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1.
Life Sci ; 230: 150-161, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125564

RESUMO

Despite novel technologies, colon cancer remains undiagnosed and 25% of patients are diagnosed with metastatic colon cancer. Resistant to chemotherapeutic agents is one of the major problems associated with treating colon cancer which creates the need to develop novel agents targeting towards newer targets. A phosphodiesterase is a group of isoenzyme, which, hydrolyze cyclic nucleotides and thereby lowers intracellular levels of cAMP and cGMP leading to tumorigenic effects. Many in vitro and in vivo studies have confirmed increased PDE expression in different types of cancers including colon cancer. cAMP-specific PDE inhibitors increase intracellular cAMP that leads to activation of effector molecules-cAMP-dependent protein kinase A, exchange protein activated by cAMP and cAMP gated ion channels. These molecules regulate cellular responses and exert its anticancer role through different mechanisms including apoptosis, inhibition of angiogenesis, upregulating tumor suppressor genes and suppressing oncogenes. On the other hand, cGMP specific PDE inhibitors exhibit anticancer effects through cGMP dependent protein kinase and cGMP dependent cation channels. Elevation in cGMP works through activation of caspases, suppression of Wnt/b-catenin pathway and TCF transcription leading to inhibition of CDK and survivin. These studies point out towards the fact that PDE inhibition is associated with anti-proliferative, anti-apoptotic and anti-angiogenic pathways involved in its anticancer effects in colon cancer. Thus, inhibition of PDE enzymes can be used as a novel approach to treat colon cancer. This review will focus on cAMP and cGMP signaling pathways leading to tumorigenesis and the use of PDE inhibitors in colon cancer.


Assuntos
Neoplasias do Colo/terapia , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Humanos , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035435

RESUMO

This research aimed to evaluate the expression and clinical implication of autotaxin (ATX)-lysophosphatidate (LPA) signaling-related proteins in breast cancer with adipose stroma. To this end, a tissue microarray (TMA) was constructed from 137 breast cancer tissues with adipose stroma and 329 breast cancer tissues with non-adipose stroma (inflammatory stroma: n = 81, 24.6%; fibrous stroma: n = 246, 75.4%). Immunohistochemical staining for ATX-LPA signaling-related proteins (ATX, LPA1, LPA2, and LPA3) was performed on the TMA. The results showed that LPA2 in tumor cells and LPA3 in stromal cells were highly expressed in breast cancer with adipose stroma and breast cancer with adipose and inflammatory stroma, respectively. Stromal LPA1 positivity (p = 0.017) and stromal LPA3 positivity (p = 0.004) were higher in breast cancer with adipose stroma containing CD68-positive crown-like structures (CLS). Stromal ATX positivity (p = 0.010) and stromal LPA3 positivity (p = 0.009) were higher in breast cancer with adipose tissue containing CD163-positive CLS. In breast cancer with adipose stroma, the number of CD163-positive macrophages was greater with stromal ATX positivity (p = 0.003), and the number of CD68-positive and CD163-positive macrophages were greater in cases with stromal LPA3 positivity. In conclusion, ATX-LPA signaling-related proteins are highly expressed in breast cancer with adipose stroma, with associated macrophage infiltration.


Assuntos
Tecido Adiposo/metabolismo , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Adulto , Idoso , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fenótipo , Prognóstico
3.
Medicine (Baltimore) ; 98(15): e15065, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30985656

RESUMO

Extensive coronary calcification without significant stenosis, described as calcific coronary artery disease (CCAD) may cause abnormal myocardial perfusion and hence generalized ischemia. There is a discrepancy in the expression pattern of CCAD compared to the well-known atherosclerotic disease which raises questions about the exact pathophysiology of coronary calcification and whether there is a genetic etiology for it.In this pilot study we studied 3 candidate genes, ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), ATP Binding Cassette Subfamily C Member 6 (ABCC6), and 5'-Nucleotidase Ecto (NT5E) involved in pyrophosphate (PPi) and inorganic phosphate (Pi) metabolism, which may predispose to coronary arterial or valvular calcification. We studied 70 patients with calcific cardiac disease; 65 with CCAD (age 43-83 years) and 5 with calcific aortic valve disease (CAVD) (age 76-82 years).Five DNA variants potentially affecting protein function were found in 6 patients. One variant is a known disease-causing mutation in the ABCC6 gene. Our findings support that disturbances in the PPi and Pi metabolism might influence the development of CCAD and CAVD. However, segregation in the families must first be performed to ascertain any damaging effect of these variants we have found.We report 4 new genetic variants potentially related to coronary calcification, through the disturbed Pi and PPi metabolism. The search for direct causative genetic variants in coronary artery and aortic valve calcification must be broadened with other genes particularly those involved with Pi and PPi metabolism.


