RESUMO
Cuproptosis is characterized by lipoylated protein aggregation and loss of iron-sulfur (Fe-S) proteins, which are crucial for a wide range of important cellular functions, including DNA replication and damage repair. Sirt2 and sirt4 are lipoamidases that remove the lipoyl moiety from lipoylated proteins using nicotinamide adenine dinucleotide (NAD+) as a cofactor. However, to date, it is not clear whether nicotinamide mononucleotide (NMN), a precursor of NAD+, affects cellular sensitivity to cuproptosis. Therefore, in the current study, cuproptosis was induced by the copper (Cu) ionophore elesclomol (Es) in HeLa cells. It was also found that Es/Cu treatment increased cellular DNA damage level. On the other hand, NMN treatment partially rescued cuproptosis in a dose-dependent manner, as well as reduced cellular DNA damage level. In addition, NMN upregulated the expression of Fe-S protein POLD1, without affecting the aggregation of lipoylated proteins. Mechanistic study revealed that NMN increased the expression of sirt2 and cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) level. Overexpression of sirt2 and sirt4 did not change the aggregation of lipoylated proteins, however, sirt2, but not sirt4, increased cellular NADPH levels and partially rescued cuproptosis. Inhibition of NAD+ kinase (NADK), which is responsible for generating NADPH, abolished the rescuing function of NMN and sirt2 for Es/Cu induced cell death. Taken together, our results suggested that DNA damage is a characteristic feature of cuproptosis. NMN can partially rescue cuproptosis by upregulating sirt2, increase intracellular NADPH content and maintain the level of Fe-S proteins, independent of the lipoamidase activity of sirt2.
Assuntos
Dano ao DNA , NADP , Mononucleotídeo de Nicotinamida , Sirtuína 2 , Regulação para Cima , Humanos , Sirtuína 2/metabolismo , Sirtuína 2/genética , Células HeLa , NADP/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Mononucleotídeo de Nicotinamida/farmacologia , Mononucleotídeo de Nicotinamida/metabolismo , Cobre/farmacologia , Cobre/metabolismo , Sirtuínas/metabolismoRESUMO
Rhein, a component derived from rhubarb, has been proven to possess anti-inflammatory properties. Here, we show that rhein mitigates obesity by promoting adipose tissue thermogenesis in diet-induced obese mice. We construct a macrophage-adipocyte co-culture system and demonstrate that rhein promotes adipocyte thermogenesis through inhibiting NLRP3 inflammasome activation in macrophages. Moreover, clues from acetylome analysis identify SIRT2 as a potential drug target of rhein. We further verify that rhein directly interacts with SIRT2 and inhibits NLRP3 inflammasome activation in a SIRT2-dependent way. Myeloid knockdown of SIRT2 abrogates adipose tissue thermogenesis and metabolic benefits in obese mice induced by rhein. Together, our findings elucidate that rhein inhibits NLRP3 inflammasome activation in macrophages by regulating SIRT2, and thus promotes white adipose tissue thermogenesis during obesity. These findings uncover the molecular mechanism underlying the anti-inflammatory and anti-obesity effects of rhein, and suggest that rhein may become a potential drug for treating obesity.
