Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.672
Filtrar
1.
Einstein (Sao Paulo) ; 18: eAO5447, 2020.
Artigo em Inglês, Português | MEDLINE | ID: mdl-33053017

RESUMO

OBJECTIVE: To investigate the possible genes that may be related to the mechanisms that modulate heparanase-1. METHODS: The analysis was conducted at Universidade Federal de São Paulo, on the data provided by: The Cancer Genome Atlas, University of California Santa Cruz Genome Browser, Kyoto Encyclopedia of Genes and Genomes Pathway Database, Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database and the softwares cBioPortal and Ingenuity Pathway Analysis. RESULTS: Using messenger RNA expression pattern of different molecular subtypes of breast cancer, we proposed that heparinase-1 was co-related with its progression. In addition, genes that were analyzed presented co-expression with heparanase-1. The results that showed that heparanase-1 co-expressed with phosphoinositide 3-kinase adapter protein 1, sialic acid-binding immunoglobulin-like lectin 7, and leukocyte-associated immunoglobulin-like receptor 1 are directed related with immune system evasion during breast cancer progression. Furthermore, cathepsin L was co-expressed with heparanase-1 and transformed inactive heparanase-1 form into active heparanase-1, triggering extracellular matrix remodeling, which contributes to enhanced tumor-host interaction of the tumor. CONCLUSION: The signaling pathway analysis using bioinformatics tools gives supporting evidence of possible mechanisms related to breast cancer development. Evasion genes of the immune system co-expressed with heparanase-1, a enzyme related with tumor progression.


Assuntos
Neoplasias da Mama/genética , Glucuronidase/genética , Simulação por Computador , Humanos
2.
Nat Commun ; 11(1): 4664, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938919

RESUMO

Cardiorenal syndrome type 4 (CRS4) is a common complication of chronic kidney disease (CKD), but the pathogenic mechanisms remain elusive. Here we report that morphological and functional changes in myocardial mitochondria are observed in CKD mice, especially decreases in oxidative phosphorylation and fatty acid metabolism. High phosphate (HP), a hallmark of CKD, contributes to myocardial energy metabolism dysfunction by downregulating peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α). Furthermore, the transcriptional factor interferon regulatory factor 1 (IRF1) is revealed as the key molecule upregulated by HP through histone H3K9 acetylation, and responsible for the HP-mediated transcriptional inhibition of PGC1α by directly binding to its promoter region. Conversely, restoration of PGC1α expression or genetic knockdown of IRF1 significantly attenuates HP-induced alterations in vitro and in vivo. These findings demonstrate that IRF1-PGC1α axis-mediated myocardial energy metabolism remodeling plays a crucial role in the pathogenesis of CRS4.


Assuntos
Síndrome Cardiorrenal/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Síndrome Cardiorrenal/patologia , Modelos Animais de Doenças , Regulação para Baixo , Metabolismo Energético , Técnicas de Silenciamento de Genes , Taxa de Filtração Glomerular , Glucuronidase/genética , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Fator Regulador 1 de Interferon/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fosfatos/metabolismo , Regiões Promotoras Genéticas , Ratos , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismo , Adulto Jovem
3.
DNA Cell Biol ; 39(9): 1478-1485, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32584609

RESUMO

Combined antiretroviral therapy has improved quality and life expectancy of people living with human immunodeficiency virus (HIV). However, this therapy increases oxidative stress (OS), which in turn causes alterations in lipid and carbon metabolism, kidney disease, liver cirrhosis, and increased risk of cardiovascular disease. The Klotho gene has been implicated in cardiovascular risk increase. Klotho protein expression at X level decreases the risk of heart disease. HIV-positive people usually present low plasma levels of Klotho; thus, contributing to some extent to an increase in cardiovascular risk for these types of patients, mostly by favoring atherosclerosis. Therefore, our aim is to provide an overview of the effect of OS on Klotho protein and its consequent cardiometabolic alterations in HIV-positive patients on antiretroviral therapy.


