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1.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916973

RESUMO

NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) is a member of a gene family with a profound effect on health/disease status. NME7 is an established member of the ciliome and contributes to the regulation of the microtubule-organizing center. We aimed to create a rat model to further investigate the phenotypic consequences of Nme7 gene deletion. The CRISPR/Cas9 nuclease system was used for the generation of Sprague Dawley Nme7 knock-out rats targeting the exon 4 of the Nme7 gene. We found the homozygous Nme7 gene deletion to be semi-lethal, as the majority of SDNme7-/- pups died prior to weaning. The most prominent phenotypes in surviving SDNme7-/- animals were hydrocephalus, situs inversus totalis, postnatal growth retardation, and sterility of both sexes. Thinning of the neocortex was histologically evident at 13.5 day of gestation, dilation of all ventricles was detected at birth, and an external sign of hydrocephalus, i.e., doming of the skull, was usually apparent at 2 weeks of age. Heterozygous SDNme7+/- rats developed normally; we did not detect any symptoms of primary ciliary dyskinesia. The transcriptomic profile of liver and lungs corroborated the histological findings, revealing defects in cell function and viability. In summary, the knock-out of the rat Nme7 gene resulted in a range of conditions consistent with the presentation of primary ciliary dyskinesia, supporting the previously implicated role of the centrosomally located Nme7 gene in ciliogenesis and control of ciliary transport.


Assuntos
Transtornos da Motilidade Ciliar/genética , Genes Letais , Predisposição Genética para Doença , Núcleosídeo-Difosfato Quinase/deficiência , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/diagnóstico , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Genótipo , Imuno-Histoquímica , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fenótipo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transcriptoma , Microtomografia por Raio-X
2.
Int J Mol Sci ; 21(10)2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32466219

RESUMO

Ablation of nucleoside diphosphate kinase B (NDPK-B) in mice causes a breakdown of the neurovascular unit in the retina, mimicking diabetic retinopathy. The NDPK-B deficiency-induced vascular damage is mediated by excessive angiopoietin 2 (Ang2). Herein, the potential involvement of its receptor, Tie2, was investigated. NDPK-B-deficient mouse retinas showed an upregulation of Tie2, specifically in the deep capillary layer. A similar upregulation of Tie2 was observed in cultured endothelial cells (ECs) from different origins upon NDPK-B depletion, whereas high glucose (HG) treatment did not alter Tie2 expression. Immunofluorescence staining and subcellular fractionation showed that the majority of Tie2 upregulation occurred at the plasma membrane. Similar to HG, however, NDPK-B depletion reduced Tie2 tyrosine phosphorylation. Compared to HG, a stronger increase of Ang2 was observed in NDPK-B depleted ECs. Treatment of ECs with soluble Tie2 or siRNA-mediated Tie2 knockdown attenuated NDPK-B depletion- but not HG-induced Ang2 upregulation. Like NDPK-B depletion, overexpression of recombinant Ang2 in ECs enhanced Ang2 secretion and concomitantly promoted the upregulation of Tie2. Thus, we identified a new mechanism showing that after reaching a threshold level of secretion, Ang2 sustains its own expression and secretion by a Tie2-dependent positive feedback loop.


Assuntos
Retinopatia Diabética/metabolismo , Receptor TIE-2/metabolismo , Ribonuclease Pancreático/metabolismo , Animais , Retinopatia Diabética/genética , Retroalimentação Fisiológica , Glucose/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos , Núcleosídeo-Difosfato Quinase/deficiência , Núcleosídeo-Difosfato Quinase/genética , Fosforilação , Receptor TIE-2/genética , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Ribonuclease Pancreático/genética , Transdução de Sinais
3.
Artif Cells Nanomed Biotechnol ; 46(sup1): 896-905, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29475390

RESUMO

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. 5-fluorouracil (5-FU)-based chemotherapeutic regimens are routinely used for the treatment of patients with CRC. However, recurrence and chemotherapeutic drug resistance limit the survival rates of patients with CRC. DNA methylation participates in diverse cellular processes by regulating the transcription of a large number of genes expression, cell division, apoptosis, cell adhesion and differentiation, and metabolism, thus it might mediate chemoresistance. Using an Illumina Infinium HD Assay, DNA methylation levels in a human 5-FU-resistant HCT-8 CRC cell line (HCT-8/FU) and its progenitor cell line HCT-8 were analysed. A total of 16,580 differentially methylated genes were identified, of which 8885 were hypermethylated and 7695 were hypomethylated in resistant cells. Among these genes, NME2 (nucleoside diphosphate kinase 2) exhibited a significant difference in methylation between cell lines and has known roles in gastric cancer and breast cancer; accordingly, we hypothesized that it plays a role in acquired resistance in CRC. Knockdown of NME2 restored 5-FU sensitivity in 5-FU-resistant CRC cells, reduced cell survival and increased cell apoptosis; and overexpression of NME2 in HCT-8 cells results in the acquisition of resistance to 5-FU, this alteration enhanced HCT-8 cells growth abilities and reduced apoptosis. These findings suggest that NME2 mediates chemoresistance to 5-FU in CRC and that specific NME2 inhibition could optimize 5-FU-based chemotherapy of CRC.


