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1.
PLoS One ; 15(7): e0227466, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678822

RESUMO

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.


Assuntos
Adenosil-Homocisteinase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Evolução Molecular , Adenosil-Homocisteinase/classificação , Adenosil-Homocisteinase/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional , Focalização Isoelétrica , Luz , Filogenia , Folhas de Planta/enzimologia , Processamento de Proteína Pós-Traducional/efeitos da radiação , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Estresse Fisiológico
2.
Nat Commun ; 11(1): 1251, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144268

RESUMO

Ferroptosis is a newly characterized form of regulated cell death mediated by iron-dependent accumulation of lipid reactive oxygen species and holds great potential for cancer therapy. However, the molecular mechanisms underlying ferroptosis remain largely elusive. In this study, we define an integrative role of DJ-1 in ferroptosis. Inhibition of DJ-1 potently enhances the sensitivity of tumor cells to ferroptosis inducers both in vitro and in vivo. Metabolic analysis and metabolite rescue assay reveal that DJ-1 depletion inhibits the transsulfuration pathway by disrupting the formation of the S-adenosyl homocysteine hydrolase tetramer and impairing its activity. Consequently, more ferroptosis is induced when homocysteine generation is decreased, which might be the only source of glutathione biosynthesis when cystine uptake is blocked. Thus, our findings show that DJ-1 determines the response of cancer cells to ferroptosis, and highlight a candidate therapeutic target to potentially improve the effect of ferroptosis-based antitumor therapy.


Assuntos
Adenosil-Homocisteinase/metabolismo , Ferroptose/fisiologia , Neoplasias/patologia , Proteína Desglicase DJ-1/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Ferroptose/efeitos dos fármacos , Fibroblastos , Glutationa/biossíntese , Células HEK293 , Homocisteína/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/tratamento farmacológico , Piperazinas/farmacologia , Cultura Primária de Células , Proteína Desglicase DJ-1/genética , Multimerização Proteica , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Proc Natl Acad Sci U S A ; 117(14): 7755-7763, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193337

RESUMO

Methionine metabolism is critical for the maintenance of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) pluripotency. However, little is known about the regulation of the methionine cycle to sustain ESC pluripotency. Here, we show that adenosylhomocysteinase (AHCY), an important enzyme in the methionine cycle, is critical for the maintenance and differentiation of mouse embryonic stem cells (mESCs). We show that mESCs exhibit high levels of methionine metabolism, whereas decreasing methionine metabolism via depletion of AHCY promotes mESCs to differentiate into the three germ layers. AHCY is posttranslationally modified with an O-linked ß-N-acetylglucosamine sugar (O-GlcNAcylation), which is rapidly removed upon differentiation. O-GlcNAcylation of threonine 136 on AHCY increases its activity and is important for the maintenance of trimethylation of histone H3 lysine 4 (H3K4me3) to sustain mESC pluripotency. Blocking glycosylation of AHCY decreases the ratio of S-adenosylmethionine versus S-adenosylhomocysteine (SAM/SAH), reduces the level of H3K4me3, and poises mESC for differentiation. In addition, blocking glycosylation of AHCY reduces somatic cell reprogramming. Thus, our findings reveal a critical role of AHCY and a mechanistic understanding of O-glycosylation in regulating ESC pluripotency and differentiation.


Assuntos
Metionina/metabolismo , Células-Tronco Pluripotentes/metabolismo , Adenosil-Homocisteinase/metabolismo , Animais , Autorrenovação Celular , Reprogramação Celular , Glicosilação , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células NIH 3T3
4.
Mol Biol Rep ; 47(3): 1821-1834, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31989428

RESUMO

An antioxidant molecule namely, adenosyl homocysteinase (AHc) was identified from the earlier constructed transcriptome database of Spirulina, where it was cultured in a sulphur deprived condition. From the AHc protein, a small peptide NL13 was identified using bioinformatics tools and was predicted to have antioxidant property. Further, the peptide was synthesised and its antioxidant mechanism was addressed at molecular level. NL13 was subjected to various antioxidant assays including DPPH assay, HARS assay, SARS Assay, NO assay and ABTS assay, where NL13 exhibited significant (P < 0.05) potential antioxidant activity compared to its antioxidant control, Trolox. Cytotoxicity was performed on Human whole blood and the cell viability was performed on VERO fibroblast cells. In both assays, it was found that NL13 did not exhibit any cytotoxic effect towards the cells. Further, the intracellular ROS was performed on Multimode reader followed by imaging on fluorescence microscope which showed scavenging activity even at lower concentration of NL13 (31.2 µM). An effective wound healing property of NL13 on VERO cells was confirmed by analysing the cell migration rate at two different time intervals (24 and 48 h). Overall, the study shows that NL13 peptide scavenges the intracellular oxidative stress.


