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1.
J Enzyme Inhib Med Chem ; 35(1): 172-186, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31752564

RESUMO

Sphingosine kinase 1 (SphK1) is a promising therapeutic target against several diseases including mammary cancer. The aim of present work is to identify a potent lead compound against breast cancer using ligand-based virtual screening, molecular docking, MD simulations, and the MMPBSA calculations. The LBVS in molecular and virtual libraries yielded 20,800 hits, which were reduced to 621 by several parameters of drug-likeness, lead-likeness, and PAINS. Furthermore, 55 compounds were selected by ADMET descriptors carried forward for molecular interaction studies with SphK1. The binding energy (ΔG) of three screened compounds namely ZINC06823429 (-11.36 kcal/mol), ZINC95421501 (-11.29 kcal/mol), and ZINC95421070 (-11.26 kcal/mol) exhibited stronger than standard drug PF-543 (-9.9 kcal/mol). Finally, it was observed that the ZINC06823429 binds tightly to catalytic site of SphK1 and remain stable during MD simulations. This study provides a significant understanding of SphK1 inhibitors that can be used in the development of potential therapeutics against breast cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Relação Estrutura-Atividade
2.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(4): 308-311, 2019 Jul 28.
Artigo em Chinês | MEDLINE | ID: mdl-31701712

RESUMO

OBJECTIVE: To observe the expressions of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate receptor 2 (S1PR2) in hippocampus of epileptic rats and to investigate the pathogenesis of SphK1 and S1PR2 in epilepsy. METHODS: One hundred and eight male Sprague-Dawley (SD) rats were randomly divided into control group (n=48) and pilocarpine (PILO) group (n=60). A robust convulsive status epilepticus (SE) was induced in PILO group rats by the application of pilocarpine. Control group rats were injected with respective of physiological saline. Pilocarpine group was randomly divided into 6 subgroups (n=8): acute group (E6 h, E1 d, E3 d), latent group (E7 d) and chronic group (E30 d, E56 d). Each subgroup has 8 control rats and 8 epileptic rats. Hippocampal tissue and brain slices were obtained from control rats and rats subjected to the Li-PILO model of epilepsy at 6 h, 1 d, 3 d,7 d,30 d and 56 d after status epilepticus (SE). Western blot technique was used to determine the expressions of SphK1 and S1PR2 in hippocampus at different point of time after pilocarpine treatment. Immunofluorescence was applied to detect the activation and proliferation of hippocampal astrocytes and the localization of SphK1 and S1PR2 in rat hippocampal astrocytes. RESULTS: Compared with control group, the levels of SphK1 in acute phase (E3 d), latent phase (E7 d) and chronic phase (E30 d, E56 d) were significantly increased while the expressions of S1PR2 were decreased in acute phase (E3 d), latent phase (E7 d) and chronic phase (E30 d, E56 d)(P<0.05 or P<0.01). Immunofluorescence results showed astrocyte activation and proliferation in hippocampus of epileptic (E7 d) rats (P<0.05). Confocal microscopy confirmed the preferential expressions of SphK1 and S1PR2 in epileptic rat(E7 d)hippocampal astrocytes. CONCLUSION: The results indicate that SphK1 and S1PR2 may play an important role in the pathogenesis of epilepsy by regulating the activation and proliferation of hippocampal astrocytes and altering neuronal excitability.


Assuntos
Epilepsia/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Animais , Astrócitos/enzimologia , Epilepsia/fisiopatologia , Hipocampo/citologia , Hipocampo/enzimologia , Masculino , Pilocarpina , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley
3.
J Agric Food Chem ; 67(42): 11805-11814, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31566383

