SIRT1 Deacetylation Research Articles

Myricetin ameliorates airway inflammation and remodeling in asthma by activating Sirt1 to regulate the JNK/Smad3 pathway

BackgroundMyricetin has various biological activities and health benefits; however, its effects on airway remodeling in asthma have not been reported. PurposeWe aimed to investigate the possibility that myricetin improves airway remodeling by activating Sirt1 and has potential as a new treatment for asthma. MethodsRAW 264.7 cells were stimulated with lipopolysaccharide and co-cultured with 3T6 cells in vitro to simulate the in vivo effects of inflammation on airway remodeling. Using an ovalbumin-induced chronic asthma mouse model, we compared changes in inflammatory factors and airway remodeling-related factors under treatment with myricetin and/or the Sirt1 inhibitor EX-527 using western blotting and quantitative PCR. Expression plasmids carrying Smad3 site mutations were transfected into 3T6 cells to identify the Sirt1 deacetylation site on Smad3 protein. ResultsMyricetin significantly reduced the infiltration of airway inflammatory cells and the production of interleukin (IL)-6 and IL-5, and inhibited mucus secretion by goblet cells, collagen fiber proliferation, and the increase in inflammatory cells in bronchoalveolar lavage fluid from asthmatic mice. Results of in vitro experiments were consistent with those conducted in vivo. Exploring the mechanism of action of myricetin, we found that myricetin downregulated the levels of phosphorylated (p)-JNK, p-Smad3, and acetylated Smad3 proteins by activating Sirt1 both in vivo and in vitro. K341 was identified as the main deacetylation site of Smad3 by myricetin-activated Sirt1. ConclusionMyricetin ameliorates airway inflammation and remodeling in asthma by activating Sirt1 to regulate the JNK/Smad3 pathway.

Aspirin-Mediated Acetylation of SIRT1 Maintains Intestinal Immune Homeostasis.

Aspirin, also named acetylsalicylate, can directly acetylate the side-chain of lysine in protein, which leads to the possibility of unexplained drug effects. Here, the study used isotopic-labeling aspirin-d3 with mass spectrometry analysis to discover that aspirin directly acetylates 10 HDACs proteins, including SIRT1, the most studied NAD+-dependent deacetylase. SIRT1 is also acetylated by aspirin in vitro. It is also identified that aspirin directly acetylates lysine 408 of SIRT1, which abolishes SIRT1 deacetylation activity by impairing the substrates binding affinity. Interestingly, the lysine 408 of SIRT1 can be acetylated by CBP acetyltransferase in cells without aspirin supplement. Aspirin can inhibit SIRT1 to increase the levels of acetylated p53 and promote p53-dependent apoptosis. Moreover, the knock-in mice of the acetylation-mimic mutant of SIRT1 show the decreased production of pro-inflammatory cytokines and maintain intestinal immune homeostasis. The study indicates the importance of the acetylated internal functional site of SIRT1 in maintaining intestinal immune homeostasis.

Angelica gigas extract inhibits acetylation of eNOS via IRE1α sulfonation/RIDD-SIRT1-mediated posttranslational modification in vascular dysfunction.

Angelica gigas NAKAI (AG) is a popular traditional medicinal herb widely used to treat dyslipidemia owing to its antioxidant activity. Vascular disease is intimately linked to obesity-induced metabolic syndrome, and AG extract (AGE) shows beneficial effects on obesity-associated vascular dysfunction. However, the effectiveness of AGE against obesity and its underlying mechanisms have not yet been extensively investigated. In this study, 40 high fat diet (HFD) rats were supplemented with 100-300 mg/kg/day of AGE to determine its efficacy in regulating vascular dysfunction. The vascular relaxation responses to acetylcholine were impaired in HFD rats, while the administration of AGE restored the diminished relaxation pattern. Endothelial dysfunction, including increased plaque area, accumulated reactive oxygen species, and decreased nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation, were observed in HFD rats, whereas AGE reversed endothelial dysfunction and its associated biochemical signaling. Furthermore, AGE regulated endoplasmic reticulum (ER) stress and IRE1α sulfonation and its subsequent sirt1 RNA decay through controlling regulated IRE1α-dependent decay (RIDD) signaling, ultimately promoting NO bioavailability via the SIRT1-eNOS axis in aorta and endothelial cells. Independently, AGE enhanced AMPK phosphorylation, additionally stimulating SIRT1 and eNOS deacetylation and its associated NO bioavailability. Decursin, a prominent constituent of AGE, exhibited a similar effect in alleviating endothelial dysfunctions. These data suggest that AGE regulates dyslipidemia-associated vascular dysfunction by controlling ROS-associated ER stress responses, especially IRE1α-RIDD/sirt1 decay and the AMPK-SIRT1 axis.

Relief of ovalbumin-induced airway remodeling by the glycyl-l-histidyl-l-lysine-Cu2+ tripeptide complex via activation of SIRT1 in airway epithelial cells

Fixed airflow limitation (FAO), prevalent in patients with severe or difficult-to-treat asthma, is mainly caused by airway remodeling. Airway remodeling is initiated by inflammation and involves subsequent pathological changes. Glycyl-l-histidyl-l-lysine (GHK) is a matrikine with anti-inflammatory and antioxidant effects, naturally existing in human tissue. At present, the GHK level in human plasma and whether it is related to airway remodeling of asthma remain unclear. This study was conducted to determine how GHK is involved in airway remodeling in asthma. Our result showed that the plasma GHK levels of patients with asthma were significantly lower than those of age-matched healthy controls. In asthma patients, plasma GHK levels display a moderate correlation with FEF25–75%, and patients with FAO had significantly lower GHK levels. Ovalbumin-induced mice of asthma model treated with PBS or GHK-Cu (a form of GHK with higher bioavailability) were used to evaluate the effect of exogenous GHK supplement on airway remodeling. GHK-Cu administration alleviated airway remodeling, as reflected by decreased peribronchial collagen deposition and airway mucus secretion, and suppressed epithelial-mesenchymal transition. The therapeutical effect related to decreased TGF-β1 level. Successively, network pharmacology and the validation data of experiments in vivo and vitro demonstrated that GHK-Cu decreased TGF-β1 level by increasing SIRT1 expression and activating SIRT1 deacetylation in airway epithelial cells, thereby alleviating airway remodeling. Collectively, decreased plasma GHK levels were related to FAO in asthma patients. Through the direct binding and activation of SIRT1, exogenous GHK-Cu administration alleviated airway remodeling in asthmatic mice.

Molecular Mechanism of Sirtuin 1 Inhibition by Human Immunodeficiency Virus 1 Tat Protein.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in metabolic regulation and aging-related dysfunctions. The nuclear isoform Sirt1 deacetylates histones and transcription factors and contributes, e.g., to brain and immune cell functions. Upon infection by human immunodeficiency virus 1 (HIV1), Sirt1 deacetylates the viral transactivator of transcription (Tat) protein to promote the expression of the viral genome. Tat, in turn, inhibits Sirt1, leading to the T cell hyperactivation associated with HIV infection. Here, we describe the molecular mechanism of Tat-dependent sirtuin inhibition. Using Tat-derived peptides and recombinant Tat protein, we mapped the inhibitory activity to Tat residues 34-59, comprising Tat core and basic regions and including the Sirt1 deacetylation site Lys50. Tat binds to the sirtuin catalytic core and inhibits Sirt1, Sirt2, and Sirt3 with comparable potencies. Biochemical data and crystal structures of sirtuin complexes with Tat peptides reveal that Tat exploits its intrinsically extended basic region for binding to the sirtuin substrate binding cleft through substrate-like β-strand interactions, supported by charge complementarity. Tat Lys50 is positioned in the sirtuin substrate lysine pocket, although binding and inhibition do not require prior acetylation and rely on subtle differences to the binding of regular substrates. Our results provide mechanistic insights into sirtuin regulation by Tat, improving our understanding of physiological sirtuin regulation and the role of this interaction during HIV1 infection.

Glycyl-l-histidyl-l-lysine-Cu2+ rescues cigarette smoking-induced skeletal muscle dysfunction via a sirtuin 1-dependent pathway.

Skeletal muscle dysfunction is an important co-morbidity in patients with chronic obstructive pulmonary disease (COPD) and is significantly associated with increased mortality. Oxidative stress has been demonstrated an important trigger for COPD-related skeletal muscle dysfunction. Glycine-histidine-lysine (GHK) is an active tripeptide, which is a normal component of human plasma, saliva, and urine; promotes tissue regeneration; and acts as an anti-inflammatory and antioxidant properties. The purpose of this study was to determine whether GHK is involved in COPD-related skeletal muscle dysfunction. The plasma GHK level in patients with COPD (n=9) and age-paired healthy subjects (n=11) were detected using reversed-phase high-performance liquid chromatography. The complex GHK with Cu (GHK-Cu) was used in in vitro (C2C12 myotubes) and in vivo experiments (cigarette smoking [CS]-exposure mouse model) to explore the involvement of GHK in CS-induced skeletal muscle dysfunction. Compared with healthy control, plasma GHK levels were decreased in patients with COPD (70.27±38.87ng/mL vs. 133.0±54.54ng/mL, P=0.009). And plasma GHK levels in patients with COPD were associated with pectoralis muscle area (R=0.684, P=0.042), inflammatory factor TNF-α (R=-0.696, P=0.037), and antioxidative stress factor SOD2 (R=0.721, P=0.029). GHK-Cu was found to rescue CSE-induced skeletal muscle dysfunction in C2C12 myotubes, as evidenced by increased expression of myosin heavy chain, reduced expression of MuRF1 and atrogin-1, elevated mitochondrial content, and enhanced resistance to oxidative stress. In CS-induced muscle dysfunction C57BL/6 mice, GHK-Cu treatment (0.2 and 2mg/kg) reduces CS-induced muscle mass loss (skeletal muscle weight (1.19±0.09% vs. 1.29±0.06%, 1.40±0.05%; P<0.05) and muscle cross-sectional area elevated (1055±552.4μm2 vs. 1797±620.9μm2 , 2252±534.0μm2 ; P<0.001), and also rescues CS-induced muscle weakness, indicated by improved grip strength (175.5±36.15g vs. 257.6±37.98g, 339.1±72.22g; P<0.01). Mechanistically, GHK-Cu directly binds and activates SIRT1(the binding energy was -6.1kcal/mol). Through activating SIRT1 deacetylation, GHK-Cu inhibits FoxO3a transcriptional activity to reduce protein degradation, deacetylates Nrf2 and contribute to its action of reducing oxidative stress by generation of anti-oxidant enzymes, increases PGC-1α expression to promote mitochondrial function. Finally, GHK-Cu could protect mice against CS-induced skeletal muscle dysfunction via SIRT1. Plasma glycyl-l-histidyl-l-lysine level in patients with chronic obstructive pulmonary disease was significantly decreased and was significantly associated with skeletal muscle mass. Exogenous administration of glycyl-l-histidyl-l-lysine-Cu2+ could protect against cigarette smoking-induced skeletal muscle dysfunction via sirtuin 1.

Astaxanthin attenuates cigarette smoking-induced oxidative stress and inflammation in a sirtuin 1-dependent manner

Oxidative stress and chronic inflammation play key roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). Astaxanthin (AXT) is a keto-carotenoid with a variety of biological functions, including antioxidant and anti-inflammatory effects This study aimed to explore the protective role and underlying mechanism of AXT in the pathogenesis of COPD. In this study, we found AXT alleviated pulmonary emphysema in a CS-exposed mouse model and regulated the expression of MMP-9/TIMP-1. And, AXT attenuates CSE-induced small airway fibrosis. Meanwhile, AXT inhibited Nrf2-modulated oxidative stress and the p65 NF-κB-regulated inflammatory pathway in both the mouse model and CSE-treated HBE cells. Mechanistically, AXT could directly bind to SIRT1 (the binding energy of the complex was −8.8 kcal/mol) and regulate the deacetylation activity of SIRT1. Finally, by activating SIRT1 deacetylation, AXT deacetylated Nrf2 and contributed to its action of reducing oxidative stress by generating antioxidant enzymes, and inhibiting p65 NF-κB transcriptional activity to suppress the inflammatory response. Our results show that treatment with AXT significantly reverses the oxidative stress and inflammation induced by cigarette smoke both in vivo and in vitro in a sirtuin 1-dependent manner.

Berberine alleviates non-alcoholic hepatic steatosis partially by promoting SIRT1 deacetylation of CPT1A in mice.

Berberine effectively alleviates non-alcoholic fatty liver disease (NAFLD). Nevertheless, the mechanism is incompletely comprehended. It has been reported that SIRT1 mediates lipid metabolism in liver and berberine promotes the expression of SIRT1 in hepatocytes. We hypothesized that SIRT1 mediated the effect of berberine on NAFLD. The effects of berberine on NAFLD were evaluated in C57BL/6J mice fed a high-fat diet (HFD) and in mouse primary hepatocytes and cell lines exposed to palmitate. The change of fatty acid oxidation (FAO) and the activity of CPT1A were observed in HepG2 cells. Quantitative real-time polymerase chain reaction and Western blot were employed to observe the expression of SIRT1 and lipid metabolism-related molecules. The interaction between SIRT1 and CPT1A was investigated by using co-immunoprecipitation assay in HEK293T cells. Berberine treatment attenuated hepatic steatosis, reduced triglyceride (190.1 ± 11.2 μmol/g liver vs 113.6 ± 7.6 μmol/g liver, P < 0.001) and cholesterol (11.3 ± 2.5 μmol/g liver vs 6.3 ± 0.4 μmol/g liver, P < 0.001) concentration in the liver, and improved lipid and glucose metabolism disorders compared with the HFD group. The expression of SIRT1 was reduced in the liver of NAFLD patients and mouse models. Berberine increased the expression of SIRT1 and promoted the protein level of CPT1A and its activity in HepG2 cells. SIRT1 overexpression mimicked the effect of berberine on reducing triglyceride levels in HepG2 cells, whereas SIRT1 knock-down attenuated the effect of berberine. Mechanistically, berberine increased the expression of SIRT1. SIRT1 deacetylated CPT1A at the Lys675 site, which suppressed its ubiquitin-dependent degradation, thereby promoting FAO and alleviating non-alcoholic liver steatosis. Berberine promoted SIRT1 deacetylation of CPT1A at the Lys675 site, which reduced the ubiquitin-dependent degradation of CPT1A and ameliorated non-alcoholic liver steatosis.

SAMHD1 deacetylation by SIRT1 promotes DNA end resection by facilitating DNA binding at double-strand breaks

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) has a dNTPase-independent function in promoting DNA end resection to facilitate DNA double-strand break (DSB) repair by homologous recombination (HR); however, it is not known if upstream signaling events govern this activity. Here, we show that SAMHD1 is deacetylated by the SIRT1 sirtuin deacetylase, facilitating its binding with ssDNA at DSBs, to promote DNA end resection and HR. SIRT1 complexes with and deacetylates SAMHD1 at conserved lysine 354 (K354) specifically in response to DSBs. K354 deacetylation by SIRT1 promotes DNA end resection and HR but not SAMHD1 tetramerization or dNTPase activity. Mechanistically, K354 deacetylation by SIRT1 promotes SAMHD1 recruitment to DSBs and binding to ssDNA at DSBs, which in turn facilitates CtIP ssDNA binding, leading to promotion of genome integrity. These findings define a mechanism governing the dNTPase-independent resection function of SAMHD1 by SIRT1 deacetylation in promoting HR and genome stability.

A Therapeutically Targetable NOTCH1-SIRT1-KAT7 Axis in T-cell Leukemia.

We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1.

Sirtuin1 attenuates acute liver failure by reducing reactive oxygen species via hypoxia inducible factor 1α

BACKGROUNDThe occurrence and development of acute liver failure (ALF) is closely related to a series of inflammatory reactions, such as the production of reactive oxygen species (ROS). Hypoxia inducible factor 1α (HIF-1α) is a key factor that regulates oxygen homeostasis and redox, and the stability of HIF-1α is related to the ROS level regulated by Sirtuin (Sirt) family. The activation of Sirt1 will lead to a powerful antioxidant defense system and therapeutic effects in liver disease. However, little is known about the relationship between HIF-1α and Sirt1 in the process of ALF and the molecular mechanism.AIMTo investigate whether HIF-1α may be a target of Sirt1 deacetylation and what the effects on ALF are.METHODSMice were administrated lipopolysaccharide (LPS)/D-gal and exposed to hypoxic conditions as animal model, and resveratrol was used as an activator of Sirt1. The cellular model was established with L02 cells stimulated by LPS. N-acetyl-L-cysteine was used to remove ROS, and the expression of Sirt1 was inhibited by nicotinamide. Western blotting was used to detect Sirt1 and HIF-1α activity and related protein expression. The possible signaling pathways involved were analyzed by immunofluorescent staining, co-immunoprecipitation, dihydroethidium staining, and Western blotting.RESULTSCompared with mice stimulated with LPS alone, the expression of Sirt1 decreased, the level of HIF-1α acetylation increased in hypoxic mice, and the levels of carbonic anhydrase 9 and Bcl-2-adenovirus E1B interacting protein 3 increased significantly, which was regulated by HIF-1α, indicating an increase of HIF-1α activity. Under hypoxia, the down-regulation of Sirt1 activated and acetylated HIF-1α in L02 cells. The inhibition of Sirt1 significantly aggravated this effect and the massive production of ROS. The regulation of ROS was partly through peroxisome proliferator-activated receptor alpha or AMP-activated protein kinase. Resveratrol, a Sirt1 activator, effectively relieved ALF aggravated by hypoxia, the production of ROS, and cell apoptosis. It also induced the deacetylation of HIF-1α and inhibited the activity of HIF-1α.CONCLUSIONSirt1 may have a protective effect on ALF by inducing HIF-1α deacetylation to reduce ROS.

Dissecting the Mechanism of Allosteric Regulation of SIRT1

The enzyme SIRT1 is a lysine deacetylase that plays a part in various pathways in the body, including insulin secretion and neurodegeneration, rendering it as a potential drug target for common diseases such as Type II diabetes and Alzheimer's disease. The activity of SIRT1 can be modulated allosterically by Sirtuin Activating Compounds (STACs), such as resveratrol, which bind to the STAC Binding Domain (SBD) of SIRT1 in the N‐terminus. End‐point activity assays have shown that resveratrol can have different effects on SIRT1 activity depending on the acetylated peptide substrate used, including an increase, decrease, or no change in deacetylation activity. The goal of our project is to dissect the activity of SIRT1 with and without resveratrol against different acetylated peptide substrates. Rather than only examining the overall catalytic efficiency, our goal is to assess the effect of resveratrol on the catalytic rate (kcat) and substrate recognition (KM) of SIRT1 against different acetylated peptide substrates. Upon addition of resveratrol, a large change in the KM of SIRT1 with a select peptide would imply that resveratrol had an effect on the substrate recognition of the enzyme, whereas a large change in the kcat may indicate that resveratrol caused an alteration in the catalytic rate of the enzyme for that specific peptide. We hypothesize that resveratrol allosterically alters SIRT1 activity by changing the conformation of the SBD region relative to the catalytic core, which would likely result in a large change in the KM, such as a two‐fold change in KM or higher, indicating a change in the efficiency of SIRT1 substrate recognition. To test this hypothesis, enzyme‐coupled assays involving SIRT1 acting on the peptide substrates Ac‐p300, Ac‐H4, and Ac‐p53 with and without resveratrol have been performed. Previous end‐point assay studies have shown that resveratrol decreases SIRT1 deacetylation activity against Ac‐p300, but increases activity against Ac‐H4, and does not affect SIRT1 activity against Ac‐p53. The Michaelis‐Menten parameters for SIRT1 activity with and without resveratrol were compared, allowing observation of the effects of resveratrol on SIRT1’s KMand kcat values. Understanding how resveratrol affects SIRT1 deacetylation activity will provide further insight into the development of drugs that can alter SIRT1 activity in a specific way to mitigate the effects of harmful diseases in humans.

D-allulose ameliorates adiposity through the AMPK-SIRT1-PGC-1α pathway in HFD-induced SD rats.

BackgroundAdiposity is a major health-risk factor, and D-allulose has beneficial effects on adiposity-related metabolic disturbances. However, the modes of action underlying anti-hyperglycemic and hypolipidemic activity are partly understood.ObjectiveThis study investigated the in vivo and in vitro effects of D-allulose involved in adipogenesis and activation of the AMPK/SIRT1/PGC-1α pathway in high-fat diet (HFD)-fed rats.DesignIn this study, 8-week-old male SD (Sprague Dawley) rats were divided into five groups (n = 8/group), (1) Control (chow diet, 3.5%); (2) 60% HFD; (3) 60% HFD supplemented with allulose powder (AP) at 0.4 g/kg; (4) 60% HFD supplemented with allulose liquid (AL) at 0.4 g/kg; (5) 60% HFD supplemented with glucose (AL) at 0.4 g/kg. All the group received the product through oral gavage for 6 weeks. Control and HFD groups were gavaged with double-distilled water.ResultsRats receiving AP and AL showed reduced body weight gain and fat accumulation in HFD-fed rats. Also, supplementation of AL/AP regulated the cytokine secretion and recovered biochemical parameters to alleviate metabolic dysfunction and hepatic injury. Additionally, AL/AP administration improved adipocyte differentiation via regulation of the PPARγ and C/EBPα signaling pathway and adipogenesis-related genes owing to the combined effect of the AMPK/SIRT1 pathway. Furthermore, AL/AP treatment mediated PGC-1α expression triggering mitochondrial genesis via activating the AMPK phosphorylation and SIRT1 deacetylation activity in adipose tissue.ConclusionThe anti-adiposity activity of D-allulose is observed on a marked alleviation in adipogenesis and AMPK/SIRT1/PGC-1α deacetylation in the adipose tissue of HFD-fed rat.

Quercetin alleviates high-fat diet-induced inflammation in brown adipose tissue

Inflammation in brown adipose tissue (BAT) causes BAT to lose its ability to regulate energy metabolism, which exacerbates obesity development. This study investigated the anti-inflammatory and antioxidant effects of quercetin on BAT in high-fat diet (HFD)-induced obese mice. Quercetin reduced proinflammatory and macrophage genes and increased antioxidant genes in the BAT of HFD-fed mice and in TNFα-induced brown adipocytes. In addition to increasing silent mating type information regulation 2 homolog 1 (SIRT1) levels, quercetin suppressed nuclear translocation and increased deacetylation of the nuclear factor kappa B (NFκB) subunit p65. The effects of quercetin on SIRT1 and NFκB p65 deacetylation disappeared in SIRT1-knockdown brown adipocytes. MicroRNAs that negatively regulate SIRT1 were downregulated in the BAT of HFDQ-fed mice. Taken together, these results suggest that quercetin improves obesity-induced inflammation in BAT by suppressing the NFκB pathway through activation of SIRT1.

Inhibition of Sirt-1 Alleviates Acute and Chronic Graft-Versus-Host-Disease While Preserving Graft-Versus-Leukemia Effect

Sirtuin-1 (Sirt-1), NAD-dependent histone deacetylase, plays a crucial role in various biological processes including cellular senescence, metabolism and inflammatory responses. Sirt-1 deacetylation regulates different transcription factors that are important for modulating immune responses. An early study showed that Sirt-1 is required to maintain T cell tolerance, but more studies provided evidences that Sirt-1 promotes Th17 responses while inhibiting function of Foxp3+regulatory T cells (Tregs). Thus, the question still remains as how Sirt-1 regulates T-cell immunity versus tolerance.Graft-versus-host-disease (GVHD) remains one of the major complications after allogeneic bone marrow transplantation (allo-BMT). In the current study, we address the role of Sirt-1 in T-cell responses to alloantigens and in the induction of GVHD taking advantages of Sirt-1 conditional knock-out (Sirt-1fl/flCD4Cre) mice on C57BL/6 (B6) background. Using MHC mismatched (B6 to BALB/c) and MHC matched (B6 to BALB.b) murine BMT models, we observed that CD25-depleted Sirt-1-/- T cells significantly reduced their ability to induce acute GVHD compared to WT T cells, as the survival and clinical scores of the mice received Sirt-1-/- T cells were remarkably improved (Fig.1A-B). Importantly, more induced Tregs (iTregs, CD4+Foxp3+) were generated from Sirt-1-/- donor T cells than from WT counterparts in recipient spleen and liver 14 days after allo-BMT. Furthermore, Sirt-1-/- donor T cells significantly reduced IFN-γ production by both CD4 and CD8 T cells in vivo . Consistently, the levels of IL-4 and IL-10 cytokines in mice serum were significantly increased in mice transplanted with Sirt-1-/- T cells in parallel with the reduction of pro-inflammatory cytokines, IFN-γ and TNF-α. Altogether, the data suggests that Sirt-1 positively regulates T cell activation, proliferation and essential for GVHD induction.We extend our study further into chronic GVHD pathophysiology, which is mainly driven by pathogenic CD4 T and B cells. We observed that splenocytes from Sirt1-/- donor remarkably reduced the clinical manifestation of cGVHD in recipient mice. Mechanistic studies revealed the significant increase of splenic B cell reconstitution with down regulation of B cell activation markers (CD86 and MHCII). Importantly, the frequency of follicular regulatory T cells (Tfr) was dramatically increased, which is known to play a critical role in suppressing T follicular helper (Tfh) and plasma B cell differentiation. Consistently, we observed a significant reduction of plasma B cell frequency in spleen of Sirt-1-/- group. Finally, we evaluated Sirt-1 as a therapeutic target by testing a specific inhibitor (Ex-527) in viv o. Using a haploidentical (B6 to BDF1) BMT model with P815 mastocytoma, we found that not only could Ex-527 significantly alleviate GVHD severity but also maintaining graft-versus-leukemia (GVL) activity against P815 tumor (Fig.1C-D). In conclusion, Sirt-1 is a promising therapeutic target for the control of aGVHD and cGVHD pathogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dissecting the Effects of Resveratrol on SIRT1 Activity Against Different Peptide Substrates

The enzyme SIRT1 is a member of a class of Sirtuins, which are NAD+-dependent protein deacylases that are involved in the metabolic and aging processes of cells. The activity of SIRT1 can be allosterically modulated by compounds known as Sirtuin Activating Compounds (STACs), such as resveratrol, which bind to the STAC Binding Domain (SBD) of SIRT1 in the N-terminus. End-point activity assays have shown that resveratrol can have different effects on SIRT1 activity depending on the acetylated peptide substrate used, including an increase, decrease, or no change in deacetylation activity. The goal of our project is to assess the effect of resveratrol on the catalytic rate (kcat) and substrate recognition (KM) of SIRT1 against different acetylated peptide substrates, rather than only examining the overall catalytic efficiency. A large change in the KM of SIRT1 with a select peptide would suggest that resveratrol had an effect on the substrate recognition of the enzyme, whereas a change in the kcat would imply that resveratrol caused an alteration in the catalytic rate of the enzyme for that specific peptide. We hypothesize that resveratrol allosterically affects SIRT1 activity by changing the conformation of the SBD region, which would likely result in a large change in the KM, such as a two-fold change in KM or higher. To test this hypothesis, enzyme-coupled assays involving SIRT1 acting on the peptide substrates Ac-CSNK, Ac-p300, Ac-H4, and Ac-p53 with and without resveratrol have been performed. Previous end-point assay studies have shown that resveratrol decreases SIRT1 deacetylation activity against Ac-CSNK as well as Ac-p300, but increases SIRT1 deacetylation activity against Ac-H4, and produces no change in deacetylation activity against the substrate Ac-p53. The Michaelis-Menten parameters for SIRT1 activity with and without resveratrol were compared, allowing observation of the effects of resveratrol on SIRT1's KM and kcat values. Understanding how resveratrol affects SIRT1 deacetylation activity will provide further insight into the development of drugs that can alter SIRT1 activity in a specific way to mitigate the effects of harmful diseases in humans.

Sirt1 deacetylates and stabilizes p62 to promote hepato-carcinogenesis

p62/SQSTM1 is frequently up-regulated in many cancers including hepatocellular carcinoma. Highly expressed p62 promotes hepato-carcinogenesis by activating many signaling pathways including Nrf2, mTORC1, and NFκB signaling. However, the underlying mechanism for p62 up-regulation in hepatocellular carcinoma remains largely unclear. Herein, we confirmed that p62 was up-regulated in hepatocellular carcinoma and its higher expression was associated with shorter overall survival in patients. The knockdown of p62 in hepatocellular carcinoma cells decreased cell growth in vitro and in vivo. Intriguingly, p62 protein stability could be reduced by its acetylation at lysine 295, which was regulated by deacetylase Sirt1 and acetyltransferase GCN5. Acetylated p62 increased its association with the E3 ligase Keap1, which facilitated its poly-ubiquitination-dependent proteasomal degradation. Moreover, Sirt1 was up-regulated to deacetylate and stabilize p62 in hepatocellular carcinoma. Additionally, Hepatocyte Sirt1 conditional knockout mice developed much fewer liver tumors after Diethynitrosamine treatment, which could be reversed by the re-introduction of exogenous p62. Taken together, Sirt1 deacetylates p62 at lysine 295 to disturb Keap1-mediated p62 poly-ubiquitination, thus up-regulating p62 expression to promote hepato-carcinogenesis. Therefore, targeting Sirt1 or p62 is a reasonable strategy for the treatment of hepatocellular carcinoma.

TP53 mutation influences the efficacy of treatment of colorectal cancer cell lines with a combination of sirtuin inhibitors and chemotherapeutic agents.

Chemoresistance of colorectal cancer (CRC) leads to tumor recurrence and metastasis and new strategies are urgently needed to improve the outcomes of conventional chemotherapy. Sirtuin (SIRT) inhibitors prevent tumor cell growth by increasing the levels of acetylated histones and non-histones, as well as disrupting survival-related pathways. The aim of the present study was to determine the effect of SIRT inhibitors on CRC chemotherapy. The CompuSyn software program was used to evaluate the synergistic or antagonistic effects of various drugs, and the status of the protein deacetylation regulatory genes in microarray datasets were analyzed using bioinformatics. In HCT116 cells expressing wild-type (wt) TP53, SIRT inhibitors were found to act antagonistically with multiple chemotherapeutic agents (cisplatin, 5-fluorouracil, oxaliplatin, gefitinib, LY294002 and metformin), and decreased the anti-tumor effects of these agents. By contrast, SIRT inhibitors sensitized TP53-mutant (mut) SW620 cells to various chemotherapeutic drugs. Bioinformatics analysis indicated that SIRT1 and protein deacetylation related genes were highly expressed in TP53wt CRC cells when compared to TP53mut cells. Therefore, it was hypothesized that the likely mechanism underlying the antagonistic effect of SIRT inhibitors on TP53wt CRC cells was a reduction in the level of stable p53 protein. The present results indicated that divergent TP53 status may translate to a different chemosensitivity profile, and suggested that a combination therapy of SIRT inhibitors and first-line chemotherapeutic drugs may be beneficial for the treatment of patients with TP53mut CRC.

Proteasome inhibitor induced SIRT1 deacetylates GLI2 to enhance hedgehog signaling activity and drug resistance in multiple myeloma.

Multiple myeloma (MM) is still incurable despite the successful application of proteasome inhibitors in clinic. Bortezomib represents the most common chemotherapy for MM, whereas acquired drug resistance and eventually developed relapse remain the major obstruction. In the current study, we established bortezomib-resistant myeloma cell lines and screened gene expression profiles using single cell RNA-sequencing. Resistant MM cells exhibited increased clonogenic potential, specific metabolic, and epigenetic signatures, along with the self-renewal signaling characteristic of MM stem-like cells. Aberrant activation of hedgehog (Hh) signaling was correlated with drug resistance and stem cell-like transcriptional program. The key transcriptional factor GLI2 of the Hh pathway was restricted in the high acetylation and low ubiquitination states in bortezomib-resistant myeloma cells. Further investigation revealed that SIRT1 deacetylates and stabilizes GLI2 protein at lysine 757 and consequentially activates the Hh signaling, and itself serves as a direct target of Hh signaling to format a positive regulating loop. Using combination screening with an epigenetic compound library, we identified the SIRT1 specific inhibitor S1541 and S2804 had very obvious synergetic antimyeloma effect. Sirt1 inhibition could partially impeded the Hh pathway and conferred bortezomib sensitivity in vitro and in vivo. Notably, elevated SIRT1 level was also a prominent hallmark for the resistant myeloma cells, and this expression pattern was confirmed in myeloma patients, but independent of RAS/RAF mutations. Clinically, SIRT1 expression in patients with complete response was suppressed but elevated in relapsed patients, and retrospective analysis showed patients with higher SIRT1 expression had poorer outcomes. In conclusion, the cooperation of SIRT1 and Hh is an important mechanism of drug resistance in myeloma, and therapeutics combining SIRT1 inhibitors will sensitize myeloma cells to proteasome inhibitors.

Correction for Morgan et al., "Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation".

Volume 10, no. 2, e00263-19, 2019, https://doi.org/10.1128/mBio.00263-19. It has come to our attention that in this study, the isogenic TERT immortalized keratinocyte lines that were used in Fig. 6 are not NOK but are in fact N/Tert-1. We apologize for this honest error that has been corrected …