Assuntos
Adenosil-Homocisteinase/química , Antioxidantes/farmacologia , Fibroblastos/citologia , Peptídeos/farmacologia , Spirulina/enzimologia , Cicatrização/efeitos dos fármacos , Animais , Antioxidantes/síntese química , Antioxidantes/química , Proteínas de Bactérias/química , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Chlorocebus aethiops , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Peptídeos/síntese química , Peptídeos/química , Espécies Reativas de Oxigênio/metabolismo , Células Vero
5.
Biochem Biophys Res Commun ; 520(1): 122-127, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31582217

RESUMO

A wealth of studies illustrate the powerful antioxidant activities and health-promoting functions of dietary phenolic compounds, e.g., anthocyanins, flavonoids, and phenolic compounds. Ferulate is methylated from caffeoyl CoA using S-adenosyl-L-methionine (SAM) as methyl donor catalyzed by caffeoyl CoA methyltransferase (CCoAOMT). Here we show that Arabidopsis CCoAOMT7 contributes to ferulate content in the stem cell wall. CCoAOMT7 was further shown to bind S-adenosyl-L-homocysteine hydrolase (SAHH), a critical step in SAM synthesis to release feedback suppression on CCoAOMT. CCoAOMT7 also bound S-adenosyl-L-methionine synthases (SAMSs) in vivo, which were mediated by SAHH1. Interruptions of endogenous SAHH1 by artificial miRNA or SAMSs by T-DNA insertion significantly reduced ferulate contents in the stem cell wall. This data reveals a novel protein complex of SAM synthesis cycle associated with O-methyltransferase and provides new insights into cellular methylation processes.


Assuntos
Adenosil-Homocisteinase/metabolismo , Arabidopsis/enzimologia , Metionina Adenosiltransferase/metabolismo , Metiltransferases/metabolismo , Fenol/química , Catálise , Parede Celular/enzimologia , Ácidos Cumáricos/química , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Genótipo , Hidrólise , Metilação , Mutação , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido
6.
Sci Rep ; 9(1): 11038, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363118

RESUMO

The activated methyl cycle (AMC) is responsible for the generation of S-adenosylmethionine (SAM), which is a substrate of N-acylhomoserine lactone (AHL) synthases. However, it is unknown whether AHL-mediated quorum sensing (QS) plays a role in the metabolic flux of the AMC to ensure cell density-dependent biosynthesis of AHL in cooperative populations. Here we show that QS controls metabolic homeostasis of the AMC critical for AHL biosynthesis and cellular methylation in Burkholderia glumae, the causal agent of rice panicle blight. Activation of genes encoding SAM-dependent methyltransferases, S-adenosylhomocysteine (SAH) hydrolase, and methionine synthases involved in the AMC by QS is essential for maintaining the optimal concentrations of methionine, SAM, and SAH required for bacterial cooperativity as cell density increases. Thus, the absence of QS perturbed metabolic homeostasis of the AMC and caused pleiotropic phenotypes in B. glumae. A null mutation in the SAH hydrolase gene negatively affected AHL and ATP biosynthesis and the activity of SAM-dependent methyltransferases including ToxA, which is responsible for the biosynthesis of a key virulence factor toxoflavin in B. glumae. These results indicate that QS controls metabolic flux of the AMC to secure the biosynthesis of AHL and cellular methylation in a cooperative population.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia/metabolismo , Homeostase , Metiltransferases/metabolismo , Percepção de Quorum , S-Adenosilmetionina/metabolismo , Adenosil-Homocisteinase/genética , Adenosil-Homocisteinase/metabolismo , Proteínas de Bactérias/genética , Burkholderia/fisiologia , Ligases/genética , Ligases/metabolismo , Metilação , Metiltransferases/genética , Mutação , S-Adenosil-Homocisteína/metabolismo
7.
Bioorg Med Chem Lett ; 29(17): 2480-2482, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31358469