Assuntos
5'-Nucleotidase/genética , Calcinose/genética , Variação Genética , Cardiopatias/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Proteínas Ligadas por GPI/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Fatores Sexuais , Suécia , Rigidez Vascular/genética
4.
Curr Top Med Chem ; 19(7): 555-564, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30931862

RESUMO

BACKGROUND: Phosphodiesterases (PDEs) are enzymes that play a key role in terminating cyclic nucleotides signalling by catalysing the hydrolysis of 3', 5'- cyclic adenosine monophosphate (cAMP) and/or 3', 5' cyclic guanosine monophosphate (cGMP), the second messengers within the cell that transport the signals produced by extracellular signalling molecules which are unable to get into the cells. However, PDEs are proteins which do not operate alone but in complexes that made up of a many proteins. OBJECTIVE: This review highlights some of the general characteristics of PDEs and focuses mainly on the Protein-Protein Interactions (PPIs) of selected PDE enzymes. The objective is to review the role of PPIs in the specific mechanism for activation and thereby regulation of certain biological functions of PDEs. METHODS: The article discusses some of the PPIs of selected PDEs as reported in recent scientific literature. These interactions are critical for understanding the biological role of the target PDE. RESULTS: The PPIs have shown that each PDE has a specific mechanism for activation and thereby regulation a certain biological function. CONCLUSION: Targeting of PDEs to specific regions of the cell is based on the interaction with other proteins where each PDE enzyme binds with specific protein(s) via PPIs.


Assuntos
Sistemas de Liberação de Medicamentos , Regulação Enzimológica da Expressão Gênica/fisiologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Ligação Proteica
5.
Org Lett ; 21(9): 3218-3222, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30995052

RESUMO

An Escherichia coli strain was constructed for the efficient import of nicotinamide adenine dinucleotide (NAD) analogues into cells by limiting extracellular degradation while expressing an efficient NAD importer. In vivo functions of three NAD analogues were characterized. Nicotinamide hypoxanthine dinucleotide was identified as an inhibitor of NAD synthesis. Nicotinamide cytosine dinucleotide had excellent biocompatibility and was used for characterizing a growth-dependent degradation of in vivo nicotinamide cofactors.


Assuntos
Escherichia coli/metabolismo , NAD/análogos & derivados , NAD/metabolismo , Niacinamida/química , Coenzimas/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Citosina/farmacologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hipoxantinas/metabolismo , Hipoxantinas/farmacologia , Estrutura Molecular , Mutação , NAD/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/genética , Pirofosfatases/metabolismo
6.
Medicine (Baltimore) ; 98(13): e14973, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30921203

RESUMO

Recent accumulating evidence indicates the biological actions of Autotaxin-Lysophosphatidic acid (ATX-LPA) signaling axis in malignant tumors. However, the role of Autotaxin-Lysophosphatidic acid signaling axis in breast cancer has not been reported. The present study aims to examine the alterations of serum autotaxin in breast cancer and discuss whether serum autotaxin could be useful as a novel parameter of breast cancer.Serum autotaxin antigen was measured in 112 patients with breast cancer and 50 healthy volunteers by ELISA. The association of serum autotaxin antigen levels with clinicopathological parameters and outcomes of breast cancer was analyzed.Serum autotaxin antigen was significantly higher in breast cancer patients than healthy volunteers (291.32 ±â€Š38.02 ng/ml vs 254.04 ±â€Š21.03 ng/ml, respectively; P < .0001). Serum autotaxin measurement successfully discriminated breast cancer patients from normal and healthy controls (AUC = 0.798, 95% CI: 0.732-0.864) with an optimal cut-off value of 267.34 ng/ml (sensitivity = 0.741, specificity = 0.800). Increased serum autotaxin was associated with breast cancer nodal status (P = .007), Tumor-Node- Metastasis (TNM) stage (P = .009) and Ki-67 index (P = .004). Univariate and multivariate Cox regression analysis revealed that elevated serum autotaxin showed an independent prognostic value for poor Disease-free survival.Our present study confirmed the elevation, potential diagnostic, and independent prognostic value of serum autotaxin for breast cancer. Serum autotaxin could serve as a reliable novel biomarker for breast cancer.


