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1.
Environ Pollut ; 291: 118214, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34740292

RESUMO

Polychlorinated biphenyls (PCBs) have been demonstrated as a kind of the persistent organic pollutants (POPs) that could exert complicated influences towards metabolism in human bodies. Since hydroxylated polychlorinated biphenyls (OH-PCBs) are important metabolites of PCBs, our study focuses on investigating the potential inhibitory capability of OH-PCBs on four human sulfotransferase (SULT) isoforms. P-nitrophenol (PNP) was utilized as nonselective probe substrate for this study, and recombinant SULT isoforms were utilized as the enzyme resources. Ultra-performance liquid chromatography (UPLC)-UV detecting system was used to analyze PNP and its metabolite PNP-sulfate. As result, 100 µM of most tested OH-PCBs significantly inhibited the activity of four SULT isoforms. Concentration-dependent inhibition of OH-PCBs towards SULTs was found, and half inhibition concentration values (IC50) of some inhibition processes were determined. Inhibition kinetics (inhibition kinetic type and parameters) were determined using 4'-OH-PCB106 as the representative OH-PCB, SULT1B1 and SULT1E1 as representative SULT isoforms. The inhibition kinetic parameters (Ki) were 1.73 µM and 1.81 µM for the inhibition of 4'-OH-PCB106 towards SULT1B1 and SULT1E1, respectively. In silico docking simulation was utilized to analyze the inhibition capability of 2'-OH-PCB5, 4'-OH-PCB9, 2'-OH-PCB12 towards SULT1A3.All these results obtained in this study are helpful for further understanding the toxicity of PCBs.


Assuntos
Bifenilos Policlorados , Cromatografia Líquida , Humanos , Hidroxilação , Bifenilos Policlorados/toxicidade , Sulfatos , Sulfotransferases/metabolismo
2.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360851

RESUMO

Endothelial cells can acquire a mesenchymal phenotype through a process called Endothelial-to-Mesenchymal transition (EndMT). This event is found in embryonic development, but also in pathological conditions. Blood vessels lose their ability to maintain vascular homeostasis and ultimately develop atherosclerosis, pulmonary hypertension, or fibrosis. An increase in inflammatory signals causes an upregulation of EndMT transcription factors, mesenchymal markers, and a decrease in endothelial markers. In our study, we show that the induction of EndMT results in an increase in long non-coding RNA AERRIE expression. JMJD2B, a known EndMT regulator, induces AERRIE and subsequently SULF1. Silencing of AERRIE shows a partial regulation of SULF1 but showed no effect on the endothelial and mesenchymal markers. Additionally, the overexpression of AERRIE results in no significant changes in EndMT markers, suggesting that AERRIE is marginally regulating mesenchymal markers and transcription factors. This study identifies AERRIE as a novel factor in EndMT, but its mechanism of action still needs to be elucidated.


Assuntos
Transição Epitelial-Mesenquimal , RNA Longo não Codificante/fisiologia , Sulfotransferases/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207476

RESUMO

Heparan sulfate proteoglycans (HSPGs) encompass a group of glycoproteins composed of unbranched negatively charged heparan sulfate (HS) chains covalently attached to a core protein. The complex HSPG biosynthetic machinery generates an extraordinary structural variety of HS chains that enable them to bind a plethora of ligands, including growth factors, morphogens, cytokines, chemokines, enzymes, matrix proteins, and bacterial and viral pathogens. These interactions translate into key regulatory activity of HSPGs on a wide range of cellular processes such as receptor activation and signaling, cytoskeleton assembly, extracellular matrix remodeling, endocytosis, cell-cell crosstalk, and others. Due to their ubiquitous expression within tissues and their large functional repertoire, HSPGs are involved in many physiopathological processes; thus, they have emerged as valuable targets for the therapy of many human diseases. Among their functions, HSPGs assist many viruses in invading host cells at various steps of their life cycle. Viruses utilize HSPGs for the attachment to the host cell, internalization, intracellular trafficking, egress, and spread. Recently, HSPG involvement in the pathogenesis of SARS-CoV-2 infection has been established. Here, we summarize the current knowledge on the molecular mechanisms underlying HSPG/SARS-CoV-2 interaction and downstream effects, and we provide an overview of the HSPG-based therapeutic strategies that could be used to combat such a fearsome virus.