RESUMO

Enantiomeric 3-deaza-1',6'-isoneplanocins (C-3 unsubstituted 7a/7b and C-3 with a bromine 8a/8b) lacking the 4'-hydroxymethyl as mechanistically designed anti-viral targets have been prepared by utilizing the Ullmann reaction. Anti-Ebola properties were found for the D-like 7a and 8a and L-like 8b. All four products showed effects against human cytomegalovirus while D-like 7a/8a affected measles; 7a was effective versus norovirus and 8a inhibited Pichinde. Both 7a and 8a produced SAHase inhibitory effects. However, the anti-EBOV activity of 7a and 8a cannot be readily correlated with this observation due with their contrasting IC50 values (8a > 7a). It is to be noted that 7b showed no effects on this enzyme and 8b was minimally inhibitory. These results offer preliminary insight into the differing mechanisms of action of D- and L- like structures and enlighten structural features to guide additional antiviral agent pursuit in the isoneplanocin series.


Assuntos
Adenosina/análogos & derivados , Antivirais/síntese química , Adenosina/síntese química , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Ebolavirus/efeitos dos fármacos , Eritrócitos/enzimologia , Humanos , Norovirus/efeitos dos fármacos , Coelhos , Estereoisomerismo
8.
J Hematol Oncol ; 12(1): 81, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31340867

RESUMO

BACKGROUND: Tamoxifen resistance remains a clinical challenge for hormone receptor-positive breast cancer. Recently, dysregulations in autophagy have been suggested as a potential mechanism for tamoxifen resistance. Although the long noncoding RNA H19 is involved in various stages of tumorigenesis, its role in tamoxifen resistance remains unknown. Here, we assessed the role of H19 in the development of tamoxifen-resistant breast cancer. METHODS: Quantitative real-time PCR analyzed expression of H19 in tamoxifen-resistant breast cancer tissues. Knockdown of H19 was used to assess the sensitivity to tamoxifen in vitro and in vivo. Both knockdown and overexpression of H19 were used to analyze the status of autophagy. Real-time quantitative methylation-specific polymerase chain reaction, chromatin immunoprecipitation, immunofluorescence, and Western blot were used to explore the tamoxifen resistance mechanism of H19. RESULTS: In this study, we observed that the expression of H19 was substantially upregulated in tamoxifen-resistant breast cancer cell line and tumor tissues, and knockdown of H19 enhanced the sensitivity to tamoxifen both in vitro and in vivo. Furthermore, knockdown of H19 significantly inhibited autophagy in MCF7 tamoxifen-resistant (MCF7/TAMR) cells. Conversely, overexpression of H19 promoted autophagy. Interestingly, overexpression of H19 in MCF7 tamoxifen-sensitive cells could recapitulate tamoxifen resistance. Moreover, an increase in methylation in the promoter region of Beclin1 was observed in MCF7/TAMR-shH19 cells. In the double knockdown groups, both shH19+shSAHH and shH19+shDNMT3B rescued the Beclin1 promoter region methylation levels and reactivated autophagy functions. A chromatin immunoprecipitation assay further validated that DNMT3B binds to the Beclin1 promoter region and the knockdown of H19 increases this binding. CONCLUSIONS: Our findings demonstrate that H19 induces autophagy activation via the H19/SAHH/DNMT3B axis, which could contribute to tamoxifen resistance in breast cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , RNA Longo não Codificante/genética , Tamoxifeno/farmacologia , Adenosil-Homocisteinase/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Doxiciclina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Distribuição Aleatória , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Med Chem ; 62(13): 6346-6362, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31244113

RESUMO

The 6'-fluorinated aristeromycins were designed as dual-target antiviral compounds aimed at inhibiting both the viral RNA-dependent RNA polymerase (RdRp) and the host cell S-adenosyl-l-homocysteine (SAH) hydrolase, which would indirectly target capping of viral RNA. The introduction of a fluorine at the 6'-position enhanced the inhibition of SAH hydrolase and the activity against RNA viruses. The adenosine and N6-methyladenosine analogues 2a-e showed potent inhibition against SAH hydrolase, while only the adenosine derivatives 2a-c exhibited potent antiviral activity against all tested RNA viruses such as Middle East respiratory syndrome-coronavirus (MERS-CoV), severe acute respiratory syndrome-coronavirus, chikungunya virus, and/or Zika virus. 6',6'-Difluoroaristeromycin (2c) showed the strongest antiviral effect for MERS-CoV, with a ∼2.5 log reduction in infectious progeny titer in viral load reduction assay. The phosphoramidate prodrug 3a also demonstrated potent broad-spectrum antiviral activity, possibly by inhibiting the viral RdRp. This study shows that 6'-fluorinated aristeromycins can serve as starting points for the development of broad-spectrum antiviral agents that target RNA viruses.