Assuntos
Neoplasias da Mama/sangue , Diester Fosfórico Hidrolases/sangue , Adulto , Idoso , Biomarcadores Tumorais , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Humanos , Lisofosfolipídeos/metabolismo , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/imunologia , Diester Fosfórico Hidrolases/metabolismo , Prognóstico , Transdução de Sinais
7.
Proc Natl Acad Sci U S A ; 116(11): 5071-5076, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30814222

RESUMO

Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2',5'-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.


Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Azacitidina/farmacologia , Desmetilação do DNA , Endorribonucleases/metabolismo , Imunidade Inata , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Morte Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Radiação Ionizante , Bibliotecas de Moléculas Pequenas/farmacologia
8.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845751

RESUMO

Lysophosphatidylcholine (LPC) is increasingly recognized as a key marker/factor positively associated with cardiovascular and neurodegenerative diseases. However, findings from recent clinical lipidomic studies of LPC have been controversial. A key issue is the complexity of the enzymatic cascade involved in LPC metabolism. Here, we address the coordination of these enzymes and the derangement that may disrupt LPC homeostasis, leading to metabolic disorders. LPC is mainly derived from the turnover of phosphatidylcholine (PC) in the circulation by phospholipase A2 (PLA2). In the presence of Acyl-CoA, lysophosphatidylcholine acyltransferase (LPCAT) converts LPC to PC, which rapidly gets recycled by the Lands cycle. However, overexpression or enhanced activity of PLA2 increases the LPC content in modified low-density lipoprotein (LDL) and oxidized LDL, which play significant roles in the development of atherosclerotic plaques and endothelial dysfunction. The intracellular enzyme LPCAT cannot directly remove LPC from circulation. Hydrolysis of LPC by autotaxin, an enzyme with lysophospholipase D activity, generates lysophosphatidic acid, which is highly associated with cancers. Although enzymes with lysophospholipase A1 activity could theoretically degrade LPC into harmless metabolites, they have not been found in the circulation. In conclusion, understanding enzyme kinetics and LPC metabolism may help identify novel therapeutic targets in LPC-associated diseases.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Lisofosfatidilcolinas/metabolismo , Doenças Metabólicas/metabolismo , Fosfolipases A2/metabolismo , Homeostase , Humanos , Hidrólise , Lipoproteínas LDL/metabolismo , Doenças Metabólicas/enzimologia , Fosfatidilcolinas/metabolismo , Diester Fosfórico Hidrolases/metabolismo
9.
EBioMedicine ; 41: 427-442, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827930

RESUMO

BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.


Assuntos
Retrovirus Endógenos/genética , Epigenômica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Redutases do Citocromo/genética , Retrovirus Endógenos/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Diester Fosfórico Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas/genética , Pirofosfatases/genética , Taxa de Sobrevida , Sequências Repetidas Terminais/genética , Enzimas de Conjugação de Ubiquitina/genética
10.
Methods Mol Biol ; 1957: 121-137, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30919351

RESUMO

The many functions of ß-arrestin proteins in the desensitization of G-protein-coupled receptors have been well characterized; however, the discovery that this scaffold protein could actually recruit phosphodiesterases (PDEs) to the site of cAMP synthesis changed the way researchers thought about the static nature of precisely localized cAMP hydrolysis by anchored PDEs. Before this discovery, the compartmentalization of cAMP gradients formed by the activation of specific receptors was generally understood to be underpinned by highly localized pools of specific PDEs that were anchored by large static anchors such as A-kinase-anchoring proteins (AKAPs). Such anchors acted to position cAMP effector proteins such as protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) in places that would allow cAMP concentrations to breach their activation threshold only when a specific receptor activation occurred. In this arrangement PDEs acted as local "sinks" for cAMP and this enforced receptor-specific function by allowing the correct activation of a distinct pool of cAMP effectors in precise localizations. The discovery that ß-arrestin could shuttle cAMP hydrolyzing activity to the membrane shortly after receptor activation added to the complexity of this process by restricting cAMP diffusion into the cell interior for some receptors. This chapter describes the methods used to identify, confirm, and test the function of PDE-ß-arrestin complexes.