Assuntos
Antraquinonas , Macrófagos , Obesidade , Sirtuína 2 , Termogênese , Animais , Masculino , Camundongos , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Antraquinonas/farmacologia , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Obesidade/metabolismo , Obesidade/tratamento farmacológico , Sirtuína 2/metabolismo , Sirtuína 2/genética , Termogênese/efeitos dos fármacosRESUMO
Introduction: Chronic obstructive pulmonary disease (COPD) is a major global cause of mortality with limited effective treatments. Sirtuins (SIRT) are histone deacetylases that are involved in the regulation of redox and inflammatory homeostasis. Hence, the present study aims to investigate the role of SIRT-2 in modulating inflammation in a murine model of COPD. Methods: COPD in mice was established by cigarette smoke (CS) exposure for 60 days, and AK-7 was used as the specific SIRT-2 inhibitor. AK-7 (100 µg/kg and 200 µg/kg body weight) was administered intranasally 1 h before CS exposure. Molecular docking was performed to analyze the binding affinity of different inflammatory proteins with AK-7. Results: Immune cell analysis showed a significantly increased number of macrophages (F4/80), neutrophils (Gr-1), and lymphocytes (CD4+, CD8+, and CD19+) in the COPD, group and their population was declined by AK-7 administration. Total reactive oxygen species, total inducible nitric oxide synthase, inflammatory mediators such as neutrophil elastase, C-reactive protein, histamine, and cytokines as IL4, IL-6, IL-17, and TNF-α were elevated in COPD and declined in the AK-7 group. However, IL-10 showed reverse results representing anti-inflammatory potency. AK-7 administration by inhibiting SIRT-2 decreased the expression of p-NF-κB, p-P38, p-Erk, and p-JNK and increased the expression of Nrf-2. Furthermore, AK-7 also declined the lung injury by inhibiting inflammation, parenchymal destruction, emphysema, collagen, club cells, and Kohn pores. AK-7 also showed good binding affinity with inflammatory proteins. Discussion: The current study reveals that SIRT-2 inhibition mitigates COPD severity and enhances pulmonary therapeutic interventions, suggesting AK-7 as a potential therapeutic molecule for COPD medication development.
Assuntos
NF-kappa B , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica , Sirtuína 2 , Animais , Sirtuína 2/metabolismo , Sirtuína 2/antagonistas & inibidores , Camundongos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Estresse Oxidativo/efeitos dos fármacos , NF-kappa B/metabolismo , Masculino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pulmão/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/efeitos dos fármacos , Modelos Animais de Doenças , Transdução de Sinais , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , CarbazóisRESUMO
Type 2 diabetes mellitus (T2DM) increases the risk of non-alcoholic fatty liver disease (NAFLD) progression to advanced stages, especially upon high-fat diet (HFD). HFD-induced hepatic fibrosis can be marked by oxidative stress, inflammation, and activation of hepatic stellate cells. Sirtuin 1/2 (SIRT1/2), NAD-dependent class III histone deacetylases, are involved in attenuation of fibrosis. In our conducted research, TGF-ß1-activated LX-2 cells, free fatty acid (FFA)-treated simultaneous co-culture (SCC) cells, and HFD-induced hepatic fibrosis in Zucker diabetic fatty (ZDF) rats, a widely used animal model in the study of metabolic syndromes, were used to evaluate the protective effect of Tenovin-1, a SIRT1/2 inhibitor. ZDF rats were divided into chow diet, HFD, and HFD + Tenovin-1 groups. Tenovin-1 reduced hepatic damage, inhibited inflammatory cell infiltration, micro/ macro-vesicular steatosis and prevented collagen deposition HFD-fed rats. Tenovin-1 reduced serum biochemical parameters, triglyceride (TG) and malondialdehyde (MDA) levels but increased glutathione, catalase, and superoxide dismutase levels. Tenovin-1 mitigated proinflammatory cytokines IL-6, IL-1ß, TNFα and fibrosis biomarkers in HFD rats, TGF-ß1-activated LX-2 and FFA treated SCC cells. Additionally, Tenovin-1 suppressed SIRT1/2 expression and inhibited JNK-1 and STAT3 phosphorylation in HFD rats and FFA-treated SCC cells. In conclusion, Tenovin-1 attenuates hepatic fibrosis by stimulating antioxidants and inhibiting inflammatory cytokines under HFD conditions in diabetic rats.