Assuntos
Doenças Cardiovasculares/metabolismo , Glucuronidase/metabolismo , Infecções por HIV/metabolismo , Estresse Oxidativo , Animais , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Glucuronidase/genética , Infecções por HIV/tratamento farmacológico , Humanos
4.
J Nutr ; 150(8): 2070-2076, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32470983

RESUMO

BACKGROUND: Dietary supplemental nicotinamide is used to treat hyperphosphatemia in humans. However, the mechanisms of its impact on body phosphorus homeostasis remain unclear. OBJECTIVE: This study was to determine effects and molecular mechanisms of 3 dietary nicotinamide concentrations on body phosphorus homeostasis in laying hens. METHODS: Hy-Line Brown layers (total = 21; 40 wk old; body weight: 1,876 ± 24 g) were individually housed (n = 7) and fed a corn-soybean meal-based diet supplemented with nicotinamide at 20 (N20), 140 (N140), and 1000 (N1000) mg/kg for 21 d. Serum phosphorus and fibroblast growth factor 23 (FGF23) concentrations, phosphorus and calcium excretion, and mRNA and/or protein of type II sodium-phosphate co-transporters (NPt2a, NPt2ab) and FGF23 and FGF23 receptors were measured in the intestines, calvaria, kidney, and liver. RESULTS: Hens in the N1000 group had a 16% lower serum phosphorus concentration and 22% greater phosphorus excretion than those in the N20 or N140 group (P ≤ 0.05). Compared with hens in the N20 group, hens in the N140 and N1000 groups, which did not differ, had 15-21% lower serum FGF23 concentrations, 19-22% greater calcium excretion, 43-56% lower ileum NPT2b protein production, and 1.5- to 1.6-fold greater kidney NPT2a protein production, respectively (all differences at P ≤ 0.05). CONCLUSIONS: Supplementing high concentrations of nicotinamide in diets for laying hens led to accelerated phosphorus and calcium excretions and decreased serum phosphorus and FGF23 concentrations, which were associated with downregulated intestinal NPt2b protein production. Our findings exclude kidney NPt2a protein production as a primary mechanism for the nicotinamide-induced body phosphorus loss.


Assuntos
Galinhas , Regulação da Expressão Gênica/efeitos dos fármacos , Niacinamida/farmacologia , Fósforo/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/metabolismo , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Dieta/veterinária , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Intestinos/efeitos dos fármacos , Intestinos/fisiologia , Rim/efeitos dos fármacos , Rim/metabolismo , Niacinamida/administração & dosagem , Oviposição , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/genética
5.
Oncogene ; 39(24): 4636-4649, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32398866

RESUMO

Klotho is a transmembrane protein, which can be shed and act as a circulating hormone and is involved in regulating cellular calcium levels and inhibition of the PI3K/AKT pathway. As a longevity hormone, it protects normal cells from oxidative stress, and as a tumor suppressor it inhibits growth of cancer cells. Mechanisms governing these differential activities have not been addressed. Altered cellular metabolism is a hallmark of cancer and dysregulation of mitochondrial activity is a hallmark of aging. We hypothesized that klotho exerts its differential effects through regulation of these two hallmarks. Treatment with klotho inhibited glycolysis, reduced mitochondrial activity and membrane potential only in cancer cells. Accordingly, global metabolic screen revealed that klotho altered pivotal metabolic pathways, amongst them glycolysis and tricarboxylic acid cycle in breast cancer cells. Alteration of metabolic activity and increased AMP/ATP ratio lead to LKB1-dependent AMPK activation. Indeed, klotho induced AMPK phosphorylation; furthermore, inhibition of LKB1 partially abolished klotho's tumor suppressor activity. By diminishing deltapsi (Δψ) klotho also inhibited mitochondria Ca2+ shuttling thereby impairing mitochondria communication with SOCE leading to reduced Ca2+ influx by SOCE channels. The reduced SOCE was followed by ER Ca2+ depletion and stress. These data delineate mechanisms mediating the differential effects of klotho toward cancer versus normal cells, and indicate klotho as a potent regulator of metabolic activity.


