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1.
Nature ; 588(7836): 174-179, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32906142

RESUMO

Mitochondria require nicotinamide adenine dinucleotide (NAD+) to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants1,2, but their existence in mammals remains controversial3-5. Here we demonstrate that mammalian mitochondria can take up intact NAD+, and identify SLC25A51 (also known as MCART1)-an essential6,7 mitochondrial protein of previously unknown function-as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial-but not whole-cell-NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as a mammalian transporter capable of importing NAD+ into mitochondria.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , NAD/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Respiração Celular/genética , Teste de Complementação Genética , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Cátions Orgânicos/deficiência , Proteínas de Transporte de Cátions Orgânicos/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
EMBO Rep ; 24(10): e56596, 2023 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-37575034

RESUMO

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.


Assuntos
Cardiolipinas , NAD , Cardiolipinas/metabolismo , Ligantes , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Humanos
3.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360789

RESUMO

The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.


Assuntos
Eritropoese , Feto/embriologia , Hematopoese Extramedular , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Animais , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Elementos de Resposta , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética
4.
Biochim Biophys Acta ; 1863(9): 2212-20, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27220534

RESUMO

A negative-pressure of 125mmHg (NP) has been widely used to treat chronic wounds in modern medicine. Keratinocytes under NP treatment have shown accelerated cell movement and decreased E-cadherin expression. However, the molecular mechanism of E-cadherin regulation under NP remains incompletely understood. Therefore, we investigated the E-cadherin regulation in keratinocytes (HaCaT cells) under NP. HaCaT cells were treated at ambient pressure (AP) and NP for 12h. Cell movement was measured by traditional and electric wound healing assays at the 2 different pressures. Mutants with overexpression of p120-catenin (p120(ctn)) were used to observe the effect of NP on p120(ctn) and E-cadherin expression during wound healing. Cell fractionation and immunoblotting data showed that NP increased Y228-phosphorylated p120(ctn) level and resulted in the translocation of p120(ctn) from the plasma membrane to cytoplasm. Immunofluorescence images revealed that NP decreased the co-localization of p120(ctn) and E-cadherin on the plasma membrane. Knockdown of p120(ctn) reduced E-cadherin expression and accelerated cell movement under AP. Overexpression of the Y228-phosphorylation-mimic p120(ctn) decreased E-cadherin membrane expression under both AP and NP. Phosphorylation-deficient mutants conferred restored adherens junctions (AJs) under NP. The Src inhibitor blocked the phosphorylation of p120(ctn) and impeded cell migration under NP. In conclusion, Src-dependent phosphorylation of p120(ctn) can respond rapidly to NP and contribute to E-cadherin downregulation. The NP-induced disassembly of the AJ further accelerates wound healing.


Assuntos
Junções Aderentes/metabolismo , Cateninas/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Pressão , Cicatrização , Caderinas/metabolismo , Linhagem Celular , Movimento Celular , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Fenótipo , Fosforilação , Fosfotirosina/metabolismo , Transporte Proteico , Frações Subcelulares/metabolismo , Quinases da Família src/metabolismo , delta Catenina
5.
J Gastroenterol Hepatol ; 32(7): 1355-1362, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27930829

RESUMO

BACKGROUND AND AIM: The effect of diabetes mellitus (DM) on the development of hepatocellular carcinoma (HCC) and all-cause mortality after HCC development in chronic hepatitis C virus (HCV)-infected patients remains inconclusive. This cohort study aimed to investigate these issues using the Taiwanese National Health Insurance Research Database. METHODS: We retrieved and enrolled newly diagnosed DM patients with HCV from the Longitudinal Cohort of Diabetes Patients database. Propensity score matching-including age, sex, alcohol-related liver disease, and baseline liver cirrhosis-was used to identify and enroll HCV patients without DM from the Longitudinal Health Insurance Database (n = 1686). A multi-state model was used to investigate transitions from "start-to-HCC," "start-to-death," and "HCC-to-death." RESULTS: The multi-state model showed higher cumulative hazards for "start-to-HCC," "start-to-death," and "HCC-to-death" transitions in the DM (vs non-DM) cohort. The cumulative probability of death with or without HCC after 10 years of follow-up was higher in the DM cohort than in the non-DM cohort. Multivariable transition-specific Cox models demonstrated that DM significantly increased the risk for transition from "start-to-HCC" (adjusted hazard ratio [aHR] 1.36; 95% confidence interval [CI] 1.16-1.59; P < 0.001), "start-to-death" (aHR 2.61; 95% CI: 2.05-3.33; P < 0.001), and "HCC-to-death" (aHR 1.36; 95% CI 1.10-1.68; P = 0.005). The effect of liver cirrhosis on "start-to-HCC" and "start-to-death" transitions decreased over time, particularly within 2 years. CONCLUSIONS: Diabetes mellitus increased the risk of HCC development in HCV-infected patients and the risk of all-cause mortality in patients with or without HCC.


Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/mortalidade , Complicações do Diabetes/complicações , Diabetes Mellitus , Hepatite C Crônica/mortalidade , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/mortalidade , Idoso , Estudos de Coortes , Feminino , Hepatite C Crônica/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Risco , Fatores de Tempo
6.
J Transl Med ; 14(1): 200, 2016 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-27370270

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC), a primary liver malignancy, is the most common cancer in males and fourth common cancer in females in Taiwan. HCC patients usually have a poor prognosis due to late diagnosis. It has been classified as a complex disease because of the heterogeneous phenotypic and genetic traits of the patients and a wide range of risk factors. Micro (mi)RNAs regulate oncogenes and tumor suppressor genes that are known to be dysregulated in HCC. Several studies have found an association between downregulation of miR-122, a liver-specific miRNA, and upregulation of paternally expressed gene 10 (PEG10) in HCC; however, the correlation between low miR-122 and high PEG10 levels still remains to be defined and require more investigations to evaluate their performance as an effective prognostic biomarker for HCC. METHODS: An in silico approach was used to isolate PEG10, a potential miR-122 target implicated in HCC development. miR-122S binding sites in the PEG10 promoter were evaluated with a reporter assay. The regulation of PEG10 by miR-122S overexpression was examined by quantitative RT-PCR, western blotting, and immunohistochemistry in miR-122 knockout mice and liver tissue from HCC patients. The relationship between PEG10 expression and clinicopathologic features of HCC patients was also evaluated. RESULTS: miR-122 downregulated the expression of PEG10 protein through binding to 3'-untranslated region (UTR) of the PEG10 transcript. In miR-122 knockout mice and HCC patients, the deficiency of miR-122 was associated with HCC progression. The expression of PEG10 was increased in 57.3 % of HCC as compared to paired non-cancerous tissue samples. However, significant upregulation was detected in 56.5 % of patients and was correlated with Okuda stage (P = 0.05) and histological grade (P = 0.001). CONCLUSIONS: miR-122 suppresses PEG10 expression via direct binding to the 3'-UTR of the PEG10 transcript. Therefore, while PEG10 could not be an ideal diagnostic biomarker for HCC but its upregulation in HCC tissue still has predictive value for HCC prognosis.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Reguladoras de Apoptose , Sequência de Bases , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/patologia , Masculino , Camundongos Knockout , MicroRNAs/genética , Pessoa de Meia-Idade , Modelos Biológicos , Gradação de Tumores , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Transcrição Gênica , Regulação para Cima/genética , alfa-Fetoproteínas/metabolismo
7.
Cell Metab ; 36(4): 808-821.e6, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38354740

RESUMO

SLC25A51 selectively imports oxidized NAD+ into the mitochondrial matrix and is required for sustaining cell respiration. We observed elevated expression of SLC25A51 that correlated with poorer outcomes in patients with acute myeloid leukemia (AML), and we sought to determine the role SLC25A51 may serve in this disease. We found that lowering SLC25A51 levels led to increased apoptosis and prolonged survival in orthotopic xenograft models. Metabolic flux analyses indicated that depletion of SLC25A51 shunted flux away from mitochondrial oxidative pathways, notably without increased glycolytic flux. Depletion of SLC25A51 combined with 5-azacytidine treatment limits expansion of AML cells in vivo. Together, the data indicate that AML cells upregulate SLC25A51 to decouple mitochondrial NAD+/NADH for a proliferative advantage by supporting oxidative reactions from a variety of fuels. Thus, SLC25A51 represents a critical regulator that can be exploited by cancer cells and may be a vulnerability for refractory AML.