Assuntos
Dieta Hiperlipídica , Cirrose Hepática , Ratos Zucker , Sirtuína 1 , Sirtuína 2 , Animais , Dieta Hiperlipídica/efeitos adversos , Ratos , Cirrose Hepática/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/induzido quimicamente , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Sirtuína 2/antagonistas & inibidores , Masculino , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacosAssuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Sirtuína 1 , Sirtuína 2 , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Sirtuína 1/metabolismo , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/metabolismo , Metabolismo Energético , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , CamundongosRESUMO
This study explores the role of SIRT2 in regulating autophagy and its interaction with AMPK in the context of acute liver failure (ALF). This study investigated the effects of SIRT2 and AMPK on autophagy in ALF mice and TAA-induced AML12 cells. The results revealed that the liver tissue in ALF model group had a lot of inflammatory cell infiltration and hepatocytes necrosis, which were reduced by SIRT2 inhibitor AGK2. In comparison to normal group, the level of SIRT2, P62, MDA, TOS in TAA group were significantly increased, which were decreased in AGK2 treatment. Compared with normal group, the expression of P-PRKAA1, Becilin1 and LC3B-II was decreased in TAA group. However, AGK2 enhanced the expression of P-PRKAA1, Becilin1 and LC3B-II in model group. Overexpression of SIRT2 in AML12 cell resulted in decreased P-PRKAA1, Becilin1 and LC3B-II level, enhanced the level of SIRT2, P62, MDA, TOS. Overexpression of PRKAA1 in AML12 cell resulted in decreased SIRT2, TOS and MDA level and triggered more autophagy. In conclusion, the data suggested the link between AMPK and SIRT2, and reveals the important role of AMPK and SIRT2 in autophagy on acute liver failure.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Falência Hepática Aguda , Sirtuína 2 , Sirtuína 2/metabolismo , Sirtuína 2/genética , Animais , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Falência Hepática Aguda/induzido quimicamente , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Masculino , Hepatócitos/metabolismo , Hepatócitos/patologia , Transdução de Sinais , Modelos Animais de Doenças , Linhagem Celular , Tioacetamida/toxicidade , Fígado/metabolismo , Fígado/patologia , Furanos , QuinolinasRESUMO
Accumulating evidence strongly support the key role of NLRP3-mediated pyroptosis in the pathogenesis and progression of vascular endothelial dysfunction associated with diabetes mellitus. Various studies have demonstrated that the activation or upregulation of Silent Information Regulation 2 homolog 2 (SIRT2) exerts inhibitory effect on the expression of NLRP3. Although 1,8-cineole has been found to protect against endothelial dysfunction and cardiovascular diseases, its role and mechanism in diabetic angiopathy remain unknown. Therefore, the aim of this study was to investigate the ameliorative effect of 1,8-cineole through SIRT2 on pyroptosis associated with diabetic angiopathy in human umbilical vein endothelial cells (HUVECs) and to elucidate the underlying mechanism. The findings revealed that 1,8-cineole exhibited a protective effect against vascular injury and ameliorated pathological alterations in the thoracic aorta of diabetic mice. Moreover, it effectively mitigated pyroptosis induced by palmitic acid-high glucose (PA-HG) in HUVECs. Treatment with 1,8-cineole effectively restored the reduced levels of SIRT2 and suppressed the elevated expression of pyroptosis-associated proteins. Additionally, our findings demonstrated the occurrence of NLRP3 deacetylation and the physical interaction between NLRP3 and SIRT2. The SIRT2 inhibitor AGK2 and siRNA-SIRT2 effectively attenuated the effect of 1,8-cineole on NLRP3 deacetylation in HUVECs and compromised its inhibitory effect against pyroptosis in HUVECs. However, overexpression of SIRT2 inhibited PA-HG-induced pyroptosis in HUVECs. 1,8-Cineole inhibited the deacetylation of NLRP3 by regulating SIRT2, thereby reducing pyroptosis in HUVECs. In conclusion, our findings suggest that PA-HG-induced pyroptosis in HUVECs plays a crucial role in the development of diabetic angiopathy, which can be mitigated by 1,8-cineole.