Assuntos
Neoplasias da Mama/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Glucuronidase/metabolismo , Mitocôndrias/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Glucuronidase/genética , Humanos , Células MCF-7 , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Neoplasias/genética
6.
Adv Exp Med Biol ; 1221: 71-96, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274707

RESUMO

Heparanase is an endo-ß-glucuronidase that cleaves at a limited number of internal sites the glycosaminoglycan heparan sulfate (HS). Heparanase enzymatic activity was first reported in 1975 and by 1983 evidence was beginning to emerge that the enzyme was a facilitator of tumor metastasis by cleaving HS chains present in blood vessel basement membranes and, thereby, aiding the passage of tumor cells through blood vessel walls. Due to a range of technical difficulties, it took another 16 years before heparanase was cloned and characterized in 1999 and a further 14 years before the crystal structure of the enzyme was solved. Despite these substantial deficiencies, there was steady progress in our understanding of heparanase long before the enzyme was fully characterized. For example, it was found as early as 1984 that activated T cells upregulate heparanase expression, like metastatic tumor cells, and the enzyme aids the entry of T cells and other leukocytes into inflammatory sites. Furthermore, it was discovered in 1989 that heparanase releases pre-existing growth factors and cytokines associated with HS in the extracellular matrix (ECM), the liberated growth factors/cytokines enhancing angiogenesis and wound healing. There were also the first hints that heparanase may have functions other than enzymatic activity, in 1995 it being reported that under certain conditions the enzyme could act as a cell adhesion molecule. Also, in the same year PI-88 (Muparfostat), the first heparanase inhibitor to reach and successfully complete a Phase III clinical trial was patented.Nevertheless, the cloning of heparanase (also known as heparanase-1) in 1999 gave the field an enormous boost and some surprises. The biggest surprise was that there is only one heparanase encoding gene in the mammalian genome, despite earlier research, based on substrate specificity, suggesting that there are at least three different heparanases. This surprising conclusion has remained unchanged for the last 20 years. It also became evident that heparanase is a family 79 glycoside hydrolase that is initially produced as a pro-enzyme that needs to be processed by proteases to form an enzymatically active heterodimer. A related molecule, heparanase-2, was also discovered that is enzymatically inactive but, remarkably, recently has been shown to inhibit heparanase-1 activity as well as acting as a tumor suppressor that counteracts many of the pro-tumor properties of heparanase-1.The early claim that heparanase plays a key role in tumor metastasis, angiogenesis and inflammation has been confirmed by many studies over the last 20 years. In fact, heparanase expression is enhanced in all major cancer types, namely carcinomas, sarcomas, and hematological malignancies, and correlates with increased metastasis and poor prognosis. Also, there is mounting evidence that heparanase plays a central role in the induction of inflammation-associated cancers. The enzymatic activity of heparanase has also emerged in unexpected situations, such as in the spread of HS-binding viruses and in Type-1 diabetes where the destruction of intracellular HS in pancreatic insulin-producing beta cells precipitates diabetes. But the most extraordinary recent discoveries have been with the realization that heparanase can exert a range of biological activities that are independent of its enzymatic function, most notably activation of several signaling pathways and being a transcription factor that controls methylation of histone tails. Collectively, these data indicate that heparanase is a truly multifunctional protein that has the additional property of cleaving HS chains and releasing from ECM and cell surfaces hundreds of HS-binding proteins with a plethora of functional consequences. Clearly, there are many unique features of this intriguing molecule that still remain to be explored and are highlighted in this Chapter.