Assuntos
Arrestinas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Sequência de Aminoácidos , Arrestinas/química , Sítios de Ligação , Células HEK293 , Humanos , Mutação/genética , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Ubiquitinação
11.
Bull Exp Biol Med ; 166(5): 661-666, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30903487

RESUMO

The antimetastatic activity of combined or individual administration of topotecan and tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitor was examined under various administration schedules in mice with Lewis lung carcinoma modeled by intravenous injection of 200,000 clone/mouse. The greatest antimetastatic effect was observed after combined use of topotecan and Tdp1 inhibitor as documented by macroscopic study of the lungs that revealed the decreased metastatic scores by 76, 91, or 74% at the respective inhibitor doses of 2, 4, or 6 mg/mouse, respectively, in parallel with inhibition of metastasis up to 98% (at inhibitor dose of 4 mg/mouse) and morphological and morphometric analyses of the lung sections, which revealed elevation of metastasis growth delay index to 86 and 63% at the respective inhibitor doses of 4 and 6 mg/mouse, respectively. The combined administration of topotecan and Tdp1 inhibitor is viewed as the most effective way to eliminate the metastatic formations with possible restitution of focal lesions.


Assuntos
Carcinoma Pulmonar de Lewis/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Diester Fosfórico Hidrolases/metabolismo , Topotecan/uso terapêutico , Animais , Carcinoma Pulmonar de Lewis/patologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
Proc Natl Acad Sci U S A ; 116(13): 6335-6340, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30862737

RESUMO

Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S Typhimurium virulence was due to overproduction of c-di-GMP-regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.


Assuntos
GMP Cíclico/análogos & derivados , Citoplasma/metabolismo , Citoplasma/microbiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Salmonella typhimurium/patogenicidade , Animais , Técnicas Biossensoriais/métodos , Celulose/metabolismo , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Fluorescência , Glucosiltransferases , Interações Hospedeiro-Patógeno/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Fagocitose , Diester Fosfórico Hidrolases/metabolismo , Salmonella typhimurium/metabolismo , Virulência
13.
Clin Epigenetics ; 11(1): 41, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846004

RESUMO

BACKGROUND: Epigenetic alternation is a common contributing factor to neoplastic transformation. Although previous studies have reported a cluster of aberrant promoter methylation changes associated with silencing of tumor suppressor genes, little is known concerning their sequential DNA methylation changes during the carcinogenetic process. The aim of the present study was to address a genome-wide search for identifying potentially important methylated changes and investigate the onset and pattern of methylation changes during the progression of colorectal neoplasia. METHODS: A three-phase design was employed in this study. In the screening phase, DNA methylation profile of 12 pairs of colorectal cancer (CRC) and adjacent normal tissues was analyzed by using the Illumina MethylationEPIC BeadChip. Significant CpG sites were selected based on a cross-validation analysis from The Cancer Genome Atlas (TCGA) database. Methylation levels of candidate CpGs were assessed using pyrosequencing in the training dataset (tumor lesions and adjacent normal tissues from 46 CRCs) and the validation dataset (tumor lesions and paired normal tissues from 13 hyperplastic polyps, 129 adenomas, and 256 CRCs). A linear mixed-effects model was used to examine the incremental changes of DNA methylation during the progression of colorectal neoplasia. RESULTS: The comparisons between normal and tumor samples in the screening phase revealed an extensive CRC-specific methylomic pattern with 174,006 (21%) methylated CpG sites, of which 22,232 (13%) were hyermethylated and 151,774 (87%) were hypomethylated. Hypermethylation mostly occurred in CpG islands with an overlap of gene promoters, while hypomethylation tended to be mapped far away from functional regions. Further cross validation analysis from TCGA dataset confirmed 265 hypermethylated promoters coupling with downregulated gene expression. Among which, hypermethylated changes in MEEPD2 promoter was successfully replicated in both training and validation phase. Significant hypermethylation appeared since precursor lesions with an extensive modification in CRCs. The linear mixed-effects modeling analysis found that a cumulative pattern of MPPED2 methylation changes from normal mucosa to hyperplastic polyp to adenoma, and to carcinoma (P < 0.001). CONCLUSIONS: Our findings indicate that epigenetic alterations of MPPED2 promoter region appear sequentially during the colorectal neoplastic progression. It might be able to serve as a promising biomarker for early diagnosis and stage surveillance of colorectal tumorigenesis.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Metilação de DNA , Estudo de Associação Genômica Ampla/métodos , Idoso , Neoplasias Colorretais/diagnóstico , Ilhas de CpG , Progressão da Doença , Detecção Precoce de Câncer , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases , Regiões Promotoras Genéticas , Análise de Sequência de DNA
14.
J Enzyme Inhib Med Chem ; 34(1): 310-321, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30734604

RESUMO

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 µM and 0.59 µM, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 µM. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.


