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1.
Nat Commun ; 11(1): 88, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900402

RESUMO

The accumulation of damaged mitochondria causes the death of dopaminergic neurons. The Parkin-mediated mitophagy pathway functions to remove these mitochondria from cells. Targeting this pathway represents a therapeutic strategy for several neurodegenerative diseases, most notably Parkinson's disease. We describe a discovery pipeline to identify small molecules that increase Parkin recruitment to damaged mitochondria and ensuing mitophagy. We show that ROCK inhibitors promote the activity of this pathway by increasing the recruitment of HK2, a positive regulator of Parkin, to mitochondria. This leads to the increased targeting of mitochondria to lysosomes and removal of damaged mitochondria from cells. Furthermore, ROCK inhibitors demonstrate neuroprotective effects in flies subjected to paraquat, a parkinsonian toxin that induces mitochondrial damage. Importantly, parkin and rok are required for these effects, revealing a signaling axis which controls Parkin-mediated mitophagy that may be exploited for the development of Parkinson's disease therapeutics.


Assuntos
Inibidores Enzimáticos/farmacologia , Mitocôndrias/metabolismo , Fármacos Neuroprotetores/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Quinases Associadas a rho/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Dípteros , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/genética , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
2.
Technol Cancer Res Treat ; 18: 1533033819871306, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31530094

RESUMO

Cancer cells undergo metabolic changes that support their malignant growth. These changes are often associated with increased expression of the rate-limiting glycolytic enzyme hexokinase 2. Hexokinase 2 is an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate. In this study, we utilized Gene Expression Profiling Interactive Analysis (GEPIA) database analysis and clinical sample analysis to find that hexokinase 2 was highly expressed in cervical cancer. Furthermore, we found that high hexokinase 2 expression in cervical cancer demonstrated a positive correlation with tumor size (P = .009696), pathological grade (P = .028551), and prognosis (P = .00069) but not with age (P = .956201) or lymph node metastasis (P = .131379). At the cellular level, we knocked down the expression of hexokinase 2 in the human cervical cancer cell line SiHa. The results demonstrated that knockdown of hexokinase 2 inhibited the proliferation and migration of SiHa cells and promoted cell apoptosis. During this process, knockdown of hexokinase 2 inhibited phosphorylation of AKT and mammalian target of rapamycin and promoted p53 expression. At the same time, overexpression of human papillomavirus 18 oncogenes E6 and E7 significantly promoted the expression of hexokinase 2. Most importantly, we discovered a novel upstream regulatory microRNA for hexokinase 2: miR-9-5p. Luciferase reporter assays and Western blot assays demonstrated that hexokinase 2 expression was inhibited by miR-9-5p by directly binding its 3'-untranslated region in SiHa cells. Next, we determined that miR-9-5p could suppress the proliferation and migration of SiHa cells and induce apoptosis. In conclusion, we found that hexokinase 2 serves a carcinogenic role in cervical cancer through the miR-9-5p/hexokinase 2/AKT pathway, which serves as the basis for potential therapeutic targets and prognostic indicators.


Assuntos
Carcinogênese/genética , Hexoquinase/genética , Neoplasias do Colo do Útero/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Transdução de Sinais , Neoplasias do Colo do Útero/patologia
3.
Mol Med Rep ; 20(3): 2774-2782, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31524259

RESUMO

MicroRNAs (miRNAs) have been proposed as potential prognostic and diagnostic biomarkers in numerous types of cancer, including osteosarcoma (OS), which is the most common bone malignancy. The present study revealed that the expression of miR­185 was downregulated in OS tissues and cells. Overexpression of miR­185 significantly suppressed the proliferation and migration of OS cells. To further investigate the functional roles of miR­185 in OS, the downstream targets of miR­185 were predicted using the microRNA.org database. The results revealed that in cancer cells, hexokinase 2 (HK2), the rate­limiting enzyme of glycolysis, was a potential target of miR­185. Molecular analysis indicated that miR­185 binds to the 3'­untranslated region of HK2 mRNA. Overexpressed miR­185 downregulated the mRNA and protein levels of HK2 in OS cells. In addition, an inverse correlation between the expression of miR­185 and HK2 was reported in OS. Consistent with the downregulation of HK2 induced by miR­185, overexpression of HK2 in OS cells significantly attenuated the inhibitory effects of miR­185 on glucose consumption and lactate production, while depletion of miR­185 promoted the glycolysis of OS cells. Additionally, restoration of HK2 abolished the inhibitory effects of miR­185 on the proliferation of OS cells. In summary, these results revealed that miR­185 suppressed the glucose metabolism of OS cells; thus, miR­185 may be considered as a promising therapeutic target for the treatment of OS.