Assuntos
Glucuronidase/história , Glucuronidase/metabolismo , Animais , Glucuronidase/genética , Heparitina Sulfato/metabolismo , História do Século XX , História do Século XXI , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/patologia , Neovascularização Patológica
7.
Adv Exp Med Biol ; 1221: 3-59, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274705

RESUMO

This review summarizes key developments in the heparanase field obtained 20 years prior to cloning of the HPSE gene and nearly 20 years after its cloning. Of the numerous publications and review articles focusing on heparanase, we have selected those that best reflect the progression in the field as well as those we regard important accomplishments with preference to studies performed by scientists and groups that contributed to this book. Apart from a general 'introduction' and 'concluding remarks', the abstracts of these studies are presented essentially as published along the years. We apologize for not being objective and not being able to include some of the most relevant abstracts and references, due to space limitation. Heparanase research can be divided into two eras. The first, initiated around 1975, dealt with identifying the enzyme, establishing the relevant assay systems and investigating its biological activities and significance in cancer and other pathologies. Studies performed during the first area are briefly introduced in a layman style followed by the relevant abstracts presented chronologically, essentially as appears in PubMed. The second era started in 1999 when the heparanase gene was independently cloned by 4 research groups [1-4]. As expected, cloning of the heparanase gene boosted heparanase research by virtue of the readily available recombinant enzyme, molecular probes, and anti-heparanase antibodies. Studies performed during the second area are briefly introduced followed by selected abstracts of key findings, arranged according to specific topics.


Assuntos
Glucuronidase/história , Pesquisa Médica Translacional/história , Glucuronidase/genética , História do Século XX , História do Século XXI , Humanos , Neoplasias
8.
Adv Exp Med Biol ; 1221: 189-229, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274711

RESUMO

In 2019, we mark the 20th anniversary of the cloning of the human heparanase gene. Heparanase remains the only known enzyme to cleave heparan sulfate, which is an abundant component of the extracellular matrix. Thus, elucidating the mechanisms underlying heparanase expression and activity is critical to understanding its role in healthy and pathological settings. This chapter provides a historical account of the race to clone the human heparanase gene, describes the intracellular and extracellular function of the enzyme, and explores the various mechanisms regulating heparanase expression and activity at the gene, transcript, and protein level.


Assuntos
Clonagem Molecular , Glucuronidase/genética , Glucuronidase/metabolismo , Matriz Extracelular , Heparitina Sulfato , Humanos , Transcrição Genética
9.
Adv Exp Med Biol ; 1221: 231-249, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274712

RESUMO

Single Nucleotide Polymorphisms (SNPs) is the substitution of a single nucleotide, stably inherited, highly abundant, and distributed throughout the genome. Up today 9746 SNPs were found in the HPSE gene. During 12 years 21 SNPs were analyzed in normal and pathological samples. The most prominent SNPs are rs4693608, rs11099592, rs4693084, and rs4364254. These SNPs were found in correlation with heparanase mRNA and protein expression among healthy persons. Moreover, an association of the HPSE gene SNPs with inflammatory processes, cancer development and progression was detected. SNP investigation allowed the identification of strong HPSE gene enhancer in the intron 2. In normal leukocytes, heparanase binds to the enhancer region and regulates HPSE gene expression via negative feedback in rs4693608 SNP-dependent manner. In malignant cells, heparanase halted self-regulation of the enhancer region. Instead of heparanase, the helicase-like transcription factor (HLTF) binds to the regulatory region. These and subsequent studies will elucidate how modification in the HPSE enhancer region could be applied to develop new approaches for cancer treatment.


Assuntos
Carcinogênese/genética , Glucuronidase/genética , Inflamação/genética , Neoplasias/genética , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único , Progressão da Doença , Humanos , Íntrons , RNA Mensageiro
10.
Adv Exp Med Biol ; 1221: 351-363, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274717

RESUMO

Heparanase is upregulated in various tumors, and its expression is closely associated with tumor growth, angiogenesis and metastasis, which accomplishes this mainly through degrading heparan sulfate and releasing heparin-binding growth factors thereby influencing multiple signaling pathways. In addition to its enzymatic degrading activity, heparanase can act via its non-enzymatic mechanisms that directly regulate various signaling. This review mainly focuses on the expression levels and role of heparanase in gastric cancer, and multiple genes and mechanisms regulating heparanase expression in gastric cancer. Furthermore, the development of heparanase-targeted immunotherapy and its potential application for treating gastric cancer are discussed.