Assuntos
COVID-19/patologia , Proteoglicanas de Heparan Sulfato/metabolismo , SARS-CoV-2/metabolismo , COVID-19/tratamento farmacológico , COVID-19/virologia , Proteoglicanas de Heparan Sulfato/química , Heparina de Baixo Peso Molecular/química , Heparina de Baixo Peso Molecular/metabolismo , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade , Sulfotransferases/metabolismo , Viroses/tratamento farmacológico , Viroses/patologia , Viroses/virologia , Internalização do Vírus/efeitos dos fármacos
4.
Methods Mol Biol ; 2342: 285-300, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272699

RESUMO

The cytosolic sulfotransferase (SULT) enzymes are found in human liver, kidney, intestine, and other tissues. These enzymes catalyze the transfer of the -SO3 group from 3'-phospho-adenosyl-5'-phosphosulfate (PAPS) to a nucleophilic hydroxyl or amine group in a drug substrate. SULTs are stable as dimers, with a highly conserved dimerization domain near the C-terminus of the protein. Crystal structures have revealed flexible loop regions in the native proteins, one of which, located near the dimerization domain, is thought to form a gate that changes position once PAPS is bound to the PAPS-binding site and modulates substrate access and enzyme properties. There is also evidence that oxidation and reduction of certain cysteine residues reversibly regulate the binding of the substrate and PAPS or PAP to the enzyme thus modulating sulfonation. Because SULT enzymes have two substrates, the drug and PAPS, it is common to report apparent kinetic constants with either the drug or the PAPS varied while the other is kept at a constant concentration. The kinetics of product formation can follow classic Michaelis-Menten kinetics, typically over a narrow range of substrate concentrations. Over a wide range of substrate concentrations, it is common to observe partial or complete substrate inhibition with SULT enzymes. This chapter describes the function, tissue distribution, structural features, and properties of the human SULT enzymes and presents examples of enzyme kinetics with different substrates.


Assuntos
Sulfotransferases/química , Sulfotransferases/metabolismo , Xenobióticos/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Distribuição Tecidual , Xenobióticos/química
5.
Bioengineered ; 12(1): 3934-3946, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34288811

RESUMO

Glioblastoma (GBM) is a common malignant tumor of the brain. Members of the carbohydrate sulfotransferase (CHST) family are deregulated in various cancer types. However, limited data are available on the role of the members of the CHST family in the development of GBM. The present study aimed to identify the role of significant members of the CHST family in GBM and explore the effects and molecular mechanisms of these significant members on GBM cell proliferation and mobility. In the current study, we demonstrated that CHST12 is the only member of CHST family that is upregulated in GBM tissues and associated with a lower survival rate according to the data obtained from The Cancer Genome Atlas. Similarly, the expression of CHST12 increased in GBM tissues than in adjacent tissues and had an important diagnostic value in distinguishing tumor tissues from adjacent tissues. The high expression of CHST12 indicated a lower overall survival rate, was negatively associated with the Karnofsky Performance Scale score, was positively associated with the KI67 expression rate, and was an independent risk factor for GBM. Knockdown of CHST12 significantly decreased GBM cell proliferation and mobility and inhibited the Wnt/ß-catenin pathway. Restoration of ß-catenin expression in GBM cells reversed the inhibitory effects of CHST12 knockdown on GBM cell proliferation and mobility. In conclusion, the present study demonstrated that CHST12 may be a novel biomarker for GBM; it regulates GBM cell proliferation and mobility via the WNT/ß-catenin pathway.