Assuntos
Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica , Hexoquinase/genética , MicroRNAs/genética , Osteossarcoma/genética , Interferência de RNA , Regiões 3' não Traduzidas , Adolescente , Adulto , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Criança , Feminino , Glucose/metabolismo , Glicólise , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Carga Tumoral , Adulto Jovem
4.
J Insect Sci ; 19(5)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31557289

RESUMO

Glucose-6-phosphatase (G6Pase) and hexokinase (HK) are two key enzymes in the glycolysis and gluconeogenesis pathways, which catalyze the synthesis and degradation of glucose in insects, respectively. G6Pase and HK play an important role in insect growth by regulating the metabolism of glucose, leading to the efficient metabolism of other macromolecules. However, it is unclear whether these genes could be investigated for pest control through their actions on chitin metabolism. We studied the potential functions of G6Pase and HK genes in the regulation of chitin metabolism pathways by RNAi technology. Interference with G6Pase expression did not affect trehalose and chitin metabolism pathways in brown planthopper, Nilaparvata lugens (Stål). However, knockdown of the HK gene resulted in a significant decrease of expression of genes associated with the trehalose metabolic pathway but had no significant effect on trehalase activity, trehalose content, or glucogen content. Additionally, HK knockdown resulting in downregulation of the genes involved in chitin metabolism in the brown planthopper. These insects also showed wing deformities and difficulty in molting to varying degrees. We suggest that the silencing of HK expression directly inhibited the decomposition of glucose, leading to impaired chitin synthesis.


Assuntos
Quitina/biossíntese , Glucose/metabolismo , Hemípteros/metabolismo , Animais , Glucose-6-Fosfatase/genética , Hemípteros/enzimologia , Hemípteros/genética , Hexoquinase/genética , Proteínas de Insetos/genética , Interferência de RNA , Trealose/metabolismo
5.
Mol Carcinog ; 58(11): 2161-2174, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31486135

RESUMO

Metabolic reprogramming (including the Warburg effect) is a hallmark of cancer, yet the association between the altered metabolism and chemoresistance remains elusive. Hexokinase II (HKII) is a key metabolic enzyme and is upregulated in multiple cancers. In this study, we examined the impact of targeting metabolism via silencing of HKII on chemoresistance in ovarian cancer (OVCA). In addition, the regulatory molecular mechanism of tumor metabolism was examined using gain- and loss-of-function approaches in epithelial OVCA cell lines of various histological subtypes. We demonstrated that cisplatin (CDDP)-induced p53-mediated HKII downregulation is a determinant of chemosensitivity in OVCA. Silencing of HKII sensitized chemoresistant OVCA cells to apoptosis in a p53-dependent manner. As a negative regulator, p53 suppressed HKII transcription by promoter binding and decreased glycolysis. Pyruvate dehydrogenase kinase-1 (PDK1) is a key regulator of cell proliferation involved in Akt signaling axis. Our Gene Expression Profiling Interactive Analysis (GEPIA) and molecular studies also revealed that PDK1, an upstream activator strongly correlates with HKII expression and regulates its metabolic activity. Finally, we demonstrated that the clinically approved drug metformin sensitizes chemoresistant OVCA cells to CDDP via PDK1-HKII pathway. Collectively, our data implicate that p53--PDK1-HKII axis is a central regulatory component of metabolism conferring chemoresistance in OVCA.


Assuntos
Carcinoma Epitelial do Ovário/tratamento farmacológico , Hexoquinase/genética , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hexoquinase/antagonistas & inibidores , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos
6.
Exp Hematol ; 78: 46-55.e3, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31560931