RESUMO

The impact of cross-breeding two low phytic acid (lpa) rice mutants on the content of phytic acid and the metabolite profile of the resulting double mutant was investigated. Progenies resulting from the cross of Os-lpa-XS110-1, a rice mutant carrying the myo-inositol kinase (OsMIK) mutated gene, and Os-lpa-XS110-2, with the multidrug resistance-associated protein ABC transporter gene 5 (OsMRP5) as the mutation target, were subjected to high-pressure ion chromatography. The reduction of the phytic acid content in the double mutant (-63%) was much more pronounced than in the single mutants (-26 and -47%). Gas chromatography-based metabolite profiling revealed a superimposition of the metabolite profiles inherited from the lpa progenitors in the double mutant progenies; the resulting metabolite signature was predominated by the OsMIK mutation effect. The study demonstrated that cross-breeding of two single lpa mutants can be employed to generate double lpa rice mutants showing both a significant reduction in the content of phytic acid and the imprinting of a specific mutation-induced metabolite signature.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Hibridização Genética , Oryza/química , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ácido Fítico/análise , Proteínas de Plantas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Mutação , Oryza/genética , Oryza/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Fítico/metabolismo , Proteínas de Plantas/metabolismo , Sementes/química , Sementes/genética , Sementes/metabolismo
4.
Nat Commun ; 10(1): 4196, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519936

RESUMO

Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3'-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3'-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3'-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3'-azido NAD+ provides an important tool for studying cellular PARylation.


Assuntos
NAD/metabolismo , ADP Ribose Transferases/metabolismo , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sirtuína 2/metabolismo
5.
Mol Cell ; 75(5): 1043-1057.e8, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31402097

RESUMO

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.


Assuntos
Proteínas de Transporte/metabolismo , Fosfatidilinositol 4,5-Difosfato/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/genética , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
PLoS Negl Trop Dis ; 13(8): e0007633, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425516

RESUMO

BACKGROUND: Amoebiasis, caused by Entamoeba histolytica infection, is a global public health problem. However, available drugs to treat amoebiasis are currently limited, and no effective vaccine exists. Therefore, development of new preventive measures against amoebiasis is urgently needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, to develop new drugs against amoebiasis, we focused on E. histolytica adenosine 5'-phosphosulfate kinase (EhAPSK), an essential enzyme in Entamoeba sulfolipid metabolism. Fatty alcohol disulfates and cholesteryl sulfate, sulfolipids synthesized in Entamoeba, play important roles in trophozoite proliferation and cyst formation. These processes are closely associated with clinical manifestation and severe pathogenesis of amoebiasis and with disease transmission, respectively. We validated a combination approach of in silico molecular docking analysis and an in vitro enzyme activity assay for large scale screening. Docking simulation ranked the binding free energy between a homology modeling structure of EhAPSK and 400 compounds. The 400 compounds were also screened by a 96-well plate-based in vitro APSK activity assay. Among fifteen compounds identified as EhAPSK inhibitors by the in vitro system, six were ranked by the in silico analysis as having high affinity toward EhAPSK. Furthermore, 2-(3-fluorophenoxy)-N-[4-(2-pyridyl)thiazol-2-yl]-acetamide, 3-phenyl-N-[4-(2-pyridyl)thiazol-2-yl]-imidazole-4-carboxamide, and auranofin, which were identified as EhAPSK inhibitors by both in silico and in vitro analyses, halted not only Entamoeba trophozoite proliferation but also cyst formation. These three compounds also dose-dependently impaired the synthesis of sulfolipids in E. histolytica. CONCLUSIONS/SIGNIFICANCE: Hence, the combined approach of in silico and in vitro-based EhAPSK analyses identified compounds that can be evaluated for their effects on Entamoeba. This can provide leads for the development of new anti-amoebic and amoebiasis transmission-blocking drugs. This strategy can also be applied to identify specific APSK inhibitors, which will benefit research into sulfur metabolism and the ubiquitous pathway terminally synthesizing essential sulfur-containing biomolecules.


Assuntos
Antiprotozoários/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Entamoeba histolytica/enzimologia , Inibidores Enzimáticos/isolamento & purificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Entamebíase/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Testes de Sensibilidade Parasitária , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores
7.
PLoS Pathog ; 15(8): e1007962, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381608

RESUMO

Enteroviruses, members of the family of picornaviruses, are the most common viral infectious agents in humans causing a broad spectrum of diseases ranging from mild respiratory illnesses to life-threatening infections. To efficiently replicate within the host cell, enteroviruses hijack several host factors, such as ACBD3. ACBD3 facilitates replication of various enterovirus species, however, structural determinants of ACBD3 recruitment to the viral replication sites are poorly understood. Here, we present a structural characterization of the interaction between ACBD3 and the non-structural 3A proteins of four representative enteroviruses (poliovirus, enterovirus A71, enterovirus D68, and rhinovirus B14). In addition, we describe the details of the 3A-3A interaction causing the assembly of the ACBD3-3A heterotetramers and the interaction between the ACBD3-3A complex and the lipid bilayer. Using structure-guided identification of the point mutations disrupting these interactions, we demonstrate their roles in the intracellular localization of these proteins, recruitment of downstream effectors of ACBD3, and facilitation of enterovirus replication. These structures uncovered a striking convergence in the mechanisms of how enteroviruses and kobuviruses, members of a distinct group of picornaviruses that also rely on ACBD3, recruit ACBD3 and its downstream effectors to the sites of viral replication.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Picornaviridae/fisiologia , Proteínas Virais/metabolismo , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Conformação Proteica , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/genética
8.
Int J Mol Sci ; 20(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295813

RESUMO

Wound healing starts with the recruitment of inflammatory cells that secrete wound-related factors. This step is followed by fibroblast activation and tissue construction. Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes angiogenesis, cell proliferation, and attracts immune cells. We investigated the roles of S1P in skin wound healing by altering the expression of its biogenic enzyme, sphingosine kinase-1 (SphK1). The murine excisional wound splinting model was used. Sphingosine kinase-1 (SphK1) was highly expressed in murine wounds and that SphK1-/- mice exhibit delayed wound closure along with less angiogenesis and inflammatory cell recruitment. Nanoparticle-mediated topical SphK1 overexpression accelerated wound closure, which associated with increased angiogenesis, inflammatory cell recruitment, and various wound-related factors. The SphK1 overexpression also led to less scarring, and the interaction between transforming growth factor (TGF)-ß1 and S1P receptor-2 (S1PR2) signaling is likely to play a key role. In summary, SphK1 play important roles to strengthen immunity, and contributes early wound healing with suppressed scarring. S1P can be a novel therapeutic molecule with anti-scarring effect in surgical, trauma, and chronic wound management.


Assuntos
Cicatriz/metabolismo , Lisofosfolipídeos/metabolismo , Neovascularização Fisiológica , Pele/metabolismo , Esfingosina/análogos & derivados , Cicatrização , Animais , Biomarcadores , Proliferação de Células , Cicatriz/genética , Cicatriz/patologia , Modelos Animais de Doenças , Expressão Gênica , Granuloma/etiologia , Granuloma/metabolismo , Granuloma/patologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pele/lesões , Pele/patologia , Esfingosina/metabolismo , /metabolismo , Cicatrização/genética
9.
J Ind Microbiol Biotechnol ; 46(8): 1047-1059, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31297713

RESUMO

L-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved L-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroGfbr and tyrAfbr from Escherichia coli to yield strain 45A12, and the L-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product L-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L L-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of L-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the L-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of L-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of L-tyrosine using B. licheniformis, a generally recognized as safe workhorse.


Assuntos
Bacillus licheniformis/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Chiquímico/metabolismo , Tirosina/biossíntese
10.
Int J Mol Sci ; 20(15)2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357484

RESUMO

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.


Assuntos
Luz , Lisofosfolipídeos/biossíntese , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos da radiação , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Esfingosina/análogos & derivados , Estresse Fisiológico/efeitos da radiação , Animais , Apoptose , Linhagem Celular , Modelos Animais de Doenças , Suscetibilidade a Doenças , Eletrorretinografia , Humanos , Luz/efeitos adversos , Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células Fotorreceptoras/patologia , Retina/metabolismo , Retina/patologia , Retina/efeitos da radiação , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/patologia , Esfingosina/biossíntese , Tomografia de Coerência Óptica
11.
Microbiol Immunol ; 63(7): 285-288, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31166044

RESUMO

Phosphatidylinositol-4 kinase III ß (PI4KB) is a host factor that is required for enterovirus (EV) replication. In this study, the importance of host proteins that interact with PI4KB in EV replication was analyzed by trans complementation with PI4KB mutants in a PI4KB-knockout cell line. Ectopically expressed PI4KB mutants, which lack binding regions for ACBD3, RAB11, and 14-3-3 proteins, rescued replication of poliovirus and enterovirus 71. These findings suggest that interaction of PI4KB with these host proteins is not essential for EV replication once PI4KB has been expressed and that PI4KB is functionally independent from these host proteins regarding EV replication.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Enterovirus/metabolismo , Domínios e Motivos de Interação entre Proteínas , Replicação Viral/fisiologia , 1-Fosfatidilinositol 4-Quinase/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Linhagem Celular , Infecções por Enterovirus , Técnicas de Inativação de Genes , Humanos , Proteínas de Membrana/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Poliovirus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
12.
J Microbiol Biotechnol ; 29(6): 923-932, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154747

RESUMO

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce L-phenylalanine (LPhe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36- fold of the strain xllp1 (45.0 g/l). Moreover, the L-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/ g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Fenilalanina/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/genética , Vias Biossintéticas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Glucose/metabolismo , Análise do Fluxo Metabólico , Mutação , Fenilalanina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas
13.
Chem Pharm Bull (Tokyo) ; 67(6): 599-603, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155566

RESUMO

The PF-543 is known as a potent and selective inhibitor of sphingosine kinase (SK) 1 amongst all the SK inhibitors known to date. In a recently reported study by Pfizer on the synthesis of PF-543 derivatives and the SK inhibitory effects, the introduction of propyl moiety into sulfonyl group of PF-543 in the case of 26b revealed an excellent result of 1.7 nM of IC50 of SK1, suggesting the potential substitution of chain structure for benzenesulfonyl structure. In the present work, we aimed for identification of antitumor activity and inhibitory effects of PF-543 derivative containing aliphatic long chain (similar to known SK inhibitors) on SK1. The synthesized compound 2 exhibited an inhibitory effect on SK1 in a manner similar to that of PF-543; the PF-543 derivative manifested similar antitumor activity on HT29, HCT116 (colorectal cancer cell line), and AGS (gastric cancer cell line) cells. Also, from the docking study conducted with PF-543 and compound 2, it was apparent that the aliphatic chain in compound 2 could probably replace benzenesulfonyl structure of PF-543.


Assuntos
Antineoplásicos/síntese química , Pirrolidinas/química , Sulfonas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pirrolidinas/síntese química , Pirrolidinas/farmacologia , Relação Estrutura-Atividade , Sulfonas/síntese química , Sulfonas/farmacologia
14.
Int J Biol Macromol ; 136: 253-265, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170491

RESUMO

Ribokinase (RK) is an ATP dependent sugar kinase that enables the entry of ribose in the metabolism. Leishmania accumulates ribose into the cytosol through hydrolysis of nucleosides and by transport from the extracellular environment. Activation by RK is critical to mobilize the ribose into the metabolism of Leishmania. To understand the catalytic role, the crystal structure of RK (LdRK) from L. donovani was determined in the apo and complex forms with several nucleotides (ATP, AMPPCP and ADP) in the presence of Na+ ion. The dual insertion of five amino acid stretches makes LdRK structurally unique from other reported structures of RKs. The structure of LdRK-ATP provided the basis for positioning of γ-phosphate of ATP by conserved -GAGD- motif. Liganded and unliganded structures of LdRK exists in similar conformation, which suggests binding of nucleotides does not make any significant conformational changes in nucleotide-bound structures. Substitution of a conserved asparagine with phenylalanine in ribose binding pocket differentiates the LdRK from other RKs. Glycerol molecule bound in the substrate binding pocket mimics the enzyme-substrate interactions but in turn, hampers the binding of ribose to LdRK. Comparative structural analysis revealed the flexibility of γ-phosphate, which adopts multiple conformations in the absence of divalent metal ion and ribose. Similar to other RKs, LdRK is also dependent on monovalent as well as divalent cations for its catalytic activity.


Assuntos
Leishmania donovani/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Nucleotídeos/metabolismo , Fosfatos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Conformação Proteica
15.
Artigo em Inglês | MEDLINE | ID: mdl-31245297

RESUMO

Phosphatidylinositol (PtdIns) metabolism is indispensable in eukaryotes. Phosphoinositides (PIs) are phosphorylated derivatives of PtdIns and consist of seven species generated by reversible phosphorylation of the inositol moieties at the positions 3, 4, and 5. Each of the seven PIs has a unique subcellular and membrane domain distribution. In the enteric protozoan parasite Entamoeba histolytica, it has been previously shown that the PIs phosphatidylinositol 3-phosphate (PtdIns3P), PtdIns(4,5)P2, and PtdIns(3,4,5)P3 are localized to phagosomes/phagocytic cups, plasma membrane, and phagocytic cups, respectively. The localization of these PIs in E. histolytica is similar to that in mammalian cells, suggesting that PIs have orthologous functions in E. histolytica. In contrast, the conservation of the enzymes that metabolize PIs in this organism has not been well-documented. In this review, we summarized the full repertoire of the PI kinases and PI phosphatases found in E. histolytica via a genome-wide survey of the current genomic information. E. histolytica appears to have 10 PI kinases and 23 PI phosphatases. It has a panel of evolutionarily conserved enzymes that generate all the seven PI species. However, class II PI 3-kinases, type II PI 4-kinases, type III PI 5-phosphatases, and PI 4P-specific phosphatases are not present. Additionally, regulatory subunits of class I PI 3-kinases and type III PI 4-kinases have not been identified. Instead, homologs of class I PI 3-kinases and PTEN, a PI 3-phosphatase, exist as multiple isoforms, which likely reflects that elaborate signaling cascades mediated by PtdIns(3,4,5)P3 are present in this organism. There are several enzymes that have the nuclear localization signal: one phosphatidylinositol phosphate (PIP) kinase, two PI 3-phosphatases, and one PI 5-phosphatase; this suggests that PI metabolism also has conserved roles related to nuclear functions in E. histolytica, as it does in model organisms.


Assuntos
Entamoeba histolytica/enzimologia , Entamoeba histolytica/metabolismo , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/classificação , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Isoformas de Proteínas , Transdução de Sinais
16.
Biochim Biophys Acta Mol Cell Res ; 1866(9): 1475-1486, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31220477

RESUMO

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Lisofosfolipídeos , Mitocôndrias/patologia , Transdução de Sinais , Esfingosina/análogos & derivados
17.
Int J Mol Sci ; 20(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035587

RESUMO

Phosphatidylinositol (PI)-related signaling plays a pivotal role in many cellular aspects, including survival, cell proliferation, differentiation, DNA damage, and trafficking. PI is the core of a network of proteins represented by kinases, phosphatases, and lipases which are able to add, remove or hydrolyze PI, leading to different phosphoinositide products. Among the seven known phosphoinositides, phosphatidylinositol 5 phosphate (PI5P) was the last to be discovered. PI5P presence in cells is very low compared to other PIs. However, much evidence collected throughout the years has described the role of this mono-phosphoinositide in cell cycles, stress response, T-cell activation, and chromatin remodeling. Interestingly, PI5P has been found in different cellular compartments, including the nucleus. Here, we will review the nuclear role of PI5P, describing how it is synthesized and regulated, and how changes in the levels of this rare phosphoinositide can lead to different nuclear outputs.


Assuntos
Núcleo Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Humanos , Metabolismo dos Lipídeos , Proteínas Nucleares/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Estresse Fisiológico
18.
Nutrients ; 11(5)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064103

RESUMO

Nonalcoholic fatty liver diseases (NAFLD) is characterized by accumulation of lipid droplets in the liver. The objective of this study was to evaluate protective effects of fermented Cordyceps militaris extract by Pediococcus pentosaceus ON188 (ONE) against hepatosteatosis and obesity in mice fed a high-fat diet (HFD). Eight-week-old male C57BL/6J mice were fed HFD mixed with ONE for four weeks and its effects on hepatosteatosis and obesity were examined. Although ONE did not change food intake, it reduced body weights of mice at administration dose of 200 mg/kg/day. Activities of lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT) as plasma parameters were reduced by ONE in a dose-dependent manner. Hepatic lipid droplets and triglyceride (TG) levels were also reduced by ONE due to upregulation of fatty acid oxidizing genes such as carnithine palmitoyltransferase (CPT1) and peroxisomal proliferator activated receptor α(PPARα) mediated by induction of sphingosine kinase 2 (SPHK2). In epididymal fat tissue, sizes of adipocytes were significantly reduced by ONE in a dose-dependent manner. This is mainly due to suppression of lipogenesis and upregulation of adipocyte browning genes. Collectively, these results suggest that fermented ONE can activate fatty acid oxidation via SPHK2 in the liver. It can also suppress lipogenesis and activate browning in adipose tissue. Thus, ONE might have potential to be used for the development of functional foods against liver dysfunction and obesity.


Assuntos
Adipócitos/efeitos dos fármacos , Produtos Biológicos/farmacologia , Cordyceps/química , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/prevenção & controle , Adenosina/química , Tecido Adiposo Branco/citologia , Animais , Produtos Biológicos/química , Desoxiadenosinas/química , Fígado Gorduroso/induzido quimicamente , Fermentação , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regulação para Cima
19.
BMC Vet Res ; 15(1): 155, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101115

RESUMO

BACKGROUND: Sphingosine kinase 1 (SPHK1) is an enzyme that converts pro-apoptotic ceramide and sphingosine into anti-apoptotic sphingosine-1-phosphate. There is growing evidence that SPHK1 activation promotes oncogenic transformation, tumor growth, chemotherapy resistance, and metastatic spread. High SPHK1 expression has been associated with a poor prognosis in several human cancers. RESULTS: In the present study, the expression level of SPHK1 was examined in feline mammary tumor (FMT) specimens, and the IHC expression level of SPHK1 was associated with the histological grade of FMTs. IHC analysis of 88 FMT cases revealed that the expression level of SPHK1 was upregulated in 53 tumor tissues (60.2%) compared to adjacent mammary tissues. SPHK1 expression in FMTs was significantly associated with histological grade, presence of lymphovascular invasion, and estrogen receptor negativity. Treatment of primary FMT cells with SPHK1 inhibitors reduced cell viability, indicating that SPHK1 acts to promote FMT cell survival. These results indicate that SPHK1 may play an important role in FMTs and may be a therapeutic target in cats with FMT. CONCLUSIONS: SPHK1 over-expression in breast cancer tissues is associated with a poor prognosis in humans. SPHK1 over-expression in more aggressive FMTs provides support for a potential role of SPHK1 inhibitors for the treatment of FMTs. Targeting SPHK1 has potent cytotoxic effects in primary FMT cells. These findings suggest that further examination of the role SPHK1 plays in FMTs will pave the way for the investigation of SPHK1 inhibitors in future clinical applications.


Assuntos
Doenças do Gato/patologia , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Vasos Sanguíneos/patologia , Doenças do Gato/enzimologia , Gatos , Feminino , Regulação Neoplásica da Expressão Gênica , Sistema Linfático/patologia , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Invasividade Neoplásica , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo
20.
EBioMedicine ; 44: 375-386, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31105034

RESUMO

BACKGROUND: Emerging evidence suggests that metabolic alterations are a hallmark of cancer cells and contribute to tumor initiation and development. Cancer cells primarily utilize aerobic glycolysis (the Warburg effect) to produce energy and support anabolic growth. The type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) is profoundly implicated in tumorigenesis, however, little is known about its role in reprogrammed energy metabolism. METHODS: Loss- and gain-of-function studies were applied to determine the oncogenic roles of PIPKIγ in colorectal cancer. Transcriptome analysis, real-time qPCR, immunohistochemical staining, Western blotting, and metabolic analysis were carried out to uncover the cellular mechanism of PIPKIγ. FINDINGS: In this study, we showed that PIPKIγ was frequently upregulated in colorectal cancer and predicted a poor prognosis. Genetic silencing of pan-PIPKIγ suppressed cell proliferation and aerobic glycolysis of colorectal cancer. In contrast, the opposite effects were observed by overexpression of PIPKIγ_i2. Importantly, PIPKIγ-induced prolific effect was largely glycolysis-dependent. Mechanistically, PIPKIγ facilitated activation of PI3K/Akt/mTOR signaling pathways to upregulate c-Myc and HIF1α levels, which regulate expression of glycolytic enzymes to enhance glycolysis. Moreover, pharmacological inhibition by PIPKIγ activity with the specific inhibitor UNC3230 significantly inhibited colorectal cancer glycolysis and tumor growth. INTERPRETATION: Our findings reveal a new regulatory role of PIPKIγ in Warburg effect and provide a key contributor in colorectal cancer metabolism with potential therapeutic potentials. FUND: National Key Research and Development Program of China, Outstanding Clinical Discipline Project of Shanghai Pudong, Natural Science Foundation of China, and Science and Technology Commission of Shanghai Municipality.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Glucose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina-Treonina Quinases TOR/metabolismo
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