Assuntos
Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Dermatopatias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Picaduras de Aranhas/tratamento farmacológico , Animais , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Humanos , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Coelhos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Aranhas
15.
Life Sci ; 220: 117-126, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710638

RESUMO

AIMS: 24-Hydroxycholesterol (24HC) is the main brain cholesterol metabolite, which level in the circulation is significantly changed under physiological and pathological conditions. Here, we have studied the effect of 24HC on the inotropic responses to ß-adrenoceptor (AR) stimulation. MAIN METHODS: Electrical stimulation-evoked contractions were recorded in isolated atria from mice. Fluorescent dyes, Fluo-4 and DAF-FM, were used for estimation of Ca2+ transient and NO production, respectively. KEY FINDINGS: We revealed that 24HC in the submicromolar range attenuated ß-AR-induced positive inotropy in isolated atria. This was accompanied by a decrease in Ca2+ transient and unchanged nitric oxide (NO) production. However, ß1-AR-induced positive inotropy and enhancement of Ca2+ transient were increased by 24HC due to suppression of NO production. Only ß2-AR-dependent inotropy and enhancement of Ca2+ transient were decreased by 24HC in a NO-independent manner. Inhibition of phosphodiesterase (PDE) suppressed effect of 24HC on ß2-AR-dependent contractility as well as on non-subtype specific ß-AR activation. Moreover, 24HC counteracted positive inopropic action of PDE inhibitors, IBMX and rolipam. Thus, 24HC modulates the effects of ß1- and ß2-AR stimulation via different mechanisms linked with change in activity of NO synthase or PDE, respectively. Under conditions of non-selective activation of ß-ARs, the depressant effect of 24HC related with ß2-AR-dependent signaling dominates. SIGNIFICANCE: We suggest that 24HC could serve as a modulator of atrial ß-AR signaling, contributing to regulation of contractility.


Assuntos
Colesterol 24-Hidroxilase/metabolismo , Hidroxicolesteróis/metabolismo , Óxido Nítrico/metabolismo , Antagonistas Adrenérgicos beta/metabolismo , Animais , Encéfalo/metabolismo , Colesterol/metabolismo , Colesterol 24-Hidroxilase/fisiologia , Átrios do Coração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/metabolismo , Oxisteróis/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Receptores Adrenérgicos beta 2/metabolismo
16.
Phys Chem Chem Phys ; 21(10): 5499-5509, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30785142

RESUMO

In this study, interactions of the catalytically active binuclear form of glycerophosphodiesterase (GpdQ) with four chemically diverse substrates, i.e. NPP (a phosphomonoester), BNPP and GPE (both phosphodiesters), and paraoxon (a phosphotriester) have been investigated using all-atom molecular dynamics (MD) simulations. The roles of metal ions and key amino acid residues, coordination flexibility, and dynamic transformations in all enzyme-substrate complexes have been elucidated. The roles of important first and second coordination shell residues in substrate binding and coordination flexibility of the enzyme suggested by simulations are supported by experimental data. The chemical nature of the substrate is found to influence the mode of binding, electrostatic surface potential, metal-metal distance, and reorganization of the active site. The experimentally proposed association between the substrate binding and coordination flexibility is analyzed using principal component analysis (PCA), movements of loops, and root-mean-square-fluctuations (RMSF) as parameters. The PCA of these substrates provides different energy basins, i.e. one, three, two and five for NPP, BNPP, GPE, and paraoxon, respectively. Additionally, the area of an irregular hexagon (268.3, 288.9, 350.8, and 362.5 Å2) formed by the residues on these loops illustrates their distinct motions. The substrate binding free energies of NPP, BNPP, and GPE are quite close (22.4-24.3 kcal mol-1), but paraoxon interacts with the smallest binding free energy (14.1 kcal mol-1). The metal binding energies in the presence of these substrates are substantially different, i.e. the lowest for NPP and the highest for paraoxon. These results thus provide deeper insight into the chemical promiscuity and coordination flexibility of this important enzyme.