Assuntos
Glucuronidase/metabolismo , Imunoterapia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/terapia , Progressão da Doença , Glucuronidase/genética , Humanos , Metástase Neoplásica , Neovascularização Patológica , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética
11.
Adv Exp Med Biol ; 1221: 787-805, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32274738

RESUMO

From 1999-2003, Oxford GlycoSciences (OGS) ran a successful drug discovery oncology programme to discover small molecule inhibitors of the Heparanase I enzyme (HPSE1). HPSE1 at the time was widely regarded as being the sole mammalian enzyme capable of cleaving Heparan Sulfate (HS). A second family protein member however called Heparanase 2 (HPSE2) including splice forms was subsequently discovered by PCR analysis based on EST sequences. HPSE2 was found to be expressed mainly in smooth muscle containing tissues, particularly bladder and brain. HPSE2 is poorly expressed in haematopoietic cells and placenta which contrasts with the HPSE1 distribution pattern. HPSE2 binds more strongly to HS than HPSE1 and is believed to out compete for substrate binding and so in effect act as a tumor suppressor. So far, all attempts to show specific HPSE2 endoglycosidase activity against HS have failed suggesting that the enzyme may act as a pseudoenzyme that has evolved to retain only certain non-catalytic heparanase like functions. A breakthrough in the elucidation of functional roles for HPSE2 came about in 2010 with the linkage of HPSE2 gene deletions and mutations to the development of Ochoa/Urofacial Syndrome. Future work into the mechanistic analysis of HPSE2's role in signalling, tumor suppression and bladder/nerve functioning are needed to fully explore the role of this family of proteins.


Assuntos
Clonagem Molecular , Glucuronidase/genética , Animais , Facies , Glucuronidase/classificação , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Síndrome , Doenças Urológicas/genética
12.
Am J Physiol Gastrointest Liver Physiol ; 318(4): G816-G826, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32146834

RESUMO

The gastrointestinal tract houses a reservoir of bacterial-derived enzymes that can directly catalyze the metabolism of drugs, dietary elements and endogenous molecules. Both host and environmental factors may influence this enzymatic activity, with the potential to dictate the availability of the biologically-active form of endogenous molecules in the gut and influence inter-individual variation in drug metabolism. We aimed to investigate the influence of the microbiota, and the modulation of its composition, on fecal enzymatic activity. Intrinsic factors related to the host, including age, sex and genetic background, were also explored. Fecalase, a cell-free extract of feces, was prepared and used in a colorimetric-based assay to quantify enzymatic activity. To demonstrate the functional effects of fecal enzymatic activity, we examined ß-glucuronidase-mediated cleavage of serotonin ß-d-glucuronide (5-HT-GLU) and the resultant production of free 5-HT by HPLC. As expected, ß-glucuronidase and ß-glucosidase activity were absent in germ-free mice. Enzymatic activity was significantly influenced by mouse strain and animal species. Sex and age significantly altered metabolic activity with implications for free 5-HT. ß-Glucuronidase and ß-glucosidase activity remained at reduced levels for nearly two weeks after cessation of antibiotic administration. This effect on fecalase corresponded to significantly lower 5-HT levels as compared with incubation with pre-antibiotic fecalase from the same mice. Dietary targeting of the microbiota using prebiotics did not alter ß-glucuronidase or ß-glucosidase activity. Our data demonstrate that multiple factors influence the activity of bacterial-derived enzymes which may have potential clinical implications for drug metabolism and the deconjugation of host-produced glucuronides in the gut.NEW & NOTEWORTHY This article explores a comprehensive range of host and environmental factors that introduce variability in the expression of bacterial-derived metabolic enzymes. Our results demonstrate that altered ß-glucuronidase activity has implications for the bioavailability of luminal serotonin. The experimental approach employed, fecalase, provides a mechanistic basis and translational platform to further delineate the functional outputs of altered metabolic activity, and the associated physiological effects of microbiota-targeted interventions on host response to drugs and host-produced glucuronides.