Assuntos
Leucemia Mieloide Aguda , NAD , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Leucemia Mieloide Aguda/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Oxirredução
8.
Metabolism ; 135: 155275, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35932995

RESUMO

INTRODUCTION: Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism and energy production. NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates the acetylation levels of mitochondrial proteins that are involved in mitochondrial homeostasis. Fasting up-regulates hepatic SIRT3 activity, which requires mitochondrial NAD+. What is the mechanism, then, to transport more NAD+ into mitochondria to sustain enhanced SIRT3 activity during fasting? OBJECTIVE: SLC25A51 is a recently discovered mitochondrial NAD+ transporter. We tested the hypothesis that, during fasting, increased expression of SLC25A51 is needed for enhanced mitochondrial NAD+ uptake to sustain SIRT3 activity. Because the fasting-fed cycle and circadian rhythm are closely linked, we further tested the hypothesis that SLC25A51 is a circadian regulated gene. METHODS: We examined Slc25a51 expression in the liver of fasted mice, and examined its circadian rhythm in wild-type mice and those with liver-specific deletion of the clock gene BMAL1 (LKO). We suppressed Slc25a51 expression in hepatocytes and the mouse liver using shRNA-mediated knockdown, and then examined mitochondrial NAD+ levels, SIRT3 activities, and acetylation levels of SIRT3 target proteins (IDH2 and ACADL). We measured mitochondrial oxygen consumption rate using Seahorse analysis in hepatocytes with reduced Slc25a51 expression. RESULTS: We found that fasting induced the hepatic expression of Slc25a51, and its expression showed a circadian rhythm-like pattern that was disrupted in LKO mice. Reduced expression of Slc25a51 in hepatocytes decreased mitochondrial NAD+ levels and SIRT3 activity, reflected by increased acetylation of SIRT3 targets. Slc25a51 knockdown reduced the oxygen consumption rate in intact hepatocytes. Mice with reduced Slc25a51 expression in the liver manifested reduced hepatic mitochondrial NAD+ levels, hepatic steatosis and hypertriglyceridemia. CONCLUSIONS: Slc25a51 is a fasting-induced gene that is needed for hepatic SIRT3 functions.


Assuntos
Sirtuína 3 , Animais , Camundongos , Acetilação , Jejum/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , NAD/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo
9.
Adv Sci (Weinh) ; 9(25): e2201409, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35822667

RESUMO

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel-like factor 1 (KLF1/EKLF), the SUMOylation-deficient Klf1K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1K74R/K74R mice exhibit a marked delay in age-related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1K74R/K74R mice extends the lifespan of the wild-type mice. The Klf1K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age-related diseases thus extending the healthspan as well as lifespan.


Assuntos
Longevidade , Sumoilação , Animais , Linfócitos T CD8-Positivos , Células-Tronco Hematopoéticas , Longevidade/genética , Camundongos
10.
Dev Cell ; 28(4): 409-22, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24576425

RESUMO

Erythropoiesis is a highly regulated process during which BFU-E are differentiated into RBCs through CFU-E, Pro-E, PolyCh-E, OrthoCh-E, and reticulocyte stages. Uniquely, most erythroid-specific genes are activated during the Pro-E to Baso-E transition. We show that a wave of nuclear import of the erythroid-specific transcription factor EKLF occurs during the Pro-E to Baso-E transition. We further demonstrate that this wave results from a series of finely tuned events, including timed activation of PKCθ, phosphorylation of EKLF at S68 by P-PKCθ(S676), and sumoylation of EKLF at K74. The latter EKLF modifications modulate its interactions with a cytoplasmic ankyrin-repeat-protein FOE and importinß1, respectively. The role of FOE in the control of EKLF nuclear import is further supported by analysis of the subcellular distribution patterns of EKLF in FOE-knockout mice. This study reveals the regulatory mechanisms of the nuclear import of EKLF, which may also be utilized in the nuclear import of other factors.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Proteínas de Transporte/metabolismo , Eritropoese , Fatores de Transcrição Kruppel-Like/genética , Proteína Quinase C/metabolismo , Animais , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Camundongos Knockout , Fatores de Tempo
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