Assuntos
Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/patologia , Movimento Celular , Técnicas de Silenciamento de Genes , Glioblastoma/enzimologia , Glioblastoma/patologia , Sulfotransferases/deficiência , Via de Sinalização Wnt , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Sulfotransferases/genética , Sulfotransferases/metabolismo , Análise de Sobrevida , Regulação para Cima/genética
6.
Sci Rep ; 11(1): 14486, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262084

RESUMO

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.


Assuntos
Inibidores Enzimáticos/farmacologia , Leucodistrofia de Células Globoides/tratamento farmacológico , Leucodistrofia Metacromática/tratamento farmacológico , N-Acilesfingosina Galactosiltransferase/antagonistas & inibidores , N-Acilesfingosina Galactosiltransferase/metabolismo , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Galactosilceramidas/metabolismo , Gangliosídeo Galactosiltransferase/genética , Gangliosídeo Galactosiltransferase/metabolismo , Humanos , Leucodistrofia de Células Globoides/mortalidade , Leucodistrofia Metacromática/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Psicosina/análogos & derivados , Psicosina/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfotransferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
7.
Commun Biol ; 4(1): 674, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083726

RESUMO

The terminal galactose residues of N- and O-glycans in animal glycoproteins are often sialylated and/or fucosylated, but sulfation, such as 3-O-sulfated galactose (3-O-SGal), represents an additional, but poorly understood modification. To this end, we have developed a novel sea lamprey variable lymphocyte receptor (VLR) termed O6 to explore 3-O-SGal expression. O6 was engineered as a recombinant murine IgG chimera and its specificity and affinity to the 3-O-SGal epitope was defined using a variety of approaches, including glycan and glycoprotein microarray analyses, isothermal calorimetry, ligand-bound crystal structure, FACS, and immunohistochemistry of human tissue macroarrays. 3-O-SGal is expressed on N-glycans of many plasma and tissue glycoproteins, but recognition by O6 is often masked by sialic acid and thus exposed by treatment with neuraminidase. O6 recognizes many human tissues, consistent with expression of the cognate sulfotransferases (GAL3ST-2 and GAL3ST-3). The availability of O6 for exploring 3-O-SGal expression could lead to new biomarkers for disease and aid in understanding the functional roles of terminal modifications of glycans and relationships between terminal sulfation, sialylation and fucosylation.


Assuntos
Epitopos/metabolismo , Galactose/análogos & derivados , Glicoproteínas/metabolismo , Lampreias/metabolismo , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Fucose/metabolismo , Galactose/metabolismo , Glicoproteínas/química , Glicosilação , Células HEK293 , Humanos , Lampreias/imunologia , Ligantes , Espectrometria de Massas/métodos , Ácido N-Acetilneuramínico/metabolismo , Sulfatos/metabolismo , Sulfotransferases/química , Sulfotransferases/genética , Sulfotransferases/metabolismo
8.
Expert Opin Drug Metab Toxicol ; 17(7): 767-784, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34107842

RESUMO

INTRODUCTION: Cytosolic sulfotransferases (SULTs)-mediated sulfation is critically involved in the metabolism of key endogenous compounds, such as catecholamines and thyroid/steroid hormones, as well as a variety of drugs and other xenobiotics. Studies performed in the past three decades have yielded a good understanding about the enzymology of the SULTs and their structural biology, phylogenetic relationships, tissue/organ-specific/developmental expression, as well as the regulation of the SULT gene expression. An emerging area is related to the functional impact of the SULT genetic polymorphisms. AREAS COVERED: The current review aims to summarize our current knowledge about the above-mentioned aspects of the SULT research. An emphasis is on the information concerning the effects of the polymorphisms of the SULT genes on the functional activity of the SULT allozymes and the associated physiological, pharmacological, and clinical implications. EXPERT OPINION: Elucidation of how SULT SNPs may influence the drug-sulfating activity of SULT allozymes will help understand the differential drug metabolism and eventually aid in formulating personalized drug regimens. Moreover, the information concerning the differential sulfating activities of SULT allozymes toward endogenous compounds may allow for the development of strategies for mitigating anomalies in the metabolism of these endogenous compounds in individuals with certain SULT genotypes.