Assuntos
Fezes/química , Glucuronidase/metabolismo , Serotonina/metabolismo , beta-Glucosidase/metabolismo , Animais , Antibacterianos , Caspase 1/genética , Caspase 1/metabolismo , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Vida Livre de Germes , Glucuronidase/química , Glucuronidase/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prebióticos , Ratos , Ratos Sprague-Dawley , Serotonina/química , Fatores Sexuais , Suínos , beta-Glucosidase/química , beta-Glucosidase/genética
13.
Talanta ; 212: 120735, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113527

RESUMO

Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Técnicas Eletroquímicas/métodos , Glucuronidase/genética , Compostos de Sulfidrila/química , Fosfatase Alcalina/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Naftalenos/química , Hibridização de Ácido Nucleico , Compostos Organofosforados/química
14.
J Biol Chem ; 295(10): 3115-3133, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32005658

RESUMO

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and ß-glucuronidase assays in vitro and distinct properties in vivo Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N'-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.


Assuntos
Glucuronidase/metabolismo , Insuficiência Renal Crônica/patologia , Animais , Células CHO , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Taxa de Filtração Glomerular/efeitos dos fármacos , Glucuronidase/química , Glucuronidase/genética , Glicopeptídeos/análise , Células HEK293 , Meia-Vida , Humanos , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Insuficiência Renal Crônica/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/veterinária , Relação Estrutura-Atividade
15.
Endocrinology ; 161(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31974587

RESUMO

Growth impairment in mucopolysaccharidoses (MPSs) is an unresolved issue as it is resistant to enzyme replacement therapy (ERT) and growth hormone therapy. C-type natriuretic peptide (CNP) is a promising agent that has growth-promoting effects. Here we investigate the effects of CNP on growth impairment of MPSs using Gusbmps-2J mice, a model for MPS type VII, with combination therapy of CNP and ERT by hydrodynamic gene delivery. Although monotherapies were not sufficient to restore short statures of treated mice, combination therapy resulted in successful restoration. The synergistic effects of CNP and ERT were not only observed in skeletal growth but also in growth plates. ERT reduced cell swelling in the resting zone and increased cell number by accelerating proliferation or inhibiting apoptosis. CNP thickened the proliferative and hypertrophic zones. Regarding changes in the bone, ERT restored bone sclerosis through decreased bone formation and increased bone resorption, and CNP did not adversely affect this process. In addition, improvement of joint deformation by ERT was suggested by analyses of joint spaces and articular cartilage. CNP additively provided restoration of the short stature of MPS VII mice in combination with ERT, which improved abnormalities of growth plates and bone metabolism.


Assuntos
Terapia Genética/métodos , Transtornos do Crescimento/terapia , Mucopolissacaridose VII/terapia , Peptídeo Natriurético Tipo C/uso terapêutico , Animais , Cartilagem Articular/anatomia & histologia , Terapia de Reposição de Enzimas , Glucuronidase/genética , Transtornos do Crescimento/etiologia , Lâmina de Crescimento/anatomia & histologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucopolissacaridose VII/complicações , Peptídeo Natriurético Tipo C/genética
16.
Am J Physiol Renal Physiol ; 318(3): F772-F792, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31984794

RESUMO

Klotho- and beclin 1-driven autophagy extends life. We examined the role of beclin 1 in modifying acute kidney injury (AKI) and whether beclin 1 mediates Klotho's known renoprotective action in AKI. AKI was induced by ischemia-reperfusion injury in mice with different levels of autophagy activity by genetic manipulation: wild-type (WT) mice with normal beclin 1 expression and function, mice with normal beclin 1 levels but high activity through knockin of gain-of-function mutant beclin 1 (Becn1F121A), mice with low beclin 1 levels and activity caused by heterozygous global deletion of beclin 1 (Becn1+/-), or mice with extremely low beclin 1 activity from knockin of the mutant constitutively active beclin 1 inhibitor Bcl-2 (Bcl2AAA). Klotho was increased by transgenic overexpression (Tg-Kl) or recombinant Klotho protein administration. After ischemia-reperfusion injury, Becn1F121A mice (high autophagy) had milder AKI and Becn1+/- and Bcl2AAA mice (low autophagy) had more severe AKI than WT mice. Tg-Kl mice had milder AKI, but its renoprotection was partially attenuated in Becn1+/-;Tg-Kl mice and was significantly reduced, although not completely abolished, in Bcl2AAA;Tg-Kl mice. Recombinant Klotho protein conferred more renoprotection from AKI in WT mice than in Becn1+/- or Bcl2AAA mice. Klotho reduced beclin 1/Bcl-2 protein complexes and increased autophagy activity, but this effect was less prominent in mice or cells with Bcl2AAA. Transfected Bcl2AAA or Becn1F123A decreased or increased autophagy activity and rendered cells more susceptible or more resistant to oxidative cytotoxicity, respectively. In conclusion, beclin 1 confers renoprotection by activating autophagy. Klotho protects the kidney partially via disruption of beclin 1/Bcl-2 interactions and enhancement of autophagy activity.


