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1.
Cell Commun Signal ; 22(1): 51, 2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-38233839

RESUMO

The dynamic changes of RNA N6-methyladenosine (m6A) during cancer progression participate in various cellular processes. However, less is known about a possible direct connection between upstream regulator and m6A modification, and therefore affects oncogenic progression. Here, we have identified that a key enzyme in N4-acetylcytidine (ac4C) acetylation NAT10 is highly expressed in human osteosarcoma tissues, and its knockdown enhanced m6A contents and significantly suppressed osteosarcoma cell growth, migration and invasion. Further results revealed that NAT10 silence inhibits mRNA stability and translation of m6A reader protein YTHDC1, and displayed an increase in glucose uptake, a decrease in lactate production and pyruvate content. YTHDC1 recognizes differential m6A sites on key enzymes of glycolysis phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) mRNAs, which suppress glycolysis pathway by increasing mRNA stability of them in an m6A methylation-dependent manner. YTHDC1 partially abrogated the inhibitory effect caused by NAT10 knockdown in tumor models in vivo, lentiviral overexpression of YTHDC1 partially restored the reduced stability of YTHDC1 caused by lentiviral depleting NAT10 at the cellular level. Altogether, we found ac4C driven RNA m6A modification can positively regulate the glycolysis of cancer cells and reveals a previously unrecognized signaling axis of NAT10/ac4C-YTHDC1/m6A-LDHA/PFKM in osteosarcoma. Video Abstract.


Assuntos
Citidina/análogos & derivados , Osteossarcoma , Fosfofrutoquinases , Humanos , Lactato Desidrogenase 5/metabolismo , Fosfofrutoquinases/metabolismo , Acetilação , RNA/metabolismo , Glicólise/genética , Osteossarcoma/patologia , Fosfofrutoquinase-1 Muscular/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Acetiltransferases N-Terminal/metabolismo
2.
Cell Death Dis ; 13(10): 876, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253358

RESUMO

Renal fibrosis is a common pathological feature and outcome of almost all chronic kidney diseases, and it is characterized by metabolic reprogramming toward aerobic glycolysis. Mesenchymal stem cell-derived exosomes (MSC-Exos) have been proposed as a promising therapeutic approach for renal fibrosis. In this study, we investigated the effect of MSC-Exos on glycolysis and the underlying mechanisms. We demonstrated that MSC-Exos significantly ameliorated unilateral ureter obstruction (UUO)-induced renal fibrosis by inhibiting glycolysis in tubular epithelial cells (TECs). miRNA sequencing showed that miR-21a-5p was highly enriched in MSC-Exos. Mechanistically, miR-21a-5p repressed the expression of phosphofructokinase muscle isoform (PFKM), a rate-limiting enzyme of glycolysis, thereby attenuating glycolysis in TECs. Additionally, knockdown of miR-21a-5p abolished the renoprotective effect of MSC-Exos. These findings revealed a novel role for MSC-Exos in the suppression of glycolysis, providing a new insight into the treatment of renal fibrosis.


Assuntos
Exossomos , Nefropatias , Células-Tronco Mesenquimais , MicroRNAs , Fosfofrutoquinase-1 Muscular , Humanos , Exossomos/genética , Exossomos/metabolismo , Fibrose , Glicólise/genética , Nefropatias/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Músculos/metabolismo , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Isoformas de Proteínas/metabolismo
3.
Genes (Basel) ; 13(3)2022 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-35328104

RESUMO

The reprogramming of energy metabolism is one of the hallmarks of cancer and is crucial for tumor progression. Altered aerobic glycolysis is a well-known characteristic of cancer cell metabolism. In the present study, the expression profiles of key metabolic genes (HK2, PFKM, and PKM2) were assessed in the breast cancer cohort of Pakistan using quantitative polymerase chain reaction (qPCR) and IHC. Expression patterns were correlated with molecular subtypes and clinical parameters in the patients. A significant upregulation of key glycolytic genes was observed in tumor samples in comparison to their adjacent controls (p < 0.0001). The expression of the studied glycolytic genes was significantly increased in late clinical stages, positive nodal involvement, and distant metastasis (p < 0.05). HK2 and PKM2 were found to be upregulated in luminal B, whereas PFKM was overexpressed in the luminal A subtype of breast cancer. The genes were positively correlated with the proliferation marker Ki67 (p < 0.001). Moreover, moderate positive linear correlations between HK2 and PKM2 (r = 0.476), HK2 and PFKM (r = 0.473), and PKM2 and PFKM (r = 0.501) were also observed (p < 0.01). These findings validate that the key regulatory genes in glycolysis can serve as potential biomarkers and/or molecular targets for breast cancer management. However, the clinical significance of these molecules needs to be further validated through in vitro and in vivo experiments.


Assuntos
Neoplasias da Mama , Idade de Início , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte , Feminino , Glicólise/genética , Hexoquinase , Humanos , Proteínas de Membrana , Metástase Neoplásica , Paquistão , Fosfofrutoquinase-1 Muscular/metabolismo , Hormônios Tireóideos , Proteínas de Ligação a Hormônio da Tireoide
4.
Dis Markers ; 2021: 8587535, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917202

RESUMO

OBJECTIVE: The purpose of this study is to explore the regulating role of microRNA-383-5p (miR-383-5p) in oxidative stress after acute myocardial infarction (AMI) through AMPK pathway via phosphofructokinase muscle-type (PFKM). METHODS: We established the AMI model, and the model mice were injected with miR-383-5p agomir to study the effect of miR-383-5p in AMPK signaling pathways. The target gene for miR-383-5p was reported to be PFKM, so we hypothesized that overexpression of miR-383-5p inhibits activation of the AMPK signaling pathway. RESULTS: In this research, we found that overexpression of miR-383-5p decreases myocardial oxidative stress, myocardial apoptosis, the expression level of PFKM malondialdehyde (MDA), and reactive oxygen species (ROS) in the myocardial tissues after AMI, and finally, AMI-induced cardiac systolic and diastolic function could be improved. CONCLUSION: This study demonstrated that miR-383-5p could reduce the oxidative stress after AMI through AMPK signaling pathway by targeting PFKM.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , MicroRNAs/metabolismo , Infarto do Miocárdio/genética , Estresse Oxidativo/genética , Fosfofrutoquinase-1 Muscular/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Cultivadas , Marcadores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Fosfofrutoquinase-1 Muscular/metabolismo , Distribuição Aleatória , Transdução de Sinais/genética , Regulação para Cima
5.
Cell Death Dis ; 12(4): 408, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33859186

RESUMO

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.


Assuntos
Carcinoma Epitelial do Ovário/genética , Glicólise/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Animais , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos
6.
Mol Cell ; 81(9): 1905-1919.e12, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33852893

RESUMO

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.


Assuntos
Glicólise , Fosforilação Oxidativa , Precursores de RNA/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Ácido Glutâmico/metabolismo , Glicogênio/metabolismo , Glicólise/genética , Células HEK293 , Células HeLa , Humanos , Células K562 , Mitocôndrias/genética , Mitocôndrias/metabolismo , Oxirredução , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U1/genética
7.
Biochem Biophys Res Commun ; 544: 52-59, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33516882

RESUMO

Dental pulp stem cells (DPSCs) can differentiate into diverse cell lineages, including odontogenic cells that are responsible for dentin formation, which is important in pulp repair and tooth regeneration. While glycolysis plays a central role in various cellular activities in both physiological and pathological conditions, its role and regulation in odontogenic differentiation are unknown. Here, we show that aerobic glycolysis is induced during odontoblastic differentiation from human DPSCs. Importantly, we demonstrate that during odontoblastic differentiation, protein expression levels of phosphofructokinase 1 muscle isoform (PFKM) and PFK2, but not other glycolytic enzymes, are mainly upregulated by AKT activation, resulting in increased total PFK enzyme activity. Increased PFK activity is essential to enhance aerobic glycolysis, which plays an important role in the odontoblastic differentiation of human DPSCs. These findings underscore that PFK activation-induced aerobic glycolysis accompanies, and participates in, human DPSCs differentiation into odontogenic lineage, and could play a role in the regulation of dental pulp repair.


Assuntos
Diferenciação Celular , Polpa Dentária/citologia , Odontogênese , Fosfofrutoquinase-1 Muscular/metabolismo , Fosfofrutoquinase-2/metabolismo , Células-Tronco/citologia , Proliferação de Células , Células Cultivadas , Polpa Dentária/metabolismo , Humanos , Transdução de Sinais , Células-Tronco/metabolismo
8.
Genomics ; 113(1 Pt 1): 135-141, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33279650

RESUMO

Head and neck squamous cell carcinoma (HNSCC) is a malignant tumor of the upper aerodigestive tract. The loss and gain of miRNA function promote cancer development through various mechanisms. RNA sequencing (RNA-seq) and miRNAs sequencing data from the Cancer Genome Atlas (TCGA) was used to show the dysfunctional miRNAs microenvironment and to provide useful biomarkers for miRNAs therapy. Seven miRNAs were found to be independent prognostic factors of HNSCC patients in the training cohort. A total of 60 target genes for these miRNAs were predicted. Nine target genes (CDCA4, CXCL14, FLNC, KLF7, NBEAL2, P4HA1, PFKM, PFN2 and SEPPINE1) were correlated with patient's overall survival (OS) outcomes. We identified novel miRNAs markers for the prognosis of head and neck squamous cell carcinoma.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Idoso , Biomarcadores Tumorais/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Feminino , Filaminas/genética , Filaminas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Profilinas/genética , Profilinas/metabolismo
9.
Biochem Biophys Res Commun ; 530(1): 67-74, 2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32828317

RESUMO

Phosphofructokinase-M (PFKM) is a key enzyme in glycolysis. The expression and activity of PFKM is closely related to the occurrence and development of malignant tumors, but its role in the regulation of renal cell carcinoma (RCC) is still unknown. We found that the expression of PFKM was lower in RCC tumor tissue than in adjacent normal tissues, and that low expression of PFKM was related to the poor overall survival of RCC patients. In addition, our results showed that FOXO3 mediated PFKM inhibited the growth, migration and invasion of RCC cells, suggesting that PFKM is a protective factor for RCC.


Assuntos
Carcinoma de Células Renais/metabolismo , Proteína Forkhead Box O3/metabolismo , Neoplasias Renais/metabolismo , Fosfofrutoquinase-1 Muscular/metabolismo , Transdução de Sinais , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteína Forkhead Box O3/análise , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/patologia , Fosfofrutoquinase-1 Muscular/análise , Prognóstico
10.
Proc Natl Acad Sci U S A ; 117(12): 6726-6732, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156725

RESUMO

The extrahypothalamic growth hormone-releasing hormone (GHRH) and its cognate receptors (GHRH-Rs) and splice variants are expressed in a variety of cancers. It has been shown that the pituitary type of GHRH-R (pGHRH-R) mediates the inhibition of tumor growth induced by GHRH-R antagonists. However, GHRH-R antagonists can also suppress some cancers that do not express pGHRH-R, yet the underlying mechanisms have not been determined. Here, using human esophageal squamous cell carcinoma (ESCC) as a model, we were able to reveal that SV1, a known splice variant of GHRH-R, is responsible for the inhibition induced by GHRH-R antagonist MIA-602. We demonstrated that GHRH-R splice variant 1 (SV1) is a hypoxia-driven promoter of tumor progression. Hypoxia-elevated SV1 activates a key glycolytic enzyme, muscle-type phosphofructokinase (PFKM), through the nuclear factor kappa B (NF-κB) pathway, which enhances glycolytic metabolism and promotes progression of ESCC. The malignant actions induced by the SV1-NF-κB-PFKM pathway could be reversed by MIA-602. Altogether, our studies demonstrate a mechanism by which GHRH-R antagonists target SV1. Our findings suggest that SV1 is a hypoxia-induced oncogenic promoter which can be an alternative target of GHRH-R antagonists.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Receptores LHRH/genética , Sermorelina/análogos & derivados , Processamento Alternativo , Animais , Apoptose , Proliferação de Células , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Feminino , Glicólise , Humanos , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Receptores LHRH/antagonistas & inibidores , Sermorelina/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Phys Biol ; 16(6): 066007, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31469100

RESUMO

The glycolytic enzyme pyruvate kinase M2 (PKM2) exists in both catalytically inactive dimeric and active tetrameric forms. In cancer cells, PKM2 dimer predominance contributes to tumor growth by triggering glycolytic reprogramming. However, the mechanism that promotes PKM2 dimer predominance over tetramer in cancer cells remains elusive. Here, we show that pulsatile phosphofructokinase (PFK-1) activity results in PKM2 dimer predominance. Mathematical simulations predict that pulsatile PFK-1 activity prevents the formation of PKM2 tetramer even under high levels of fructose-1,6-bisphosphate (FBP), a PKM2 tetramer-promoting metabolite produced by PFK-1. We experimentally confirm these predictions at the single-molecule level by providing evidence for pulsatile PFK-1 activity-induced synchronized dissociation of PKM2 tetramers and the subsequent accumulation of PKM2 dimers under high levels of FBP in HeLa cells. Moreover, we show that pulsatile PFK-1 activity-induced PKM2 dimer predominance also controls cell proliferation. Thus, our study reveals the significance of pulsatile PFK-1 activity in cancer cell metabolism.


Assuntos
Proteínas de Transporte/genética , Frutosedifosfatos/metabolismo , Glicólise , Proteínas de Membrana/genética , Fosfofrutoquinase-1 Muscular/genética , Hormônios Tireóideos/genética , Proteínas de Transporte/metabolismo , Reprogramação Celular , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Fosfofrutoquinase-1 Muscular/metabolismo , Hormônios Tireóideos/metabolismo , Proteínas de Ligação a Hormônio da Tireoide
12.
Biochim Biophys Acta Proteins Proteom ; 1866(5-6): 602-607, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29563071

RESUMO

PURPOSE: Cancer cells consume more glucose than normal human cells and convert most glucose into lactate. It has been proposed that deregulated glycolysis is triggered by the posttranslational modification of 85 kDa muscle-type 6-phosphofructo-1-kinase (PFK-M) which is cleaved by a specific protease to form shorter, highly active, feedback-inhibition-resistant PFK-M fragments. PRINCIPAL RESULTS: To find the protease involved in PFK-M modification, analyses of the protease target sites on the human PFK-M enzyme yielding 45-47 kDa fragments were performed in silico. The results suggested that an enzyme in the kallikrein (KLK) family may be involved. Kallikreins can be self-activated in the cytosol and are often overexpressed in cancer cells. After incubating the internally quenched FRET peptide with a sequence characteristic of the target site, along with the active KLK6, the cleavage of the peptide was observed. The ability of KLK6 to cleave native PFK-M and form highly active citrate-resistant 45 kDa fragments was further confirmed by enzymatic tests and SDS-PAGE. A role of KLK6 in the posttranslational modification of native PFK-M was ultimately confirmed in vivo. A yeast strain that encoded native human PFK-M as the only PFK1 enzyme was additionally transformed with proKLK6 or KLK6 genes under the control of an inducible promoter. The transformants growth rate was found to increase after the induction of proKLK6 gene expression as compared to the strain with the native PFK-M enzyme. CONCLUSION: KLK6 may be the key protease involved in the modification of PFK-M and trigger deregulated glycolytic flux in cancer cells.


Assuntos
Calicreínas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfofrutoquinase-1 Muscular/metabolismo , Processamento de Proteína Pós-Traducional , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Humanos , Calicreínas/genética , Cinética , Fragmentos de Peptídeos/genética , Fosfofrutoquinase-1 Muscular/genética , Proteólise , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato
13.
Diabetes ; 67(3): 351-359, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29463575

RESUMO

Insulin secretion from pancreatic islet ß-cells occurs in a pulsatile fashion, with a typical period of ∼5 min. The basis of this pulsatility in mouse islets has been investigated for more than four decades, and the various theories have been described as either qualitative or mathematical models. In many cases the models differ in their mechanisms for rhythmogenesis, as well as other less important details. In this Perspective, we describe two main classes of models: those in which oscillations in the intracellular Ca2+ concentration drive oscillations in metabolism, and those in which intrinsic metabolic oscillations drive oscillations in Ca2+ concentration and electrical activity. We then discuss nine canonical experimental findings that provide key insights into the mechanism of islet oscillations and list the models that can account for each finding. Finally, we describe a new model that integrates features from multiple earlier models and is thus called the Integrated Oscillator Model. In this model, intracellular Ca2+ acts on the glycolytic pathway in the generation of oscillations, and it is thus a hybrid of the two main classes of models. It alone among models proposed to date can explain all nine key experimental findings, and it serves as a good starting point for future studies of pulsatile insulin secretion from human islets.


Assuntos
Sinalização do Cálcio , Glicólise , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Modelos Biológicos , Regulação Alostérica , Animais , Glicemia/metabolismo , Simulação por Computador , Ativação Enzimática , Frutosedifosfatos/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/enzimologia , Cinética , Fosfofrutoquinase-1 Muscular/metabolismo , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/metabolismo
14.
J Cell Biol ; 216(8): 2305-2313, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28646105

RESUMO

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.


Assuntos
Glucose/metabolismo , Fígado/enzimologia , Fosfofrutoquinase-1 Hepática/metabolismo , Plaquetas/enzimologia , Membrana Celular/enzimologia , Glicólise , Células HEK293 , Humanos , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Músculo Esquelético/enzimologia , Fosfofrutoquinase-1 Hepática/genética , Fosfofrutoquinase-1 Hepática/ultraestrutura , Fosfofrutoquinase-1 Muscular/metabolismo , Fosfofrutoquinase-1 Muscular/ultraestrutura , Fosfofrutoquinase-1 Tipo C/metabolismo , Fosfofrutoquinase-1 Tipo C/ultraestrutura , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Relação Estrutura-Atividade , Especificidade por Substrato , Imagem com Lapso de Tempo
15.
Biosci Biotechnol Biochem ; 81(8): 1478-1483, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28508704

RESUMO

Glucose metabolism is a basic biological process that shows substantial variation within and between species. Using pig as a model organism, we investigated differences in glucose metabolic genes in seven tissues from domesticated pigs (Rongchang pig and Tibetan pig, meanwhile, the Tibetan pig just as a special case of the domesticated pig under plateau condition) and wild boar. We found large differences in the expression of genes involved in multiple aspects of glucose metabolism, including genes associated with glucose transport, gluconeogenesis, and glycolysis. In addition, we identified microRNAs (miRNAs) that may be involved in the divergence of glucose metabolism in pig. A combined analysis of mRNA and miRNA expression indicated that some miRNA:mRNA pairs showed ab facto function in it. Our results provide a valuable resource for further determination of miRNA regulatory roles in pig glucose metabolism and reveal the divergence of glucose metabolism in pigs under domestication.


Assuntos
Regulação da Expressão Gênica , Glucose/metabolismo , Músculo Esquelético/metabolismo , Sus scrofa/genética , Suínos/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Transporte Biológico , Domesticação , Perfilação da Expressão Gênica , Gluconeogênese/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Glicólise/genética , Hexoquinase/genética , Hexoquinase/metabolismo , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Especificidade de Órgãos , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Especificidade da Espécie , Sus scrofa/metabolismo , Suínos/metabolismo
16.
Skelet Muscle ; 6: 12, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27486508

RESUMO

BACKGROUND: Diabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1 (-/-) mouse (iMSBmal1 (-/-)). RESULTS: Progressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1 (-/-) mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1 (-/-) muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1 (-/-) muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle. CONCLUSIONS: These data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.


Assuntos
Fatores de Transcrição ARNTL/fisiologia , Glicemia/metabolismo , Ritmo Circadiano , Insulina/metabolismo , Músculo Esquelético/metabolismo , Tecido Adiposo/metabolismo , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/análogos & derivados , Animais , Glicemia/análise , Peso Corporal , Feminino , Transportador de Glucose Tipo 4/metabolismo , Hexoquinase/metabolismo , Homeostase , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Insulina/sangue , Masculino , Camundongos , Camundongos Knockout , Atividade Motora , Fosfofrutoquinase-1 Muscular/metabolismo , RNA Mensageiro/metabolismo , Ribonucleotídeos/administração & dosagem
17.
Appl Biochem Biotechnol ; 173(7): 1640-51, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24840038

RESUMO

Glycolytic potential is a hot aspect to meat quality research in recent years. Phosphofructokinase, muscle type (PFKM), is a key regulatory enzyme used to catalyze the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate in glycolysis. The present study was designed to investigate the association of PFKM SNP and meat quality traits in pigs. In this study, the 2,864-bp full-length cDNA sequence of the porcine PFKM gene was obtained, which contained 30 bp of 5' UTR, 2,343 bp of coding region, and 491 bp of 3' UTR. The porcine PFKM mRNA was predominantly expressed in skeletal muscle and heart. One single nucleotide polymorphism (SNP) T129C in exon 13 of PFKM gene was detected, with its allele frequencies significantly different between Chinese indigenous pig breed and Western pig breeds. The SNP was significantly associated with meat color value (m. biceps femoris), meat marbling (m. longissimus dorsi), meat marbling (m. biceps femoris), intramuscular fat (m. longissimus dorsi) (P<0.01), and water moisture (m. longissimus dorsi) in the Large White×Meishan F2 population. These results laid a foundation for further investigations on the detailed physiological function of porcine PFKM gene.


Assuntos
Regulação Enzimológica da Expressão Gênica , Carne , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Suínos/anatomia & histologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fosfofrutoquinase-1 Muscular/química , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Análise de Sequência
18.
Am J Physiol Cell Physiol ; 304(2): C180-93, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23114964

RESUMO

The hypothesis was tested that the variation of in vivo glycolytic flux with contraction frequency in skeletal muscle can be qualitatively and quantitatively explained by calcium-calmodulin activation of phosphofructokinase (PFK-1). Ischemic rat tibialis anterior muscle was electrically stimulated at frequencies between 0 and 80 Hz to covary the ATP turnover rate and calcium concentration in the tissue. Estimates of in vivo glycolytic rates and cellular free energetic states were derived from dynamic changes in intramuscular pH and phosphocreatine content, respectively, determined by phosphorus magnetic resonance spectroscopy ((31)P-MRS). Computational modeling was applied to relate these empirical observations to understanding of the biochemistry of muscle glycolysis. Hereto, the kinetic model of PFK activity in a previously reported mathematical model of the glycolytic pathway (Vinnakota KC, Rusk J, Palmer L, Shankland E, Kushmerick MJ. J Physiol 588: 1961-1983, 2010) was adapted to contain a calcium-calmodulin binding sensitivity. The two main results were introduction of regulation of PFK-1 activity by binding of a calcium-calmodulin complex in combination with activation by increased concentrations of AMP and ADP was essential to qualitatively and quantitatively explain the experimental observations. Secondly, the model predicted that shutdown of glycolytic ATP production flux in muscle postexercise may lag behind deactivation of PFK-1 (timescales: 5-10 s vs. 100-200 ms, respectively) as a result of accumulation of glycolytic intermediates downstream of PFK during contractions.


Assuntos
Glicólise/fisiologia , Músculo Esquelético/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/análise , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Simulação por Computador , Concentração de Íons de Hidrogênio , Isquemia/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Masculino , Modelos Biológicos , Contração Muscular/fisiologia , Fosfocreatina/análise , Fosfocreatina/metabolismo , Fosfofrutoquinase-1 Muscular/química , Fosfofrutoquinase-1 Muscular/metabolismo , Condicionamento Físico Animal/fisiologia , Ratos , Ratos Wistar
19.
FASEB J ; 26(11): 4710-21, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22767230

RESUMO

Glycolysis is the initial step of glucose catabolism and is up-regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H(2)O(2)-treated myotubes, we show up-regulation of the rate-limiting glycolytic enzyme muscle-type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P<0.05). Using microRNA profiling, we identify miR-320a as a regulator of PFKm expression. Reduced miR-320a levels (to ∼50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR-320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR-320a appears to regulate oxidative stress-induced PFKm expression, and reduced miR-320a allows greater induction of glycolysis in response to H(2)O(2) treatment. We show that this microRNA-mediated regulation occurs through PFKm's 3' untranslated region and that Ets proteins are involved in the regulation of PFKm via miR-320a. These findings suggest that oxidative stress-responsive microRNA-320a may regulate glycolysis broadly within nature.


Assuntos
Glicólise/fisiologia , MicroRNAs/metabolismo , Estresse Oxidativo/fisiologia , Adenocarcinoma/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo , Reação em Cadeia da Polimerase , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
20.
J Biol Chem ; 287(21): 17546-17553, 2012 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-22474333

RESUMO

6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution).


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Fosfofrutoquinase-1 Muscular/química , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/fisiologia , Sítios de Ligação , Evolução Molecular , Humanos , Mutação , Fosfofrutoquinase-1 Muscular/genética , Fosfofrutoquinase-1 Muscular/metabolismo
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