Assuntos
Adenosina/análogos & derivados , Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus de RNA/efeitos dos fármacos , Adenosina/síntese química , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Animais , Antivirais/síntese química , Chlorocebus aethiops , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Halogenação , Humanos , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Células Vero
10.
Fish Shellfish Immunol ; 88: 284-292, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30849500

RESUMO

SAHH is an enzyme, playing a significant role in the catalyzation of the S-adenosyl homocysteine (SAH) into homocysteine (Hcy) and adenosine (Ado). However, little is known information of the enzyme in crustaceans. In the present study, SAHH cDNA was cloned from Litopenaeus vannamei (LvSAHH). The full length of the LvSAHH was found, containing a 5' UTR of 119 bp, an ORF of 1236 bp and a 3' UTR of 549 bp. The LvSAHH gene encoded a polypeptide of 411 amino acids with an estimated molecular mass of 45.55 kD and a predicted isoelectronic point (pI) of 5.63. Comparison of the deduced amino acid sequence showed that LvSAHH has high identity (70 %-82%) with other known species. qRT-PCR analysis revealed that LvSAHH mRNA was broadly expressed in all of the examined tissues, while the highest expression level was observed in muscle, followed by the expression in stomach, gill, pleopod, hepatopancreas, heart, eye and intestine. Subcellular localization analysis revealed that LvSAHH was predominantly localized in the cytoplasm and nucleus. LvSAHH mRNA expression levels in hepatopancreas and gill were significantly up-regulated from 6 to 48 h after V. alginolyticus injection and reached the highest level (15-fold and 8-fold, p < 0.01) at 24 h, respectively. Additionally, the Toll-like receptors (TLR) and interleukins-16 (IL-16) were detected in hepatopancreas and gill of LvSAHH-knockdown SAHH. LvRack1, LvToll1, LvToll2, LvToll3 and LvIL-16 transcripts were decreased significantly in LvSAHH-knockdown shrimp at 24 h post V. alginolyticus stimulation in hepatopancreas and gill. But LvToll3 was no significant difference in gill. In summary, these results indicated that LvSAHH may play a regulatory role in the invertebrate innate immune defense by regulating TLR and IL-16 expression.


Assuntos
Adenosil-Homocisteinase/metabolismo , Penaeidae/imunologia , Vibrio alginolyticus , Adenosil-Homocisteinase/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Expressão Gênica , Técnicas de Silenciamento de Genes , Imunidade Inata/genética , Interleucina-16/metabolismo , Penaeidae/enzimologia , Penaeidae/microbiologia , RNA Mensageiro/metabolismo , Receptores Toll-Like/metabolismo
11.
Sci Adv ; 5(3): eaav2448, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30854431

RESUMO

Profiling the chromatin-bound proteome (chromatome) in a simple, direct, and reliable manner might be key to uncovering the role of yet uncharacterized chromatin factors in physiology and disease. Here, we have designed an experimental strategy to survey the chromatome of proliferating cells by using the DNA-mediated chromatin pull-down (Dm-ChP) technology. Our approach provides a global view of cellular chromatome under normal physiological conditions and enables the identification of chromatin-bound proteins de novo. Integrating Dm-ChP with genomic and functional data, we have discovered an unexpected chromatin function for adenosylhomocysteinase, a major one-carbon pathway metabolic enzyme, in gene activation. Our study reveals a new regulatory axis between the metabolic state of pluripotent cells, ribosomal protein production, and cell division during the early phase of embryo development, in which the metabolic flux of methylation reactions is favored in a local milieu.


Assuntos
Adenosil-Homocisteinase/genética , Adenosil-Homocisteinase/metabolismo , Cromatina/genética , Células-Tronco/metabolismo , Animais , Biologia Computacional/métodos , Epigênese Genética , Genoma , Genômica/métodos , Humanos , Camundongos , Células-Tronco/citologia
12.
Neuroscience ; 406: 38-49, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30849448

RESUMO

Enhancing the migration and phagocytosis of microglial cells is of great significance for the reducing of the risk of the neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The effect of mouse selenoprotein K (mSELENOK) on the migration and phagocytosis of BV2 microglial cells and its mechanism were studied. The results showed that the over-expression of mSELENOK can increase the migratory and phagocytic abilities of the microglial cells, while the knockdown of mSELENOK can decrease the migratory and phagocytic abilities of the cells. The cytosolic free Ca2+ level and inositol trisphosphate receptor (IP3R) mRNA transcript and protein expression were also increased significantly as the consequence of the over-expression of mSELENOK in the microglial cells. On the contrary, the level of cytosolic free Ca2+ and the mRNA transcript and protein expression of IP3R in mSELENOK knockdown cells were decreased significantly. 2-aminoethoxydiphenyl borate (2-APB), an antagonist of IP3R, could prevent the increased migration, phagocytosis, and cytosolic free Ca2+ level of mSELENOK over-expressed microglial cells, and knockdown of IP3R3 could reduce the increased cytosolic Ca2+ level in mSELENOK over-expressed microglial cells. Further studies revealed that selenium supplement (Na2SeO3) can increase the expression of mSELENOK in microglial cells significantly. In summary, these data suggest that mSELENOK can increase cytosolic free Ca2+ level of microglial cells by up-regulating the expression of IP3R, thus enhancing the migration and phagocytosis of microglial cells. Our results indicated that mSELENOK is an important selenoprotein, which plays a role in trace element selenium's functions and can enhance the migration and phagocytosis of microglial cells.


Assuntos
Adenosil-Homocisteinase/biossíntese , Movimento Celular/fisiologia , Citosol/metabolismo , Microglia/metabolismo , Fagocitose/fisiologia , Selenoproteínas/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Camundongos , Regulação para Cima/fisiologia
13.
Circulation ; 139(19): 2260-2277, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30773021

RESUMO

BACKGROUND: Elevated levels of S-adenosylhomocysteine (SAH), the precursor of homocysteine, are positively associated with the risk of cardiovascular disease and with the development and progression of atherosclerosis. However, the role of SAH in endothelial dysfunction is unclear. METHODS: Apolipoprotein E-deficient ( apoE-/-) mice received dietary supplementation with the SAH hydrolase (SAHH) inhibitor adenosine dialdehyde or were intravenously injected with a retrovirus expressing SAHH shRNA. These 2 approaches, along with the heterozygous SAHH gene knockout ( SAHH+/-) mouse model, were used to elevate plasma SAH levels and to examine the role of SAH in aortic endothelial dysfunction. The relationship between plasma SAH levels and endothelial dysfunction was also investigated in human patients with coronary artery disease and healthy control subjects. RESULTS: Plasma SAH levels were increased in SAHH+/- mice and in apoE-/- mice after dietary administration of adenosine dialdehyde or intravenous injection with SAHH shRNA. SAHH+/- mice or apoE-/- mice with SAHH inhibition showed impaired endothelium-dependent vascular relaxation and decreased nitric oxide bioavailability after treatment with acetylcholine; this was completely abolished by the administration of the endothelial nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. Furthermore, SAHH inhibition induced production of reactive oxygen species and p66shc expression in the mouse aorta and human aortic endothelial cells. Antioxidants and p66shc siRNA prevented SAHH inhibition-induced generation of reactive oxygen species and attenuated the impaired endothelial vasomotor responses in high-SAH mice. Moreover, inhibition of SAHH induced hypomethylation in the p66shc gene promoter and inhibited expression of DNA methyltransferase 1. Overexpression of DNA methyltransferase 1, induced by transduction of an adenovirus, was sufficient to abrogate SAHH inhibition-induced upregulation of p66shc expression. Finally, plasma SAH levels were inversely associated with flow-mediated dilation and hypomethylation of the p66shc gene promoter and positively associated with oxidative stress levels in patients with coronary artery disease and healthy control subjects. CONCLUSIONS: Our findings indicate that inhibition of SAHH results in elevated plasma SAH levels and induces endothelial dysfunction via epigenetic upregulation of the p66shc-mediated oxidative stress pathway. Our study provides novel molecular insight into mechanisms of SAH-associated endothelial injury that may contribute to the development of atherosclerosis. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT03345927.