Assuntos
Autofagia/fisiologia , Proteína Beclina-1/metabolismo , Glucuronidase/metabolismo , Nefropatias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismo por Reperfusão , Animais , Proteína Beclina-1/genética , Linhagem Celular , Regulação da Expressão Gênica , Genótipo , Glucuronidase/genética , Peróxido de Hidrogênio , Nefropatias/etiologia , Camundongos , Gambás , Proteínas Proto-Oncogênicas c-bcl-2/genética
17.
FASEB J ; 34(2): 2087-2104, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907991

RESUMO

Proteinuria is associated with renal function decline and cardiovascular mortality. This association may be attributed in part to alterations of Klotho expression induced by albuminuria, yet the underlying mechanisms are unclear. The presence of albumin decreased Klotho expression in the POD-ATTAC mouse model of proteinuric kidney disease as well as in kidney epithelial cell lines. This downregulation was related to both decreased Klotho transcription and diminished protein half-life, whereas cleavage by ADAM proteases was not modified. The regulation was albumin specific since it was neither observed in the analbuminemic Col4α3-/- Alport mice nor induced by exposure of kidney epithelial cells to purified immunoglobulins. Albumin induced features of ER stress in renal tubular cells with ATF3/ATF4 activation. ATF3 and ATF4 induction downregulated Klotho through altered transcription mediated by their binding on the Klotho promoter. Inhibiting ER stress with 4-PBA decreased the effect of albumin on Klotho protein levels without altering mRNA levels, thus mainly abrogating the increased protein degradation. Taken together, albuminuria decreases Klotho expression through increased protein degradation and decreased transcription mediated by ER stress induction. This implies that modulating ER stress may improve proteinuria-induced alterations of Klotho expression, and hence renal and extrarenal complications associated with Klotho loss.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Albuminúria/metabolismo , Regulação para Baixo , Estresse do Retículo Endoplasmático , Glucuronidase/biossíntese , Túbulos Renais/metabolismo , Transcrição Genética , Fator 3 Ativador da Transcrição/genética , Albuminúria/genética , Albuminúria/patologia , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Glucuronidase/genética , Humanos , Túbulos Renais/patologia , Camundongos , Camundongos Knockout
18.
FASEB J ; 34(2): 3129-3150, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908069

RESUMO

Aging-related organ degeneration is driven by multiple factors including the cell maintenance mechanisms of autophagy, the cytoprotective protein αKlotho, and the lesser known effects of excess phosphate (Pi), or phosphotoxicity. To examine the interplay between Pi, autophagy, and αKlotho, we used the BK/BK mouse (homozygous for mutant Becn1F121A ) with increased autophagic flux, and αKlotho-hypomorphic mouse (kl/kl) with impaired urinary Pi excretion, low autophagy, and premature organ dysfunction. BK/BK mice live longer than WT littermates, and have heightened phosphaturia from downregulation of two key NaPi cotransporters in the kidney. The multi-organ failure in kl/kl mice was rescued in the double-mutant BK/BK;kl/kl mice exhibiting lower plasma Pi, improved weight gain, restored plasma and renal αKlotho levels, decreased pathology of multiple organs, and improved fertility compared to kl/kl mice. The beneficial effects of heightened autophagy from Becn1F121A was abolished by chronic high-Pi diet which also shortened life span in the BK/BK;kl/kl mice. Pi promoted beclin 1 binding to its negative regulator BCL2, which impairs autophagy flux. Pi downregulated αKlotho, which also independently impaired autophagy. In conclusion, Pi, αKlotho, and autophagy interact intricately to affect each other. Both autophagy and αKlotho antagonizes phosphotoxicity. In concert, this tripartite system jointly determines longevity and life span.