RESUMO

Hexokinase II (HXKII) is a key regulator of glucose metabolism that converts glucose to glucose 6-phosphate. Furthermore, HXKII blocks mitochondria-dependent apoptosis by inhibiting the release of cytochrome c. HXKII overexpression is frequently observed in several types of cancer and confers chemoresistance to cancer cells. In the present study, we found that compared with cell lines generated from diffuse large-B-cell lymphoma (DLBCL) patients, cell lines with features of Burkitt lymphoma have higher levels of HXKII because of the activation of both c-MYC and HIF-1. Under normoxia, HXKII levels were correlated with the growth ability of each B-cell lymphoma cell line. HXKII levels were further enhanced when the B-cell lymphoma cells were cultured under hypoxia. The high levels of HXKII induced by hypoxia conferred cisplatin resistance in all tested B-cell lymphoma cell lines. The HDAC inhibitor panobinostat significantly suppressed HXKII expression under both normoxic and hypoxic conditions. Importantly, panobinostat reversed the anti-lymphoma action of cisplatin, and this effect was diminished by hypoxia. These data suggest that HXKII plays different roles, including in the regulation of glycolysis and inhibition of apoptosis, depending on its expression levels. Furthermore, inhibition of HXKII expression by panobinostat may represent a new and attractive strategy to overcome cisplatin resistance.


Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hexoquinase/biossíntese , Linfoma Difuso de Grandes Células B , Panobinostat/farmacologia , Idoso , Idoso de 80 Anos ou mais , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Glicólise/efeitos dos fármacos , Glicólise/genética , Hexoquinase/genética , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/enzimologia , Linfoma Difuso de Grandes Células B/genética , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
7.
Int J Mol Med ; 44(4): 1377-1387, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432102

RESUMO

The retina is sensitive to injury resulting from oxidative stress (OS) due to its high oxygen consumption. Patients with retinitis pigmentosa suffer from excessive OS. N­acetylcysteine (NAC) is used as a mucolytic agent for the clinical treatment of disorders, such as chronic bronchitis and other pulmonary diseases. The aim of the present study was to investigate the role of hexokinase 2 (HKII) in retinal OS injury. Amyloid ß (Aß)1­40 was used to establish a cellular model of OS. Cell viability was measured with a Cell Counting Kit­8 assay, and the apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) of cells were analyzed via flow cytometry with corresponding kits. The mRNA and protein levels were detected by reverse transcription­quantitative PCR and western blot analyses, respectively. It was observed that Aß1­40 reduced the expression of HKII in the mitochondria of retinal pigment epithelial ARPE cells and impaired mitochondrial antioxidant functions. Additionally, knockdown of HKII promoted apoptosis, and increased ROS levels and the MMP. NAC attenuated the inhibition of mitochondrial functions induced by Aß1­40. The knockdown of HKII was revealed to decrease the levels of Bcl­2, manganese superoxide dismutase (SOD) and copper­zinc­SOD, and increase the levels of cleaved caspase­3, Bax and cytochrome c. The present findings suggested that the dissociation of HKII induced by OS induces apoptosis and mitochondrial damage. This study provided improved understanding of the mechanisms underlying the effects of OS on retinal epithelial cells.


Assuntos
Hexoquinase/genética , Estresse Oxidativo , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Apoptose , Biomarcadores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Inativação de Genes , Hexoquinase/metabolismo , Humanos , Mitocôndrias , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/efeitos dos fármacos
8.
Oncol Rep ; 42(3): 1125-1132, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322265

RESUMO

Disturbed mitochondrial dynamics are closely associated with the progression of different types of cancer including hepatocellular carcinoma (HCC). However, the manner in which mitochondrial dynamics are regulated in HCC remains largely unclear. In the present study, via immunofluorescence, real­time PCR and western blot analysis, the effects of dynamin­1­like (DNM1L) on mitochondrial translocation and the mitochondrial permeability transition pore (mPTP) were investigated in HCC cells under hypoxic conditions, and the underlying molecular mechanisms were explored. Our data revealed that hypoxic treatment decreased the mitochondrial membrane potential, elevated generation of reactive oxygen species, and promoted mitochondrial fission in a DNM1L­dependent manner. Instead of changing the levels of DNMlL, hypoxia increased the translocation of DNM1L to mitochondria, leading to excessive mitochondrial fission and decreased the viability of HCC cells. In addition to the effects on mitochondrial dynamics, DNM1L also regulated mPTP opening in HCC. IP analysis revealed that DNM1L interacted with the enzyme hexokinase 2 (HK2), and was involved in its phosphorylation, resulting in HK2 detachment from the mitochondria and consequently mPTP opening.