Assuntos
Diester Fosfórico Hidrolases , Domínio Catalítico , Simulação de Dinâmica Molecular , Organofosfatos/química , Paraoxon/química , Ácidos Fosfóricos/química , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Eletricidade Estática , Especificidade por Substrato
17.
Proc Natl Acad Sci U S A ; 116(11): 5144-5153, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30796193

RESUMO

G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (GT*s), each then activating a cGMP-phosphodiesterase catalytic subunit (GT*·PDE*). This high gain at the Rho*-to-GT* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single GT*·PDE* molecular complex in intact mouse rods. Comparing the single-GT*·PDE* effect to the WT single-photon response, both in Gcaps -/- background, gives an effective gain of only ∼12-14 GT*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transducina/metabolismo , Motivos de Aminoácidos , Animais , GMP Cíclico/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinal Luminoso , Camundongos Transgênicos , Mutação/genética , Diester Fosfórico Hidrolases/metabolismo , Fótons , Rodopsina/química , Rodopsina/metabolismo
18.
J Biol Chem ; 294(10): 3432-3443, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622140

RESUMO

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.


Assuntos
Membrana Celular/enzimologia , Coanoflagelados/enzimologia , Diester Fosfórico Hidrolases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Mutação , Domínios Proteicos , Rodopsina/genética , Análise Espectral
19.
Expert Opin Investig Drugs ; 28(3): 261-266, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30678501

RESUMO

INTRODUCTION: Severe, inadequately-controlled asthma remains a clinical challenge. For this reason, clinical trials and preclinical experimental studies on novel agents as an add-on therapies continue emerge. Phosphodiesterases (PDEs) are enzymes that regulate the function of immune cells by hydrolyzing cyclic guanosine monophosphate/cGMP and cyclic adenosine monophosphate/cAMP. PDEs are divided into subfamilies [PDE3, PDE4, PDE5 and PDE7] which are mainly found in the respiratory tract. Inhibitors of PDEs have already been approved for COPD and pulmonary hypertension. AREAS COVERED: The role of PDE inhibitors in asthma treatment and the possible mechanism of action via their anti-inflammatory and/or bronchodilating effect are discussed. EXPERT OPINION: Novel PDE inhibitors exhibiting fewer adverse events may have a role as add-on therapies in asthma treatment in the future. More clinical trials are necessary to prove their efficacy and evaluate their safety profile before approval by regulatory bodies is granted.


Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Inibidores de Fosfodiesterase/farmacologia , Animais , Antiasmáticos/administração & dosagem , Antiasmáticos/efeitos adversos , Asma/fisiopatologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Desenvolvimento de Medicamentos/métodos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/efeitos adversos , Drogas em Investigação/farmacologia , Humanos , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/efeitos adversos , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/metabolismo
20.
J Biochem ; 165(3): 269-275, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629186

RESUMO

Autotaxin (ATX) is a secreted enzyme that produces a bioactive lysophospholipid, lysophosphatidic acid (LPA). ATX plays a role in vascular and neural development in embryos but its mechanisms remain unclear. At the beginning of this study, only one zebrafish atx gene (atxa) was known and had been investigated. In this study, we generated ATX knockout (KO) fish by TALEN targeting atxa. Unexpectedly, atxa KO fish showed neither vascular defects nor reduction of ATX activity, implying the existence of one or more other ATXs in the genome. By a BLAST search using ATXa protein fragments as a query, we found a genomic sequence that closely resembled atxa exons 13, 14 and 15. Consequently, we cloned a cDNA encoding a second zebrafish autotaxin (ATXb), and found that it was transcribed in various tissues. The atxb gene encoded a protein of 832 amino acids (compared to 850 amino acids in ATXa) with 60% amino acid identity to ATXa and clustered with ATXs from other species. A recombinant ATXb protein showed lysophospholipase D (lysoPLD) activities with substrate specificities similar to those of ATXa and mammalian ATXs. These results indicate that ATXb is a second zebrafish ATX, which possibly shares redundant roles with ATXa in embryonic development.


Assuntos
Diester Fosfórico Hidrolases/genética , Peixe-Zebra/genética , Animais , Clonagem Molecular , Mutação , Diester Fosfórico Hidrolases/deficiência , Diester Fosfórico Hidrolases/metabolismo
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