Assuntos
Envelhecimento/metabolismo , Autofagia , Glucuronidase/metabolismo , Fosfatos/metabolismo , Animais , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Feminino , Glucuronidase/genética , Células HEK293 , Humanos , Rim/metabolismo , Masculino , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
19.
PLoS One ; 15(1): e0226382, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31929539

RESUMO

Klotho is an age-extending, cognition-enhancing protein found to be down-regulated in aged mammals when age-related diseases start to appear. Low levels of Klotho occur in neurodegenerative diseases, kidney disease and many cancers. Many normal and pathologic processes involve the proteolytic shedding of membrane proteins. Transmembrane (TM) Klotho contains two homologous domains, KL1 and KL2 with homology to glycosidases. After shedding by ADAM 10 and 17, a shed Klotho isoform is released into serum and urine by the kidney, and into the CSF by the choroid plexus. We previously reported that human Klotho contains two major cleavage sites. However, the exact cleavage site responsible for the cleavage between the KL1 and KL2 domains remains unknown for the human Klotho, and both sites are unknown for mouse Klotho. In this study, we aimed to identify the cleavage sites leading to the shed forms of human and mouse Klotho. Mutations in the region close to the TM domain of mouse Klotho result in the reduced shedding of the 130 kD (KL1+KL2) and 70 kD (KL1) fragments, suggesting that the cleavage site lies within the mutated region. We further identified the cleavage sites responsible for the cleavage between KL1 and KL2 of human and mouse Klotho. Moreover, mutated Klotho proteins have similar subcellular localization patterns as wild type Klotho. Finally, in an FGF23 functional assay, all Klotho mutants with a nine amino acid deletion can also function as an FGFR1 co-receptor for FGF23 signaling, however, the signaling activity was greatly reduced. The study provides new and important information on Klotho shedding, and paves the way for studies aimed to distinguish between the distinct roles of the various isoforms of Klotho.


Assuntos
Glucuronidase/metabolismo , Proteína ADAM10/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/química , Glucuronidase/genética , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Mutagênese , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Alinhamento de Sequência , Transdução de Sinais
20.
PLoS One ; 14(12): e0225115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800586

RESUMO

WRI1 is a plant-specific transcription factor that enhances the accumulation of oils through the upregulation of the expression of genes involved in glycolysis and fatty acid biosynthesis. In this study, the EgWRI1 promoter from oil palm was isolated and characterized in transgenic Arabidopsis. The sequence analysis results revealed that various putative plant regulatory elements are present in the EgWRI1 promoter region. The EgWRI1 promoter and beta-glucuronidase (GUS) reporter gene were transcriptionally fused and transformed into Arabidopsis thaliana. Histochemical analysis revealed that GUS staining was very strong in whole seedlings, especially the stems, leaves, and siliques. Moreover, GUS staining was strong in the silique coats but weak in the seeds. Furthermore, to detect whether EgWRI1 was induced by environmental stress, we detected the expression efficiency of the EgWRI1 promoter in transgenic Arabidopsis treated with low temperature, darkness, and exogenous ethylene. The results showed that the activity of the EgWRI1 promoter was induced by darkness but suppressed significantly when exposed to exogenous ethylene. When treated with low temperature, the activity of the EgWRI1 promoter was first reduced after 24 hours but recovered after 48 hours. Taken together, these results reveal the features of the EgWRI1 promoter from oil palm, which will be helpful for improving oil accumulation in oil palm via reasonable cultivation methods.


Assuntos
Arecaceae/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Fatores de Transcrição/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Etilenos/farmacologia , Genes Reporter , Glucuronidase/genética , Glucuronidase/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA