drugs.

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.

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.

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.