Assuntos
Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Fluoruracila/farmacologia , Núcleosídeo-Difosfato Quinase/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Núcleosídeo-Difosfato Quinase/deficiência , Núcleosídeo-Difosfato Quinase/genética
4.
Mol Cell Biochem ; 329(1-2): 45-50, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19381783

RESUMO

Nm23/NDP kinases A and B encoded by the Nme1/Nme2 genes are multifunctional enzymes responsible for the majority of NDP kinase activity in mammals. This review summarizes recent studies on their physiological roles using a mouse model in which both Nme1 and Nme2 genes have been deleted. The double knockout mice are stunted in growth and die perinatally. Additionally, these mice display hematologic phenotypes, including severe anemia, abnormal erythroid cell development, loss of the iron transport receptor molecule TfR1, and reduced iron uptake by Nme1 ( -/- ) /Nme2 ( -/- ) erythroid cells. We hypothesize that Nm23/NDP kinases regulate TfR1 gene expression in erythroid cells in some manner, and that defective iron transport into these cells is responsible for the anemia and death. This Nme1/Nme2 mouse model also links nucleotide metabolism with erythropoiesis, suggesting alternative or additional mechanisms that may explain the observed phenomena.


Assuntos
Eritropoese , Nucleosídeo NM23 Difosfato Quinases/genética , Núcleosídeo-Difosfato Quinase/deficiência , Núcleosídeo-Difosfato Quinase/metabolismo , Animais , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/enzimologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Modelos Animais , Núcleosídeo-Difosfato Quinase/genética
6.
Mol Cell Biol ; 28(6): 1964-73, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18212059

RESUMO

Border cell migration during Drosophila melanogaster oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. Here we show that the metastasis suppressor gene homolog Nm23/awd is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion.


Assuntos
Proteínas de Drosophila/fisiologia , Células Epiteliais/fisiologia , Núcleosídeo-Difosfato Quinase/fisiologia , Ovário/citologia , Animais , Animais Geneticamente Modificados , Movimento Celular , Regulação para Baixo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Dinaminas/deficiência , Dinaminas/genética , Dinaminas/fisiologia , Endocitose , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Sistema de Sinalização das MAP Quinases/fisiologia , Núcleosídeo-Difosfato Quinase/biossíntese , Núcleosídeo-Difosfato Quinase/deficiência , Núcleosídeo-Difosfato Quinase/genética , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Interleucina/genética , Receptores de Interleucina/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/fisiologia
7.
Cell Signal ; 18(10): 1595-603, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16466905

RESUMO

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.


Assuntos
Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Complexos Multienzimáticos/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Pulmão/citologia , Camundongos , Modelos Biológicos , Núcleosídeo-Difosfato Quinase/deficiência , Nucleotídeos/metabolismo , Ligação Proteica
8.
J Bacteriol ; 178(14): 4115-21, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8763939

RESUMO

Bacteriophage T4 encodes nearly all of its own enzymes for synthesizing DNA and its precursors. An exception is nucleoside diphosphokinase (ndk gene product), which catalyzes the synthesis of ribonucleoside triphosphates and deoxyribonucleoside triphosphates (dNTPs) from the corresponding diphosphates. Surprisingly, an Escherichia coli ndk deletion strain grows normally and supports T4 infection. As shown elsewhere, these ndk mutant cells display both a mutator phenotype and deoxyribonucleotide pool abnormalities. However, after T4 infection, both dNTP pools and spontaneous mutation frequencies are near normal. An E. coli strain carrying deletions in ndk and pyrA and pyrF, the structural genes for both pyruvate kinases, also grows and supports T4 infection. We examined anaerobic E. coli cultures because of reports that in anaerobiosis, pyruvate kinase represents the major route for nucleoside triphosphate synthesis in the absence of nucleoside diphosphokinase. The dNTP pool imbalances and the mutator phenotype are less pronounced in the anaerobic than in the corresponding aerobic ndk mutant strains. Anaerobic dNTP pool data, which have not been reported before, reveal a disproportionate reduction in dGTP, relative to the other pools, when aerobic and anaerobic conditions are compared. The finding that mutagenesis and pool imbalances are mitigated in both anaerobic and T4-infected cultures provides strong, if circumstantial, evidence that the mutator phenotype of ndk mutant cells is a result of the dNTP imbalance. Also, the viability of these cells indicates the existence of a second enzyme system in addition to nucleoside diphosphokinase for nucleoside triphosphate synthesis.


Assuntos
Bacteriófago T4/crescimento & desenvolvimento , Escherichia coli/virologia , Mutagênese , Núcleosídeo-Difosfato Quinase/deficiência , Nucleotídeos/metabolismo , Trifosfato de Adenosina , Aerobiose , Anaerobiose , Bacteriófago T4/genética , Desoxirribonucleotídeos/análise , Piruvato Quinase/deficiência
9.
Proc Natl Acad Sci U S A ; 93(12): 5720-5, 1996 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-8650159

RESUMO

Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.


Assuntos
Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Núcleosídeo-Difosfato Quinase/deficiência , Difosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Catálise , Linhagem Celular , Dados de Sequência Molecular
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