Assuntos
Preparações Farmacêuticas/metabolismo , Sulfotransferases/genética , Animais , Citosol/metabolismo , Regulação Enzimológica da Expressão Gênica , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Sulfatos/metabolismo , Sulfotransferases/metabolismo
9.
Biochem Pharmacol ; 190: 114662, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34157297

RESUMO

Circulating estrogens levels significantly decrease in menopause and levels off in postmenopausal women. Accordingly, the liver represses levels of enzymes and membrane transporters, thereby decreasing capability of inactivating and excreting estrogens. Women increasingly develop type 2 diabetes during or after menopause. Estrogens are known to promote liver diseases in these women. Here, we have found that the estrogen inactivating sulfotransferase (SULT1E1) and an ATP-binding cassette subfamily G member 2 (ABCG2), a gene encoding breast cancer resistance protein that exports sulfated estrogens, increased their expression levels in diabetic women but not men. For the sulfotransferase gene, phosphorylated nuclear receptors ERα and RORα, at Ser212 and Ser100, respectively, bind their response elements to activate the SULT1E1 promoter in women. This coordinated increase in estrogen inactivation and excretion, and the phosphorylated nuclear receptor-mediated gene activation could be a defense mechanism against toxicities of estrogens through inactivation and excretion in the livers of women.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptor alfa de Estrogênio/metabolismo , Proteínas de Neoplasias/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Sulfotransferases/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Animais , Células COS , Chlorocebus aethiops , Receptor alfa de Estrogênio/genética , Feminino , Regulação da Expressão Gênica , Humanos , Fígado , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Fatores Sexuais , Sulfotransferases/genética
10.
Sci Rep ; 11(1): 10926, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035403

RESUMO

Renal cell carcinoma (RCC) is the deadliest primary genitourinary malignancy typically associated with asymptomatic initial presentation and poorly predictable survival. Next to established risk factors, tumor microenvironment may alter metastatic capacity and immune landscape. Due to their high concentrations, sulfoglycolipids (sulfatides) were among the first well-described antigens in RCC that are associated with worse prognosis. As sulfatide detection in routine diagnostics is not possible, we aimed to test the prognostic value of its protein counterpart, sulfatide-producing enzyme Gal3ST1. We performed retrospective long-term follow up analysis of Gal3ST1 expression as prognostic risk factor in a representative RCC patient cohort. We observed differentially regulated Gal3ST1 expression in all RCC types, being significantly more associated with clear cell RCC than to chromophobe RCC (p = 0.001). Surprisingly, in contrast to published observations from in vitro models, we could not confirm an association between Gal3ST1 expression and a malignant clinical behaviour of the RCC. In our cohort, Gal3ST1 did not significantly influence progression-free survival (Hazard Ratio (HR): 1.7 95% CI (0.6-4.9), p = 0.327). Particularly after adjusting for histology, T-stage, N-status and M-status at baseline, we observed no independent prognostic effect (HR = 1.0 95% CI (0.3-3.3), p = 0.96). The analysis of Gal3ST1 mRNA expression in a TCGA dataset supported the results of our cohort. Thus, Gal3ST1 might help to differentiate between chromophobe RCC and other frequent RCC entities but-despite previously published data from cell culture models-does not qualify as a prognostic marker for RCC. Further investigation of regulatory mechanisms of sulfatide metabolism in human RCC microenvironment is necessary to understand the role of this quantitatively prominent glycosphingolipid in RCC progression.


Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Sulfotransferases/genética , Sulfotransferases/metabolismo , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida
11.
PLoS One ; 16(5): e0250805, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33951064

RESUMO

A FLAG tag consisting of DYKDDDDK is an epitope tag that is frequently and widely used to detect recombinant proteins of interest. In this study, we performed a CRISPR-based genetic screening to identify factors involved in the detection of a FLAG-tagged misfolded model protein at the cell surface. In the screening, SLC35B2, which encodes 3'-phosphoadenosine-5'-phosphosulfate transporter 1, was identified as the candidate gene. The detection of FLAG-tagged misfolded proteins at the cell surface was significantly increased in SLC35B2-knockout cells. Furthermore, protein tyrosine sulfation mediated by tyrosyl-protein sulfotransferase 2 (TPST2) suppressed FLAG-tagged protein detection. Localization analysis of the FLAG-tagged misfolded proteins confirmed that defects in tyrosine sulfation are only responsible for enhancing anti-FLAG staining on the plasma membrane but not inducing the localization change of misfolded proteins on the plasma membrane. These results suggest that a FLAG tag on the misfolded protein would be sulfated, causing a reduced detection by the M2 anti-FLAG antibody. Attention should be required when quantifying the FLAG-tagged proteins in the secretory pathway.


Assuntos
Anticorpos/metabolismo , Proteínas de Membrana/metabolismo , Transportadores de Sulfato/metabolismo , Sulfotransferases/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Epitopos/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes/metabolismo
12.
Biochem Biophys Res Commun ; 562: 15-20, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34030040

RESUMO

Bile acids play essential roles in facilitating the intestinal absorption of lipophilic nutrients as well as regulation of glucose, lipid, and energy homeostasis via activation of some receptors. Bile acids are cytotoxic, and consequently their concentrations are tightly controlled. A critical pathway for bile acid elimination and detoxification is sulfation. The pattern of bile acid sulfation differs by species. Sulfation preferentially occurs at the 3α-OH of bile acids in humans, but at the 7α-OH in mice. A recent study identified mouse cytosolic sulfotransferase 2A8 (mSULT2A8) as the major hepatic 7α-hydroxyl bile acid-sulfating enzyme. To elucidate the 7α-OH specific sulfation mechanism of mSULT2A8, instead of 3α-OH specific sulfation in humans, we determined a crystal structure of mSULT2A8 in complex with cholic acid, a major bile acid, and 3'-phosphoadenosine-5'-phosphate, the sulfate donor product. Our study shows that bile acid-binding mode of mSULT2A8 and how the enzyme holds the 7α-OH group of bile acids at the catalytic center, revealing that the mechanism underlying 7α-OH specific sulfation. The structure shows the substrate binds to mSULT2A8 in an orientation perpendicular to that of human 3α-hydroxyl bile acid-sulfotransferase (hSULT2A1). The structure of the complex provides new insight into species different bile acid metabolism.


Assuntos
Ácidos e Sais Biliares/metabolismo , Sulfatos/metabolismo , Sulfotransferases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Humanos , Cinética , Camundongos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Especificidade por Substrato , Sulfotransferases/metabolismo
13.
J Exp Bot ; 72(15): 5508-5521, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34028532

RESUMO

Tyrosine-sulfated peptides are key regulators of plant growth and development. The disulfated pentapeptide phytosulfokine (PSK) mediates growth via leucine-rich repeat receptor-like kinases, PSKR1 and PSKR2. PSK receptors (PSKRs) are part of a response module at the plasma membrane that mediates short-term growth responses, but downstream signaling of transcriptional regulation remains unexplored. In Arabidopsis, tyrosine sulfation is catalyzed by a single-copy gene (TPST; encoding tyrosylprotein sulfotransferase). We performed a microarray-based transcriptome analysis in the tpst-1 mutant background that lacks sulfated peptides to identify PSK-regulated genes and genes that are regulated by other sulfated peptides. Of the 169 PSK-regulated genes, several had functions in root growth and development, in agreement with shorter roots and a higher lateral root density in tpst-1. Further, tpst-1 roots developed higher numbers of root hairs, and PSK induced expression of WEREWOLF (WER), its paralog MYB DOMAIN PROTEIN 23 (MYB23), and At1g66800 that maintain non-hair cell fate. The tpst-1 pskr1-3 pskr2-1 mutant showed even shorter roots, and higher lateral root and root hair density than tpst-1, revealing unexpected synergistic effects of ligand and PSKR deficiencies. While residual activities may exist, overexpression of PSKR1 in the tpst-1 background induced root growth, suggesting that PSKR1 may be active in the absence of sulfated ligands.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Receptores de Superfície Celular/genética , Transdução de Sinais , Sulfotransferases/genética , Sulfotransferases/metabolismo
14.
J Biochem ; 170(3): 419-426, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33950190

RESUMO

Doxorubicin is a chemotherapeutic drug widely utilized in cancer treatment. An enzyme critical to doxorubicin metabolism is the cytosolic sulfotransferase (SULT) SULT1C4. This study investigated the functional impact of SULT1C4 single nucleotide polymorphisms (SNPs) on the sulfation of doxorubicin by SULT1C4 allozymes. A comprehensive database search was performed to identify various SULT1C4 SNPs. Ten nonsynonymous SULT1C4 SNPs were selected, and the corresponding cDNAs, packaged in pGEX-2TK expression vector, were generated via site-directed mutagenesis. Respective SULT1C4 allozymes were bacterially expressed and purified by affinity chromatography. Purified SULT1C4 allozymes, in comparison with the wild-type enzyme, were analysed for sulphating activities towards doxorubicin and 4-nitrophenol, a prototype substrate. Results obtained showed clearly differential doxorubicin-sulphating activity of SULT1C4 allozymes, implying differential metabolism of doxorubicin through sulfation in individuals with distinct SULT1C4 genotypes.


Assuntos
Doxorrubicina/metabolismo , Polimorfismo de Nucleotídeo Único , Sulfotransferases/genética , Sulfotransferases/metabolismo , Citosol/metabolismo , Genótipo , Humanos , Isoenzimas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/metabolismo , Sulfatos/metabolismo
15.
Sci Rep ; 11(1): 9036, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907224

RESUMO

Sulfotransferase enzymes (SULT) catalyse sulfoconjugation of drugs, as well as endogenous mediators, gut microbiota metabolites and environmental xenobiotics. To address the limited evidence on sulfonation activity from clinical research, we developed a clinical metabolic phenotyping method using paracetamol as a probe substrate. Our aim was to estimate sulfonation capability of phenolic compounds and study its intraindividual variability in man. A total of 36 healthy adult volunteers (12 men, 12 women and 12 women on oral contraceptives) received paracetamol in a 1 g-tablet formulation on three separate occasions. Paracetamol and its metabolites were measured in plasma and spot urine samples using liquid chromatography-high resolution mass spectrometry. A metabolic ratio (Paracetamol Sulfonation Index-PSI) was used to estimate phenol SULT activity. PSI showed low intraindividual variability, with a good correlation between values in plasma and spot urine samples. Urinary PSI was independent of factors not related to SULT activity, such as urine pH or eGFR. Gender and oral contraceptive intake had no impact on PSI. Our SULT phenotyping method is a simple non-invasive procedure requiring urine spot samples, using the safe and convenient drug paracetamol as a probe substrate, and with low intraindividual coefficient of variation. Although it will not give us mechanistic information, it will provide us an empirical measure of an individual's sulfonator status. To the best of our knowledge, our method provides the first standardised in vivo empirical measure of an individual's phenol sulfonation capability and of its intraindividual variability. EUDRA-CT 2016-001395-29, NCT03182595 June 9, 2017.