Assuntos
Diabetes Mellitus Experimental , Eucaliptol , Células Endoteliais da Veia Umbilical Humana , Inflamassomos , Piroptose , Animais , Humanos , Masculino , Camundongos , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Angiopatias Diabéticas/tratamento farmacológico , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/prevenção & controle , Angiopatias Diabéticas/patologia , Eucaliptol/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/efeitos dos fármacos , Sirtuína 2/metabolismo , Sirtuína 2/antagonistas & inibidoresRESUMO
Asthma is a chronic pulmonary disease with the worldwide prevalence. The structural alterations of airway walls, termed as "airway remodeling", are documented as the core contributor to the airway dysfunction during chronic asthma. Forkhead box transcription factor FOXK2 is a critical regulator of glycolysis, a metabolic reprogramming pathway linked to pulmonary fibrosis. However, the role of FOXK2 in asthma waits further explored. In this study, the chronic asthmatic mice were induced via ovalbumin (OVA) sensitization and repetitive OVA challenge. FOXK2 was upregulated in the lungs of OVA mice and downregulated after adenovirus-mediated FOXK2 silencing. The lung inflammation, peribronchial collagen deposition, and glycolysis in OVA mice were obviously attenuated after FOXK2 knockdown. Besides, the expressions of FOXK2 and SIRT2 in human bronchial epithelial cells (BEAS-2B) were increasingly upregulated upon TGF-ß1 stimulation and downregulated after FOXK2 knockdown. Moreover, the functional loss of FOXK2 remarkably suppressed TGF-ß1-induced epithelial-mesenchymal transition (EMT) and glycolysis in BEAS-2B cells, as manifested by the altered expressions of EMT markers and glycolysis enzymes. The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) inhibited the EMT in TGF-ß1-induced cells, making glycolysis a driver of EMT. The binding of FOXK2 to SIRT2 was validated, and SIRT2 overexpression blocked the FOXK2 knockdown-mediated inhibition of EMT and glycolysis in TGF-ß1-treated cells, which suggests that FOXK2 regulates EMT and glycolysis in TGF-ß1-treated cells in a SIRT2-dependnet manner. Collectively, this study highlights the protective effect of FOXK2 knockdown on airway remodeling during chronic asthma.
Assuntos
Remodelação das Vias Aéreas , Asma , Fatores de Transcrição Forkhead , Glicólise , Sirtuína 2 , Asma/metabolismo , Asma/patologia , Animais , Sirtuína 2/metabolismo , Sirtuína 2/genética , Camundongos , Remodelação das Vias Aéreas/fisiologia , Humanos , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Transição Epitelial-Mesenquimal , Camundongos Endogâmicos BALB C , Feminino , Fator de Crescimento Transformador beta1/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Linhagem CelularRESUMO
NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2.
Assuntos
Fígado , Piruvato Quinase , Sirtuína 2 , Estresse Fisiológico , Ubiquitinação , Animais , Humanos , Masculino , Camundongos , Acetilação , Glucose/metabolismo , Glicólise , Resistência à Insulina , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Piruvato Quinase/metabolismo , Sirtuína 2/metabolismoRESUMO
As a sirtuin (SIR2) family protein, defense-associated sirtuin2 (DSR2) has been demonstrated to participate in bacterial anti-phage resistance via depleting nicotinamide adenine dinucleotide (NAD+) of infected cells, which can be activated by tail tube protein (TTP) and inhibited by DSR anti-defense 1 (DSAD1) of diverse phages. However, the regulating mechanism remains elusive. Here, we determined the cryo-electron microscopy structure of apo DSR2, as well as the respective complex structures with TTP and DSAD1. Structural analyses and biochemical studies reveal that DSR2 forms a tetramer with a SIR2 central core and two distinct conformations. Monomeric TTP preferentially binds to the closed conformation of DSR2, inducing conformational distortions on SIR2 tetramer assembly to activate its NADase activity. DSAD1 combines with the open conformation of DSR2, directly or allosterically inhibiting TTP activation on DSR2 NAD+ hydrolysis. Our findings decipher the detailed molecule mechanisms for DSR2 NADase activity regulation and lay a foundation for in-depth understanding of the DSR2 anti-phage defense system.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , Bacteriófagos/metabolismo , NAD+ Nucleosidase/metabolismo , NAD+ Nucleosidase/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Modelos Moleculares , NAD/metabolismo , Ligação Proteica , Conformação Proteica , Sirtuína 2/metabolismo , Sirtuína 2/química , Multimerização ProteicaRESUMO
The NAD+-dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. Furthermore, the orthologue from Schistosoma mansoni (SmSirt2) has been considered for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the "Kinetobox" library as a dual inhibitor of human Sirt2 (hSirt2) and SmSirt2. Herein, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.
Assuntos
Oxidiazóis , Sirtuína 2 , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/metabolismo , Oxidiazóis/farmacologia , Oxidiazóis/química , Oxidiazóis/síntese química , Humanos , Relação Estrutura-Atividade , Simulação de Acoplamento Molecular , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/enzimologia , Movimento Celular/efeitos dos fármacosRESUMO
Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and "long" prokaryotic Ago proteins (pAgos) are well understood, those for short pAgos remain enigmatic. Here, we determine two cryoelectron microscopy structures of short pAgos in association with the NADase-domain-containing protein Sir2-APAZ from Geobacter sulfurreducens (GsSir2/Ago): the guide RNA-target DNA-loaded GsSir2/Ago quaternary complex (2.58 Å) and the dimer of the quaternary complex (2.93Å). These structures show that the nucleic acid binding causes profound conformational changes that result in disorder or partial dissociation of the Sir2 domain, suggesting that it adopts a NADase-active conformation. Subsequently, two RNA-/DNA-loaded GsSir2/Ago complexes form a dimer through their MID domains, further enhancing NADase activity through synergistic effects. The findings provide a structural basis for short-pAgo-mediated defense against invading nucleic acids.
Assuntos
Proteínas Argonautas , Proteínas Argonautas/metabolismo , Proteínas Argonautas/química , Geobacter/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Sirtuína 2/metabolismo , Multimerização Proteica , Ligação Proteica , Microscopia Crioeletrônica , Ativação Enzimática , Modelos Moleculares , Ácidos Nucleicos/metabolismoRESUMO
INTRODUCTION: The early diagnosis of kidney injury in type 2 diabetes (T2DM) is important to prevent the long-term damaging effects of kidney loss and is decisive for patient outcomes. While SIRT2 is implicated in diabetes pathogenesis, its correlation with diabetic nephropathy remains unexplored. This study was designed to evaluate the association of urine SIRT2 levels with diabetic kidney injury, as well as potential underlying mechanisms. METHODS: In T2DM patients, db/db mice, and high glucose plus palmitic acid treated HK2 cell models, ELISA, Immunoturbidimetry, Immunohistochemistry, Western blot, and Quantitative real-time polymerase chain reaction were used to detect SIRT2 levels and kidney damage. According to urinary albumin/creatinine ratio (UACR), 163 T2DM patients were divided into three groups. Spearman correlation analysis was used to investigate the relationship between urinary sirtuin2/creatinine ratio (USCR) and biomarkers of kidney injury. The influencing factors of albuminuria in T2DM patients were analyzed by logistic regression model. RESULTS: In our findings, the Macro group exhibited the highest USCR levels as UACR increased. There was a positive association between USCR and UACR, α1-microglobulin/creatinine ratio (UαCR), ß2-microglobulin/creatinine ratio (UßCR), and retinol-binding protein/creatinine ratio (URCR), with a negative correlation observed with eGFR. Logistic ordered multiclassification regression analysis, adjusting for confounding variables, confirmed that USCR remained a significant risk factor for the severity of albuminuria in T2DM patients. In the db/db mice kidney SIRT2 protein level increased significantly. Increased SIRT2 protein levels were also observed in renal tubular epithelial cells treated with high glucose plus palmitic acid. Moreover, SIRT2 promotes the expression of proinflammatory factors TNF-α and IL-6 by modulating the phosphorylation of p38 MAPK and p-JNK in renal tubular cells induced by high glucose and palmitic acid. CONCLUSION: Urinary SIRT2 is closely related to eGFR, renal tubule injury, and urinary albumin excretion in T2DM patients, which is expected to be an important indicator to comprehensively reflect renal injury.
Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Sirtuína 2 , Sirtuína 2/urina , Diabetes Mellitus Tipo 2/urina , Diabetes Mellitus Tipo 2/complicações , Animais , Humanos , Camundongos , Nefropatias Diabéticas/urina , Nefropatias Diabéticas/diagnóstico , Masculino , Pessoa de Meia-Idade , Feminino , Biomarcadores/urina , Albuminúria/urina , Creatinina/urina , Linhagem CelularRESUMO
Human sirtuin-2 (SIRT2) has emerged as an attractive drug target for a variety of diseases. The enzyme is a deacylase that can remove chemically different acyl modifications from protein lysine residues. Here, we developed a high-throughput screen based on a homogeneous time-resolved fluorescence (HTRF) binding assay to identify inhibitors of SIRT2's demyristoylase activity, which is uncommon among many ligands that only affect its deacetylase activity. From a test screen of 9600 compounds, we identified a small molecule that inhibited SIRT2's deacetylase activity (IC50 = 7 µM) as well as its demyristoylase activity (IC50 = 37 µM). The inhibitor was composed of two small fragments that independently inhibited SIRT2: a halogenated phenol fragment inhibited its deacetylase activity, and a tricyclic thiazolobenzimidazole fragment inhibited its demyristoylase activity. The high-throughput screen also detected multiple deacetylase-specific SIRT2 inhibitors.
Assuntos
Ensaios de Triagem em Larga Escala , Sirtuína 2 , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/metabolismo , Humanos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , FluorescênciaRESUMO
The mammalian cytoplasmic protein SIRT2, a class III histone deacetylase family member, possesses NAD+-dependent lysine deacetylase/deacylase activity. Dysregulation of SIRT2 has been implicated in the pathogenesis of several diseases, including neurological and metabolic disorders and cancer; thus, SIRT2 emerges as a potential therapeutic target. Herein, we identified a series of diaryl acetamides (ST61-ST90) by the structural optimization of our hit STH2, followed by enhanced SIRT2 inhibitory potency and selectivity. Among them, ST72, ST85, and ST88 selectively inhibited SIRT2 with IC50 values of 9.97, 5.74, and 8.92 µM, respectively. Finally, the entire study was accompanied by in silico prediction of binding modes of docked compounds and the stability of SIRT2-ligand complexes. We hope our findings will provide substantial information for designing selective inhibitors of SIRT2.
Assuntos
Acetamidas , Sirtuína 2 , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/química , Sirtuína 2/metabolismo , Humanos , Acetamidas/química , Acetamidas/farmacologia , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/químicaRESUMO
BACKGROUND: Sirtuins (SIRTs) are NAD+-dependent deacetylases that play various roles in numerous pathophysiological processes, holding promise as therapeutic targets worthy of further investigation. Among them, the SIRT2 subtype is closely associated with tumorigenesis and malignancies. Dysregulation of SIRT2 activation can regulate the expression levels of related genes in cancer cells, leading to tumor occurrence and metastasis. METHODS: In this study, we used computer simulations to screen for novel SIRT2 inhibitors from the FDA database, based on which 10 compounds with high docking scores and good interactions were selected for in vitro anti-pancreatic cancer metastasis testing and enzyme binding inhibition experiments. The results showed that fluvastatin sodium may possess inhibitory activity against SIRT2. Subsequently, fluvastatin sodium was subjected to molecular docking experiments with various SIRT isoforms, and the combined results from Western blotting experiments indicated its potential as a SIRT2 inhibitor. Next, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were performed, revealing the binding mode of fluvastatin sodium at the SIRT2 active site, further validating the stability and interaction of the ligand-protein complex under physiological conditions. RESULTS: Overall, this study provides a systematic virtual screening workflow for the discovery of SIRT2 activity inhibitors, identifies the potential inhibitory effect of fluvastatin sodium as a lead compound on SIRT2, and opens up a new direction for developing highly active and selectively targeted SIRT2 inhibitors.
Assuntos
Fluvastatina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Sirtuína 2 , Fluvastatina/farmacologia , Fluvastatina/química , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/química , Sirtuína 2/metabolismo , Humanos , Ligação Proteica , Domínio Catalítico , Simulação por ComputadorRESUMO
The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished. Recovery of cellular uptake could be achieved by deacetylation of the masked acetylated L17E peptide using the NAD-dependent sirtuin 2 (SirT2) deacetylase in vitro. Finally, trastuzumab-SirT2 and anti-B7H3-SirT2 antibody-enzyme conjugates were generated for the conditional and selective delivery of a cryptophycin cytotoxin by the L17E peptide. While the masked peptide still demonstrated some cytotoxicity, selective cell killing mediated by the antibody-enzyme conjugates was observed.
Assuntos
Peptídeos Penetradores de Células , Humanos , Células HeLa , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Sirtuína 2/metabolismo , Sistemas de Liberação de Medicamentos , Trastuzumab/química , Trastuzumab/farmacologiaRESUMO
The NLRP3 inflammasome functions as an inflammatory driver, but its relationship with lipid metabolic changes in early sepsis remains unclear. Here, we found that GITR expression in monocytes/macrophages was induced by lysophosphatidylcholine (LPC) and was positively correlated with the severity of sepsis. GITR is a costimulatory molecule that is mainly expressed on T cells, but its function in macrophages is largely unknown. Our in vitro data showed that GITR enhanced LPC uptake by macrophages and specifically enhanced NLRP3 inflammasome-mediated macrophage pyroptosis. Furthermore, in vivo studies using either cecal ligation and puncture (CLP) or LPS-induced sepsis models demonstrated that LPC exacerbated sepsis severity/lethality, while conditional knockout of GITR in myeloid cells or NLRP3/caspase-1/IL-1ß deficiency attenuated sepsis severity/lethality. Mechanistically, GITR specifically enhanced inflammasome activation by regulating the posttranslational modification (PTM) of NLRP3. GITR competes with NLRP3 for binding to the E3 ligase MARCH7 and recruits MARCH7 to induce deacetylase SIRT2 degradation, leading to decreasing ubiquitination but increasing acetylation of NLRP3. Overall, these findings revealed a novel role of macrophage-derived GITR in regulating the PTM of NLRP3 and systemic inflammatory injury, suggesting that GITR may be a potential therapeutic target for sepsis and other inflammatory diseases. GITR exacerbates LPC-induced macrophage pyroptosis in sepsis via posttranslational regulation of NLRP3. According to the model, LPC levels increase during the early stage of sepsis, inducing GITR expression on macrophages. GITR not only competes with NLRP3 for binding to the E3 ligase MARCH7 but also recruits MARCH7 to induce the degradation of the deacetylase SIRT2, leading to decreasing ubiquitination but increasing acetylation of NLRP3 and therefore exacerbating LPC-induced NLRP3 inflammasome activation, macrophage pyroptosis and systemic inflammatory injury.
Assuntos
Proteína Relacionada a TNFR Induzida por Glucocorticoide , Lisofosfatidilcolinas , Macrófagos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Processamento de Proteína Pós-Traducional , Piroptose , Sepse , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Sepse/imunologia , Macrófagos/metabolismo , Macrófagos/imunologia , Lisofosfatidilcolinas/metabolismo , Camundongos , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Inflamassomos/metabolismo , Masculino , Camundongos Knockout , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Sirtuína 2/metabolismo , Sirtuína 2/genética , AcetilaçãoRESUMO
BACKGROUND: Aging is associated with learning and memory disorder, affecting multiple brain areas, especially the hippocampus. Previous studies have demonstrated trilobatin (TLB), as a natural food additive, can extend the life of Caenorhabditis elegans and exhibit neuroprotection in Alzheimer's disease mice. However, the possible significance of TLB in anti-aging remains elusive. PURPOSE: This study aimed to delve into the physiological mechanism by which TLB ameliorated aging-induced cognitive impairment in senescence-accelerated mouse prone 8 (SAMP8) mice. METHODS: 6-month-old SAMP8 mice were administrated with TLB (5, 10, 20 mg/kg/day, i.g.) for 3 months. The therapeutic effect of TLB on aging-induced cognitive impairment was assessed in mice using behavioral tests and aging score. The gut microbiota composition in fecal samples was analyzed by metagenomic analysis. The protective effects of TLB on blood-brain barrier (BBB) and intestinal barrier were detected by transmission electron microscope, H&E staining and western blot (WB) assay. The inhibitive effects of TLB on inflammation in brain and intestine were assessed using immunofluorescence, WB and ELISA assay. Molecular docking and surface plasma resonance (SPR) assay were utilized to investigate interaction between TLB and sirtuin 2 (SIRT2). RESULTS: Herein, the findings exhibited TLB mitigated aging-induced cognitive impairment, neuron injury and neuroinflammation in hippocampus of aged SAMP8 mice. Moreover, TLB treatment repaired imbalance of gut microbiota in aged SAMP8 mice. Furthermore, TLB alleviated the damage to BBB and intestinal barrier, concomitant with reducing the expression of SIRT2, phosphorylated levels of c-Jun NH2 terminal kinases (JNK) and c-Jun, and expression of MMP9 protein in aged SAMP8 mice. Molecular docking and SPR unveiled TLB combined with SIRT2 and down-regulated SIRT2 protein expression. Mechanistically, the potential mechanism of SIRT2 in TLB that exerted anti-aging effect was validated in vitro. As expected, SIRT2 deficiency attenuated phosphorylated level of JNK in HT22 cells treated with d-galactose. CONCLUSION: These findings reveal, for the first time, SIRT2-mediated brain-gut barriers contribute to aging and aging-related diseases, and TLB can rescue aging-induced cognitive impairment by targeting SIRT2 and restoring gut microbiota disturbance to mediate the brain-gut axis. Overall, this work extends the potential application of TLB as a natural food additive in aging-related diseases.
Assuntos
Envelhecimento , Eixo Encéfalo-Intestino , Disfunção Cognitiva , Microbioma Gastrointestinal , Sirtuína 2 , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Disfunção Cognitiva/tratamento farmacológico , Camundongos , Envelhecimento/efeitos dos fármacos , Sirtuína 2/metabolismo , Masculino , Eixo Encéfalo-Intestino/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Simulação de Acoplamento Molecular , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Modelos Animais de DoençasRESUMO
BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevalent metabolic and endocrine condition in females of reproductive age. This work was to discover the underlying role of Dickkopf 1 (DKK1) and its putative regulating mechanism in P COS. METHODS: Mice recieved dehydroepiandrosterone (DHEA) injection to establish the in vivo P COS model.Hematoxylin and eosin (H&E) staining was performed for histological analysis. RT-qP CR and Western blotting were used to detect gene and protein expression. CCK-8 and flow cytometry assays were applied to detect cell viability and apoptosis. Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP) were applied to assess association between DKK1 and SIRT2. RESULTS: In this work, DKK1 is downregulated in P COS rats. It was revealed that DKK1 knockdown induced apoptosis and suppressed proliferation in KGN cells, whereas DKK1 overexpression had exactly the opposite effects. In addition, DKK1 deactivates the T GF-ß1/SMad3 signaling pathway, thereby controlling KGN cell proliferation and apoptosis. Besides, SIRT2 inhibition reversed the impact of DKK1 overexpression on KGN cell proliferation and apoptosis. Furthermore, SIRT2 downregulated DKK1 expression by deacetylating DKK1 in KGN cells. DISCUSSION: Altogether, we concluded that SIRT2-induced deacetylation of DKK1 triggers T GF-ß1/Smad3 hyperactivation, thereby inhibiting proliferation and promoting apoptosis of KGN cells. The above results indicated that DKK1 might function as a latent target for P COS treatment.