Assuntos
Carcinoma Hepatocelular/patologia , Dinaminas/metabolismo , Hexoquinase/metabolismo , Hipóxia/fisiopatologia , Neoplasias Hepáticas/patologia , Dinâmica Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Dinaminas/genética , Hexoquinase/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas
9.
Gene ; 711: 143932, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31202905

RESUMO

Hexokinase (HXK) is a multifunctional protein that serves as a sugar sensor for glucose signaling and a catalyst for glycolysis. It has been well studied in many species, however, there is far less information about this family in pear. To investigate the roles of HXK in the growth and development of pear fruit, we performed a genome-wide analysis and identified the HXK gene family members in pear. In addition, we functionally characterized a glucose sensor gene, PbHXK1, in P. bretschneideri. In total, 10 HXK genes were identified in pear, and a multiple sequence alignment and phylogenetic analysis showed that PbHXK1 is a Type B HXK that contains four conserved domains, phosphate 1 and 2, sugar binding and adenosine, which are specific to plant HXKs and essential for enzymatic functions. A qRT-PCR analysis revealed that the relative expression levels of PbHXK1 were negatively correlated with sugar content but significantly positively correlated with HXK activity during pear fruit development. Furthermore, the overexpression of PbHXK1 in tomatoes significantly enhanced the HXK activity and decreased the sugar content. In addition, the growth of transgenic tomato plants overexpressing PbHXK1 was inhibited, leading to shortened internodes and smaller leaves. Thus, in pear, PbHXK1 encodes HXK, which regulated the sugar content in fruit and affected the growth and development of plants.


Assuntos
Hexoquinase/genética , Hexoquinase/metabolismo , Pyrus/crescimento & desenvolvimento , Sequenciamento Completo do Genoma/métodos , Sítios de Ligação , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hexoquinase/química , Família Multigênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Pyrus/enzimologia , Pyrus/genética , Açúcares/metabolismo
10.
BMC Pharmacol Toxicol ; 20(1): 22, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053173

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies, with an increasing incidence. Despite the fact that systematic chemotherapy with a doxorubicin provides only marginal improvements in survival of the HCC patients, the doxorubicin is being used in transarterial therapies or combined with the target drug - sorafenib. The aim of the study was to evaluate the effect of natural flavonoids on the cytotoxicity of the doxorubicin against human hepatocellular carcinoma cell line HepG2. METHODS: The effect of apigenin and its glycosides - cosmosiin, rhoifolin; baicalein and its glycosides - baicalin as well as hesperetin and its glycosides - hesperidin on glycolytic genes expression of HepG2 cell line, morphology and cells' viability at the presence of doxorubicin have been tested. In an attempt to elucidate the mechanism of observed results, the fluorogenic probe for reactive oxygen species (ROS), the DNA oxidative damage, the lipid peroxidation and the double strand breaks were evaluated. To assess impact on the glycolysis pathway, the mRNA expression for a hexokinase 2 (HK2) and a lactate dehydrogenase A (LDHA) enzymes were measured. The results were analysed statistically with the one-way analysis of variance (ANOVA) and post hoc multiple comparisons. RESULTS: The apigenin and the hesperidin revealed the strongest effect on the toxicity of doxorubicin. Both flavonoids simultaneously changed the expression of the glycolytic pathway genes - HK2 and LDHA, which play a key role in the Warburg effect. Although separate treatment with doxorubicin, apigenin and hesperidin led to a significant oxidative DNA damage and double strand breaks, simultaneous administration of doxorubicin and apigenin or hesperidin abolished these damage with the simultaneous increase in the doxorubicin toxicity. CONCLUSION: The obtained results indicate the existence of a very effective cytotoxic mechanism in the HepG2 cells of the combined effect of doxorubicin and apigenin (or hesperidin), not related to the oxidative stress. To explain this synergy mechanism, further research is needed, The observed intensification of the cytotoxic effect of doxorubicin by this flavonoids may be a promising direction of the research on the therapy of hepatocellular carcinoma, especially in a chemoembolization.


Assuntos
Antineoplásicos/farmacologia , Apigenina/farmacologia , Doxorrubicina/farmacologia , Hesperidina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Sinergismo Farmacológico , Células Hep G2 , Hexoquinase/genética , Humanos , L-Lactato Desidrogenase/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
11.
Mol Med Rep ; 19(6): 4955-4963, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059031

RESUMO

In most cases, exogenous oestradiol benzoate (EB) inhibits spermatogenesis, however, the mechanism underlying this process has not been fully elucidated. The present study investigated the effect of EB on redox equilibrium and glycometabolism in mouse testes. Male Kunming mice were divided into 3 groups and injected with 0, 5 and 10 mg/kg EB, respectively. Histological analysis revealed no sperm and far fewer spermatogenic cells in the testes of EB­treated mice. Additionally, transmission electron microscopy revealed that mitochondria in Sertoli cells were transformed to vacuoles with irregular cristae in the EB­treated group. EB also significantly decreased the activities and mRNA expression of catalase, superoxide dismutase, and glutathione peroxidase and increased the activity of nitric oxide synthase and nitric oxide concentration in the testes compared with the control. These results indicated that oxidative damage was caused by EB treatment. With regard to glycometabolism, ATP content and activities of hexokinase and pyruvate kinase were significantly reduced in the EB­treated group. Although glucose and pyruvate concentrations were significantly increased by EB treatment, levels of lactate, the main energy source of spermatogenic cells, were unchanged. Monocarboxylate transporter 2 (MCT2) and MCT4, which are responsible for lactate transportation, were downregulated by EB. In conclusion, the results of the present study indicated that azoospermia induced by EB in male mice was associated with oxidative damage and the disorder of testicular metabolic cooperation.


Assuntos
Azoospermia/patologia , Estradiol/análogos & derivados , Metaboloma/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Testículo/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Azoospermia/induzido quimicamente , Azoospermia/veterinária , Cromatografia Líquida de Alta Pressão , Regulação para Baixo/efeitos dos fármacos , Estradiol/farmacologia , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Masculino , Camundongos , Microscopia Eletrônica , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Fosforilação/efeitos dos fármacos , Células de Sertoli/ultraestrutura , Espermatogênese/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Testículo/metabolismo , Testículo/ultraestrutura
12.
Molecules ; 24(10)2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-31126138

RESUMO

Guanine-rich DNA strands can adopt tertiary structures known as G-quadruplexes (G4s) that form when Hoogsteen base-paired guanines assemble as planar stacks, stabilized by a central cation like K+. In this study, we investigated the conformational heterogeneity of a G-rich sequence from the 5' untranslated region of the Zea mays hexokinase4 gene. This sequence adopted an extensively polymorphic G-quadruplex, including non-canonical bulged G-quadruplex folds that co-existed in solution. The nature of this polymorphism depended, in part, on the incorporation of different sets of adjacent guanines into a quadruplex core, which permitted the formation of the different conformations. Additionally, we showed that the maize homolog of the human nucleoside diphosphate kinase (NDPK) NM23-H2 protein-ZmNDPK1-specifically recognizes and promotes formation of a subset of these conformations. Heteromorphic G-quadruplexes play a role in microorganisms' ability to evade the host immune system, so we also discuss how the underlying properties that determine heterogeneity of this sequence could apply to microorganism G4s.


Assuntos
DNA de Plantas/química , Hexoquinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Zea mays/enzimologia , Regiões 5' não Traduzidas , Sítios de Ligação , Dicroísmo Circular , DNA de Plantas/metabolismo , Quadruplex G , Hexoquinase/química , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espectrofotometria Ultravioleta , Zea mays/genética
13.
Cell ; 177(5): 1201-1216.e19, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31031005

RESUMO

Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR.


Assuntos
Microambiente Celular/imunologia , Células Dendríticas/imunologia , Imunidade Inata , Mitocôndrias/imunologia , Espécies Reativas de Oxigênio/imunologia , Resposta a Proteínas não Dobradas/imunologia , Animais , Microambiente Celular/genética , Ciclo do Ácido Cítrico/genética , Ciclo do Ácido Cítrico/imunologia , Células Dendríticas/patologia , Hexoquinase/genética , Hexoquinase/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/imunologia
14.
Mol Genet Genomics ; 294(4): 811-847, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30945019

RESUMO

Gestational glucose homeostasis influences mother's metabolic health, pregnancy outcomes, fetal development and offspring growth. To understand the genetic roles in pregnant glucose metabolism and genetic predisposition for gestational diabetes (GDM), we reviewed the recent literature up to Jan, 2018 and evaluated the influence of T2DM-related genetic variants on gestational glycemic traits and glucose tolerance. A total of 140 variants of 89 genes were integrated. Their associations with glycemic traits in and outside pregnancy were compared. The genetic circumstances underlying glucose metabolism exhibit a similarity between pregnant and non-pregnant populations. While, not all of the T2DM-associated genetic variants are related to pregnant glucose tolerance, such as genes involved in fasting insulin/C-peptide regulation. Some genetic variants may have distinct effects on gestational glucose homeostasis. And certain genes may be particularly involved in this process via specific mechanisms, such as HKDC1, MTNR1B, BACE2, genes encoding cell cycle regulators, adipocyte regulators, inflammatory factors and hepatic factors related to gestational glucose sensing and insulin signaling. However, it is currently difficult to evaluate these associations with quantitative synthesis due to inadequate data, different analytical methods, varied measurements for glycemic traits, controversies in diagnosis of GDM, and unknown ethnicity- and/or sex-related influences on pregnant maternal metabolism. In conclusion, different genetic associations with glycemic traits may exist between pregnant and non-pregnant conditions. Comprehensive research on specific genetic regulation in gestation is necessary.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Gestacional/genética , Redes Reguladoras de Genes , Variação Genética , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Feminino , Predisposição Genética para Doença , Hexoquinase/genética , Humanos , Gravidez , Receptor MT2 de Melatonina/genética
15.
PLoS Genet ; 15(4): e1007739, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30990817

RESUMO

Sleep disordered breathing (SDB)-related overnight hypoxemia is associated with cardiometabolic disease and other comorbidities. Understanding the genetic bases for variations in nocturnal hypoxemia may help understand mechanisms influencing oxygenation and SDB-related mortality. We conducted genome-wide association tests across 10 cohorts and 4 populations to identify genetic variants associated with three correlated measures of overnight oxyhemoglobin saturation: average and minimum oxyhemoglobin saturation during sleep and the percent of sleep with oxyhemoglobin saturation under 90%. The discovery sample consisted of 8,326 individuals. Variants with p < 1 × 10(-6) were analyzed in a replication group of 14,410 individuals. We identified 3 significantly associated regions, including 2 regions in multi-ethnic analyses (2q12, 10q22). SNPs in the 2q12 region associated with minimum SpO2 (rs78136548 p = 2.70 × 10(-10)). SNPs at 10q22 were associated with all three traits including average SpO2 (rs72805692 p = 4.58 × 10(-8)). SNPs in both regions were associated in over 20,000 individuals and are supported by prior associations or functional evidence. Four additional significant regions were detected in secondary sex-stratified and combined discovery and replication analyses, including a region overlapping Reelin, a known marker of respiratory complex neurons.These are the first genome-wide significant findings reported for oxyhemoglobin saturation during sleep, a phenotype of high clinical interest. Our replicated associations with HK1 and IL18R1 suggest that variants in inflammatory pathways, such as the biologically-plausible NLRP3 inflammasome, may contribute to nocturnal hypoxemia.


Assuntos
Hexoquinase/genética , Subunidade alfa de Receptor de Interleucina-18/genética , Oxiemoglobinas/metabolismo , Sono/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Moléculas de Adesão Celular Neuronais/genética , Biologia Computacional , Proteínas da Matriz Extracelular/genética , Feminino , Redes Reguladoras de Genes , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Hipóxia/sangue , Hipóxia/genética , Masculino , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas do Tecido Nervoso/genética , Oxigênio/sangue , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Serina Endopeptidases/genética , Síndromes da Apneia do Sono/sangue , Síndromes da Apneia do Sono/genética , Adulto Jovem
16.
Toxicol Lett ; 310: 23-30, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30980912

RESUMO

The aim of this study was to determine whether Pb affects glucose metabolism in the hippocampus of rats. Male Sprague-Dawley rats aged 21 days were orally administered a 0.1%, 0.2%, or 0.3% lead acetate solution in deionized water for 65 days. Then, the weight of the rats; brain Pb content; brain glucose levels; activities of hexokinase, fructose-6-phosphate kinase, pyruvate kinase, glucose-6-phosphate dehydrogenase; expression of genes related to the insulin signaling pathway; as well as the gene and protein expression of glucose transporter (GLUT)-1 and GLUT-3 in the hippocampus were evaluated. The results showed that Pb content in the brain tissue of rats in the dose groups significantly increased, whereas the body weight gain, activities of glucose metabolism-related enzymes, and expression of the insulin signaling pathway-related genes significantly decreased compared to the corresponding values in the control group. In comparison with the control group, the brain glucose levels increased significantly in the low-dose group, but there were no significant differences with the middle- and high-dose groups. Furthermore, the mRNA of GLUT-1 in the three dose groups and the GLUT-3 in the middle- and high-dose groups rose markedly, while the GLUT-1 and GLUT-3 protein expression significantly increased in the middle- and high-dose groups and in the high-dose group, respectively. Taken together, the results showed that Pb exposure resulted in a lower body weight gain, higher brain Pb content and also affected brain glucose metabolism and the insulin signaling pathway.


Assuntos
Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Hipocampo/efeitos dos fármacos , Insulina/metabolismo , Compostos Organometálicos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fosfofrutoquinase-2/genética , Fosfofrutoquinase-2/metabolismo , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Ratos Sprague-Dawley , Medição de Risco , Ganho de Peso/efeitos dos fármacos
17.
Genes (Basel) ; 10(3)2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818878

RESUMO

Recent studies show an association of microRNA (miRNA) polymorphisms (miRSNPs) in different cancer types, including non-Hodgkin lymphoma (NHL). The identification of miRSNPs that are associated with NHL susceptibility may provide biomarkers for early diagnosis and prognosis for patients who may not respond well to current treatment options, including the immunochemotherapy drug combination that includes rituximab, cyclophosphamide, doxorubicin, vincristine and prednisome (R-CHOP). We developed a panel of miRSNPs for genotyping while using multiplex PCR and chip-based mass spectrometry analysis in an Australian NHL case-control population (300 cases, 140 controls). Statistical association with NHL susceptibility was performed while using Chi-square (χ²) and logistic regression analysis. We identified three SNPs in MIR143 that are to be significantly associated with reduced risk of NHL: rs3733846 (odds ratio (OR) [95% confidence interval (CI)] = 0.54 [0.33 ⁻ 0.86], p = 0.010), rs41291957 (OR [95% CI] = 0.61 [0.39 ⁻ 0.94], p = 0.024), and rs17723799 (OR [95% CI] = 0.43 [0.26 ⁻ 0.71], p = 0.0009). One SNP, rs17723799, remained significant after correction for multiple testing (p = 0.015). Subsequently, we investigated an association between the rs17723799 genotype and phenotype by measuring target gene Hexokinase 2 (HKII) expression in cancer cell lines and controls. Our study is the first to report a correlation between miRSNPs in MIR143 and a reduced risk of NHL in Caucasians, and it is supported by significant SNPs in high linkage disequilibrium (LD) in a large European NHL genome wide association study (GWAS) meta-analysis.


Assuntos
Grupo com Ancestrais do Continente Europeu/genética , Linfoma não Hodgkin/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Idoso , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Hexoquinase/genética , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade
18.
Mol Cancer ; 18(1): 33, 2019 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-30825877

RESUMO

BACKGROUND: The long non-coding RNA PVT1 (lncRNA PVT1) has been reported to act as an oncogenic regulator of several cancers. However, its expression and function in gallbladder cancer (GBC) remain largely unknown. METHODS: In situ hybridization (ISH) and quantitative real-time PCR (qPCR) were performed to detect the expression of PVT1 and miR-143 in GBC tissues and cell lines. Immunohistochemistry (IHC) assays were performed to assess the expression of the hexokinase 2 (HK2) protein. The relationships among PVT1, miR-143 and HK2 were evaluated using dual-luciferase reporter, RNA immunoprecipitation (RIP) and biotin pull-down assays. The biological functions of PVT1, miR-143 and HK2 in GBC cells were explored with cell counting kit 8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU), colony formation, transwell, wound healing and glucose metabolism assays in vitro. For in vivo experiments, a xenograft model was used to investigate the effects of PVT1 and HK2 on GBC. RESULTS: PVT1 was upregulated in GBC tissues and cells and was positively associated with malignancies and worse overall survival. PVT1 knockdown inhibited cell proliferation, migration, and invasion in vitro and restrained tumor growth in vivo. Further studies demonstrated that PVT1 positively regulated HK2 expression via its competing endogenous RNA (ceRNA) activity on miR-143. Additionally, HK2 expression and function were positively correlated with PVT1. Furthermore, we observed that the PVT1/miR-143/HK2 axis promoted cell proliferation and metastasis by regulating aerobic glucose metabolism in GBC cells. CONCLUSIONS: The results of our study reveal a potential ceRNA regulatory pathway in which PVT1 modulates HK2 expression by competitively binding to endogenous miR-143 in GBC cells, which may provide new insights into novel molecular therapeutic targets for GBC.


Assuntos
Carcinoma/genética , Neoplasias da Vesícula Biliar/genética , Regulação Neoplásica da Expressão Gênica , Hexoquinase/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Adolescente , Adulto , Animais , Carcinoma/metabolismo , Carcinoma/mortalidade , Carcinoma/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/mortalidade , Neoplasias da Vesícula Biliar/patologia , Glucose/metabolismo , Glicólise/genética , Hexoquinase/metabolismo , Humanos , Metástase Linfática , Masculino , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Parasitol Res ; 118(5): 1511-1518, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30863897

RESUMO

The secretion of hexokinases (HKs) by microsporidia followed by their accumulation in insect host nuclei suggests that these enzymes play regulatory and catalytic roles in infected cells. To confirm whether HKs exert catalytic functions in insect cells, we expressed in E. coli the functionally active HKs of two entomopathogenic microsporidia, Nosema bombycis and Nosema ceranae, that cause silkworm and honey bee nosematoses. N. bombycis HK with C-terminal polyHis tag and N. ceranae enzyme with N-terminal polyHis tag were cloned into pOPE101 and pRSET vectors, respectively, and overexpressed. Specific activities of N. bombycis and N. ceranae enzymes isolated by metal chelate affinity chromatography were 29.2 ± 0.5 and 60.2 ± 1.2 U/mg protein at an optimal pH range of 8.5-9.5. The kinetic characteristics of the recombinant enzymes were similar to those of HKs from other parasitic and free-living organisms. N. bombycis HK demonstrated Km 0.07 ± 0.01 mM and kcat 1726 min-1 for glucose, and Km 0.39 ± 0.05 mM and kcat 1976 min-1 for ATP, at pH 8.8. N. ceranae HK showed Km 0.3 ± 0.04 mM and kcat 3293 min-1 for glucose, and Km 1.15 ± 0.11 mM and kcat 3732 min-1 for ATP, at the same pH value. These data demonstrate the capability of microsporidia-secreted HKs to phosphorylate glucose in infected cells, suggesting that they actively mediate the effects of the parasite on host metabolism. The present findings justify further study of the enzymes as targets to suppress the intracellular development of silkworm and honey bee pathogens.


Assuntos
Abelhas/parasitologia , Bombyx/parasitologia , Hexoquinase/biossíntese , Nosema/metabolismo , Animais , Escherichia coli/genética , Glucose/metabolismo , Hexoquinase/genética , Nosema/classificação , Nosema/isolamento & purificação , Fosforilação
20.
Int J Mol Sci ; 20(6)2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30875800

RESUMO

This study aimed to examine whether inhibition of hexokinase (HK)-II activity enhances the efficacy of sorafenib in in-vivo models of hepatocellular carcinoma (HCC), and to evaluate the prognostic implication of HK-II expression in patients with HCC. We used 3-bromopyruvate (3-BP), a HK-II inhibitor to target HK-II. The human HCC cell line was tested as both subcutaneous and orthotopic tumor xenograft models in BALB/c nu/nu mice. The prognostic role of HK-II was evaluated in data from HCC patients in The Cancer Genome Atlas (TCGA) database and validated in patients treated with sorafenib. Quantitative real-time PCR, western blot analysis, and immunohistochemical staining revealed that HK-II expression is upregulated in the presence of sorafenib. Further analysis of the endoplasmic reticulum-stress network model in two different murine HCC models showed that the introduction of additional stress by 3-BP treatment synergistically increased the in vivo/vitro efficacy of sorafenib. We found that HCC patients with increased HK-II expression in the TCGA database showed poor overall survival, and also confirmed similar results for TCGA database HCC patients who had undergone sorafenib treatment. These results suggest that HK-II is a promising therapeutic target to enhance the efficacy of sorafenib and that HK-II expression might be a prognostic factor in HCC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Hexoquinase/genética , Hexoquinase/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Piruvatos/administração & dosagem , Sorafenibe/administração & dosagem , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Prognóstico , Piruvatos/farmacologia , Sorafenibe/farmacologia , Análise de Sobrevida , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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