Assuntos
Acetaminofen/metabolismo , Fenol/metabolismo , Sulfotransferases/metabolismo , Acetaminofen/sangue , Acetaminofen/urina , Adulto , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino
16.
Toxicol Appl Pharmacol ; 425: 115553, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-33915121

RESUMO

p-Cresol sulfate, the primary metabolite of p-cresol, is a uremic toxin that has been associated with toxicities and mortalities. The study objectives were to i) characterize the contributions of human sulfotransferases (SULT) catalyzing p-cresol sulfate formation using multiple recombinant SULT enzymes (including the polymorphic variant SULT1A1*2), pooled human liver cytosols, and pooled human kidney cytosols; and ii) determine the potencies and mechanisms of therapeutic inhibitors capable of attenuating the production of p-cresol sulfate. Human recombinant SULT1A1 was the primary enzyme responsible for the formation of p-cresol sulfate (Km = 0.19 ±â€¯0.02 µM [with atypical kinetic behavior at lower substrate concentrations; see text discussion], Vmax = 789.5 ±â€¯101.7 nmol/mg/min, Ksi = 2458.0 ±â€¯332.8 µM, mean ±â€¯standard deviation, n = 3), while SULT1A3, SULT1B1, SULT1E1, and SULT2A1 contributed negligible or minor roles at toxic p-cresol concentrations. Moreover, human recombinant SULT1A1*2 exhibited reduced enzyme activities (Km = 81.5 ±â€¯31.4 µM, Vmax = 230.6 ±â€¯17.7 nmol/mg/min, Ksi = 986.0 ±â€¯434.4 µM) compared to the wild type. The sulfonation of p-cresol was characterized by Michaelis-Menten kinetics in liver cytosols (Km = 14.8 ±â€¯3.4 µM, Vmax = 1.5 ±â€¯0.2 nmol/mg/min) and substrate inhibition in kidney cytosols (Km = 0.29 ±â€¯0.02 µM, Vmax = 0.19 ±â€¯0.05 nmol/mg/min, Ksi = 911.7 ±â€¯278.4 µM). Of the 14 investigated therapeutic inhibitors, mefenamic acid (Ki = 2.4 ±â€¯0.1 nM [liver], Ki = 1.2 ±â€¯0.3 nM [kidney]) was the most potent in reducing the formation of p-cresol sulfate, exhibiting noncompetitive inhibition in human liver cytosols and recombinant SULT1A1, and mixed inhibition in human kidney cytosols. Our novel findings indicated that SULT1A1 contributed an important role in p-cresol sulfonation (hence it can be considered a probe reaction) in liver and kidneys, and mefenamic acid may be utilized as a potential therapeutic agent to attenuate the generation of p-cresol sulfate as an approach to detoxification.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cresóis/metabolismo , Cresóis/toxicidade , Ácido Mefenâmico/farmacologia , Sulfotransferases/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Ésteres do Ácido Sulfúrico/toxicidade , Catálise , Citosol/enzimologia , Humanos , Rim , Fígado , Proteínas Recombinantes , Sulfotransferases/antagonistas & inibidores , Sulfotransferases/genética
17.
Chem Res Toxicol ; 34(6): 1612-1618, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33872499

RESUMO

Labetalol hydrochloride (LHCl), an α- and ß-adrenoreceptor blocker, is widely used for the treatment of hypertension as well as angina pectoris. Previous reports have demonstrated the adverse events during clinical application of LHCl, such as liver injury and acute renal failure. The present study aimed to investigate metabolic activation of LHCl to initiate the elucidation of the mechanisms of its liver toxicity. One glutathione (GSH) conjugate was detected in rat and human primary hepatocytes as well as bile of rats after exposure to LHCl. The GSH conjugate was chemically synthesized and characterized by Q-TOF and 1H NMR. Pretreatment of 2,6-dichloro-4-nitrophenol (DCNP), a broad-spectrum sulfotransferase (SULT) inhibitor, significantly attenuated the formation of the GSH conjugate in LHCl-treated hepatocytes and animals, indicating the participation of SULTs in metabolic activation of LHCl. Moreover, pretreatment with DCNP displayed significant protection against the observed cytotoxicity in rat primary hepatocytes, which suggests a correlation of the bioactivation of LHCl mediated by SULTs with LHCl-induced hepatotoxicity.


Assuntos
Hepatócitos/efeitos dos fármacos , Labetalol/farmacologia , Sulfotransferases/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Labetalol/química , Labetalol/metabolismo , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley
18.
Protein J ; 40(2): 192-204, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33665770

RESUMO

Cytosolic estrogen sulfotransferase (SULT1E) mainly catalyzes the sulfate conjugation of estrogens, which decrease atherosclerosis progression. Recently we reported that a YKEG sequence in human SULT1E1 (hSULT1E1) corresponding to residues 61-64 can bind specifically to oxidized low-density lipoprotein (Ox-LDL), which plays a major role in the pathogenesis of atherosclerosis; its major oxidative lipid component lysophosphatidylcholine (LPC), and its structurally similar lipid, platelet-activating factor (PAF). In this study, we investigated the effect of Ox-LDL on the sulfating activity of hSULT1E1. In vivo experiments using a mouse model of atherosclerosis showed that the protein expression of SULT1E1 was higher in the aorta of mice with atherosclerosis compared with that in control animals. Results from a sulfating activity assay of hSULT1E1 using 1-hydroxypyrene as the substrate demonstrated that Ox-LDL, LPC, and PAF markedly decreased the sulfating activity of hSULT1E1, whereas native LDL and 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) as one of the oxidized phosphatidylcholines showed the opposite effect. The sulfating activity greatly changed in the presence of LPC, PAF, and POVPC in their concentration-dependen manner (especially above their critical micelle concentrations). Moreover, Ox-LDL specifically recognized dimeric hSULT1E1. These results suggest that the effects of Ox-LDL and native LDL on the sulfating activity of hSULT1E1 might be helpful in elucidating the novel mechanism underlying the pathogenesis of atherosclerosis, involving the relationship between estrogen metabolism, LDL, and Ox-LDL.


Assuntos
Lipoproteínas LDL , Sulfotransferases , Animais , Aterosclerose , Humanos , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Lisofosfatidilcolinas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fator de Ativação de Plaquetas/metabolismo , Ligação Proteica , Sulfotransferases/química , Sulfotransferases/metabolismo
19.
Nat Commun ; 12(1): 1389, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654100

RESUMO

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production-chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 µg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.


Assuntos
Vias Biossintéticas , Sulfatos de Condroitina/biossíntese , Escherichia coli/metabolismo , Transporte Biológico , Escherichia coli/enzimologia , Fermentação , Oxirredutases/metabolismo , Sulfotransferases/metabolismo
20.
Nat Commun ; 12(1): 1923, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772011

RESUMO

Chronic demyelination in the human CNS is characterized by an inhibitory microenvironment that impairs recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) leading to failed remyelination and axonal atrophy. By network-based transcriptomics, we identified sulfatase 2 (Sulf2) mRNA in activated human primary OPCs. Sulf2, an extracellular endosulfatase, modulates the signaling microenvironment by editing the pattern of sulfation on heparan sulfate proteoglycans. We found that Sulf2 was increased in demyelinating lesions in multiple sclerosis and was actively secreted by human OPCs. In experimental demyelination, elevated OPC Sulf1/2 expression directly impaired progenitor recruitment and subsequent generation of oligodendrocytes thereby limiting remyelination. Sulf1/2 potentiates the inhibitory microenvironment by promoting BMP and WNT signaling in OPCs. Importantly, pharmacological sulfatase inhibition using PI-88 accelerated oligodendrocyte recruitment and remyelination by blocking OPC-expressed sulfatases. Our findings define an important inhibitory role of Sulf1/2 and highlight the potential for modulation of the heparanome in the treatment of chronic demyelinating disease.


Assuntos
Diferenciação Celular/genética , Microambiente Celular/genética , Doenças Desmielinizantes/genética , Perfilação da Expressão Gênica/métodos , Células Precursoras de Oligodendrócitos/metabolismo , Remielinização/genética , Animais , Axônios/metabolismo , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Células Precursoras de Oligodendrócitos/citologia , Sulfatases/genética , Sulfatases/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo
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