Assuntos
Adenosil-Homocisteinase/metabolismo , Aterosclerose/metabolismo , Doença da Artéria Coronariana/metabolismo , Endotélio Vascular/fisiologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Adenosina/administração & dosagem , Adenosina/análogos & derivados , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/genética , Idoso , Animais , Metilação de DNA , Modelos Animais de Doenças , Epigênese Genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Estresse Oxidativo , RNA Interferente Pequeno/genética , S-Adenosil-Homocisteína/sangue , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética
14.
Arthritis Res Ther ; 21(1): 40, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696480

RESUMO

BACKGROUND: Glomerulonephritis is one of the major complications and causes of death in systemic lupus erythematosus (SLE) and is characterized by glomerulosclerosis, interstitial fibrosis, and tubular atrophy, along with severe persistent proteinuria. DZ2002 is a reversible S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitor with potent therapeutic activity against lupus nephritis in mice. However, the molecular events underlying the renal protective effects of DZ2002 remained unclear. This study is designed to uncover the molecular mechanisms of DZ2002 on glomerulonephritis of lupus-prone mice. METHODS: We conducted a twice-daily treatment of DZ2002 on the lupus-prone NZB/WF1 mice, and the progression of lupus nephritis and alteration of renal function were monitored. The LC-MS-based label-free quantitative (LFQ) proteomic approach was applied to analyze the kidney tissue samples from the normal C57BL/6 mice and the NZB/WF1 mice treated with DZ2002 or vehicle. KEGG pathway enrichment and direct protein-protein interaction (PPI) network analyses were used to map the pathways in which the significantly changed proteins (SCPs) are involved. The selected proteins from proteomic analysis were validated by Western blot analysis and immunohistochemistry in the kidney tissues. RESULTS: The twice-daily regimen of DZ2002 administration significantly ameliorated the lupus nephritis and improved the renal function in NZB/WF1 mice. A total of 3275 proteins were quantified, of which 253 proteins were significantly changed across normal C57BL/6 mice and the NZB/WF1 mice treated with DZ2002 or vehicle. Pathway analysis revealed that 13 SCPs were involved in tight junction and focal adhesion process. Further protein expression validation demonstrated that DZ2002-treated NZB/WF1 mice exhibited downregulation of α-actinin-4 and integrin-linked kinase (ILK), as well as the restoration of ß1-integrin activation in the kidney tissues compared with the vehicle-treated ones. CONCLUSIONS: Our study demonstrated the first evidence for the molecular mechanism of SAHH inhibitor on glomerulonephritis in SLE via the modulation of α-actinin-4 expression and focal adhesion-associated signaling proteins in the kidney.


Assuntos
Actinina/metabolismo , Adenina/análogos & derivados , Adenosil-Homocisteinase/antagonistas & inibidores , Butiratos/farmacologia , Citoesqueleto/metabolismo , Integrinas/metabolismo , Rim/efeitos dos fármacos , Nefrite Lúpica/tratamento farmacológico , Adenina/farmacologia , Adenosil-Homocisteinase/metabolismo , Animais , Regulação para Baixo/efeitos dos fármacos , Humanos , Rim/metabolismo , Nefrite Lúpica/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Substâncias Protetoras/farmacologia , Ligação Proteica/efeitos dos fármacos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteômica/métodos
15.
Bioorg Med Chem Lett ; 28(23-24): 3674-3675, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30385162

RESUMO

A convenient stereospecific synthesis of 6'-fluoro-3-deazaneplanocin (6) has been accomplished from d-ribose in 15 steps. It is reported to possess significant activity towards Ebola (Zaire, Vero, µM: EC50 < 0.36; CC50 125; SI > 347) with moderate inhibition of the target enzyme (S-adenosylhomocysteine hydrolase), which did not correlate directly with its anti-Ebola effects. Compound 6, with limited cytotoxicity, also displayed activity against measles, H1N1 and Pichinde.


Assuntos
Adenosina/análogos & derivados , Antivirais/síntese química , Adenosina/síntese química , Adenosina/química , Adenosina/farmacologia , Adenosil-Homocisteinase/antagonistas & inibidores , Adenosil-Homocisteinase/metabolismo , Animais , Antivirais/química , Antivirais/farmacologia , Chlorocebus aethiops , Ebolavirus/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Concentração Inibidora 50 , Células Vero
16.
Sci Signal ; 11(554)2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30377224

RESUMO

IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl- in Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl- in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl- in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3 - secretion.


Assuntos
Adenosil-Homocisteinase/metabolismo , Calcineurina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cloro/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Biotinilação , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Mutação , Oócitos/metabolismo , Fosforilação , Domínios Proteicos , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Serina/química , Transdução de Sinais , Simportadores de Sódio-Bicarbonato/metabolismo , Fatores de Transcrição/metabolismo , Xenopus
17.
Sci Rep ; 8(1): 14012, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30228286

RESUMO

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.


Assuntos
Adenosina/deficiência , Adenosil-Homocisteinase/antagonistas & inibidores , Biomarcadores Tumorais/análise , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Neoplasias Hepáticas/patologia , Adenosil-Homocisteinase/genética , Adenosil-Homocisteinase/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mutação , Proteoma , RNA Interferente Pequeno/genética , Transcriptoma , Células Tumorais Cultivadas
18.
Neonatology ; 114(4): 337-340, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30121674

RESUMO

A late-preterm infant with a prenatal diagnosis of non-immune hydrops was born with hypotonia, poor respiratory effort, chylothorax, encephalopathy, coagulopathy, progressive hepatic failure, and refractory pulmonary hypertension. Life support was withdrawn at 7 days of life due to multisystem organ failure. Rapid whole exome sequencing revealed novel compound heterozygous mutations in the gene encoding S-adenosylhomocysteine hydrolase (AHCY); each novel variant was carried by an asymptomatic parent. Reports of neonates with other AHCY mutations describe a pathology of varying severity. AHCY mutations should be considered when seeking an etiology for neonates with the combination of non-immune hydrops, hypotonia, encephalopathy, and liver failure.


Assuntos
Adenosil-Homocisteinase/genética , Hidropisia Fetal/genética , Hidropisia Fetal/fisiopatologia , Mutação , Encefalopatias/etiologia , Quilotórax/etiologia , Evolução Fatal , Feminino , Humanos , Hipertensão Pulmonar/etiologia , Recém-Nascido , Falência Hepática/etiologia , Hipotonia Muscular/etiologia , Diagnóstico Pré-Natal
19.
Cell Signal ; 52: 65-73, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30165103

RESUMO

Long noncoding RNAs (lncRNAs) are emerging as important regulators in molecular processes and may play vital roles in odontogenic differentiation of human dental pulp stem cells (hDPSCs). However, their functions remain to be elucidated. As lncRNA H19 is one of the most classical lncRNA, which plays essential roles in cellular differentiation, thus we explored the effects and mechanisms of H19 in odontogenic differentiation of hDPSCs. Stable overexpression and knockdown of H19 in hDPSCs were constructed using recombinant lentiviruses containing H19 and short hairpin-H19 expression cassettes, respectively. Alkaline phosphatase (ALP) assay, Alizarin red staining assay, von kossa staining, quantitative polymerase chain reaction (qPCR), Western blot analysis, and immunofluorescent staining results indicated that overexpression of H19 in hDPSCs positively regulates the odontogenic differentiation of hDPSCs, while knockdown of H19 in hDPSCs inhibits odontogenic differentiation of hDPSCs. Further, we found that H19 promotes the odontogenic differentiation of hDPSCs through S-adenosylhomocysteine hydrolase (SAHH) epigenetically regulates the methylation and expression of distal-less homeobox (DLX3) gene. Herein, for the first time, we determined that H19/SAHH axis epigentically regulates odontogenic differentiaiton of hDPSCs by inhibiting the DNA methyltransferase 3B (DNMT3B)-mediated methylation of DLX3. Our findings provide a new insight into how H19/SAHH axis play its role in odontogenic differentiation of hDPSCs, and would be helpful in developing therapeutic approaches for dentin regeneration based on stem cells.


Assuntos
Adenosil-Homocisteinase/genética , Diferenciação Celular/genética , Polpa Dentária/citologia , Epigênese Genética , Odontogênese/genética , RNA Longo não Codificante/fisiologia , Células-Tronco/metabolismo , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , RNA Longo não Codificante/genética , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Sci Rep ; 8(1): 11334, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30054521

RESUMO

S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K+ ion and one Zn2+ ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K+ ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn2+ ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K+ or Rb+ ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a 23Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn2+ ion is bound in the area of a molecular gate that regulates access to the active site. Zn2+ coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.


Assuntos
Adenosil-Homocisteinase/metabolismo , Metais/farmacologia , Pseudomonas aeruginosa/enzimologia , Adenosil-Homocisteinase/química , Sequência de Aminoácidos , Sítios de Ligação , Cátions , Sequência Conservada , Inibidores Enzimáticos/farmacologia , Glutamina/metabolismo , Cinética , Ligantes , Potássio/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Termodinâmica , Fatores de Tempo , Zinco/farmacologia
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