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1.
Biometals ; 34(5): 987-1006, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34236558

RESUMO

The effects of both Tl+ and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO3 and KNO3. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl+ and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl+ in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.


Assuntos
Mitocôndrias Hepáticas , Proteínas de Transporte da Membrana Mitocondrial , Animais , Cálcio/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/farmacologia , Permeabilidade , Ratos , Ratos Wistar , Respiração , Ácido Succínico/metabolismo , Ácido Succínico/farmacologia , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/farmacologia , Reagentes de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologia , Tálio/metabolismo , Tálio/farmacologia
2.
Ecotoxicol Environ Saf ; 188: 109858, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31706236

RESUMO

Cultivar-dependent cadmium (Cd) accumulation was principal in developing Cd-pollution safe cultivars (PSCs). Proteins related to different Cd accumulations of the low-Cd-accumulating (SJ19) and high-Cd-accumulating (CX4) cultivars were investigated by iTRAQ analysis. Higher Cd bioaccumulation factors and translocation factor in CX4 than in SJ19 were consistent with the cultivar-dependent Cd accumulations. The Cd uptake was promoted in CX4 due to its higher expression of Cd-binding proteins and the lower expression of Cd-efflux proteins in roots. What's more, significantly elevated thiol groups (PC2 and PC3) in CX4 under Cd stress might contribute to the high Cd accumulation in roots and the root-to-shoot translocation of Cd-PC complex. Up-regulated proteins involved in cellulose biosynthesis and pectin de-esterification in SJ19 enhanced the Cd sequestration of root cell walls, which was considered as the predominant strategy for reducing Cd accumulation in shoots. The present study provided novel insights in the cultivar-dependent Cd accumulation in shoots of B. parachinensis.


Assuntos
Brassica/metabolismo , Cádmio/metabolismo , Proteínas de Plantas/metabolismo , Poluentes do Solo/metabolismo , Transporte Biológico , Brassica/genética , Celulose/metabolismo , Pectinas/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Proteômica , Reagentes de Sulfidrila/metabolismo
3.
Biochemistry ; 56(23): 2921-2927, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28520393

RESUMO

Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30 000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure-activity relationship information.


Assuntos
Compostos de Boro/metabolismo , Descoberta de Drogas/métodos , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/metabolismo , Maleimidas/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Reagentes de Sulfidrila/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Biocatálise , Compostos de Boro/química , Domínio Catalítico , Cisteína/química , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Ligantes , Maleimidas/química , Conformação Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera , Reagentes de Sulfidrila/química
4.
FEBS J ; 283(22): 4113-4127, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27685835

RESUMO

After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKa of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.


Assuntos
Cisteína/química , Dissulfetos/química , Albumina Sérica/química , Reagentes de Sulfidrila/química , Animais , Sítios de Ligação , Bovinos , Cisteína/metabolismo , Dissulfetos/metabolismo , Cães , Glutationa/química , Glutationa/metabolismo , Cabras , Cavalos , Humanos , Cinética , Modelos Moleculares , Domínios Proteicos , Dobramento de Proteína , Albumina Sérica/metabolismo , Ovinos , Especificidade da Espécie , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/metabolismo
5.
Bioorg Med Chem ; 24(12): 2631-40, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27132865

RESUMO

Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes.


Assuntos
Cisteína/análogos & derivados , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Sulfonas/química , Sulfonas/farmacologia , Amidas/química , Amidas/metabolismo , Domínio Catalítico/efeitos dos fármacos , Cisteína/química , Cisteína/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Ditioeritritol/metabolismo , Humanos , Insulina/metabolismo , Modelos Moleculares , Oxirredução/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Reagentes de Sulfidrila/metabolismo
6.
Mol Microbiol ; 98(4): 625-35, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26234817

RESUMO

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.


Assuntos
Arsenitos/metabolismo , Campylobacter jejuni/enzimologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Arseniatos/metabolismo , Arsenicais/metabolismo , Arsenicais/farmacologia , Arsenitos/farmacologia , Campylobacter jejuni/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/metabolismo , Roxarsona/química , Roxarsona/farmacologia , Reagentes de Sulfidrila/metabolismo
7.
J Microbiol Biotechnol ; 23(3): 329-34, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23462005

RESUMO

Uridinediphospho-N-acetylglucosamine enolpyruvyl transferase (MurA, E.C. 2.5.1.7) is an essential bacterial enzyme that catalyzes the first step of the cell wall biosynthetic pathway, which involves the transfer of an enolpyruvyl group from phosphoenolpyruvate to uridinediphospho-Nacetylglucosamine. In this study, novel inhibitors of Haemophilus influenzae MurA (Hi MurA) were identified using high-throughput screening of a chemical library from the Korea Chemical Bank. The identified compounds contain a quinoline moiety and have much lower effective inhibitory concentrations (IC(50)) than fosfomycin, a wellknown inhibitor of MurA. These inhibitors appear to covalently modify the sulfhydryl group of the active site cysteine (C117), since the C117D mutant Hi MurA was not inhibited by these compounds and excess dithiothreitol abolished their inhibitory activities. The increased mass value of Hi MurA after treatment with the identified inhibitor further confirmed that the active-site cysteine residue of Hi MurA is covalently modified by the inhibitor.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Haemophilus influenzae/enzimologia , Cisteína/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Coreia (Geográfico) , Bibliotecas de Moléculas Pequenas , Reagentes de Sulfidrila/metabolismo
8.
Arch Biochem Biophys ; 521(1-2): 102-10, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22450170

RESUMO

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ∼threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.


Assuntos
Ácidos Graxos/farmacologia , Albumina Sérica/química , Cristalografia por Raios X , Ácido Ditionitrobenzoico/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Ácidos Graxos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Modelos Moleculares , Oxirredução , Ligação Proteica , Estabilidade Proteica , Albumina Sérica/efeitos dos fármacos , Albumina Sérica/metabolismo , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologia
9.
Biol Chem ; 393(12): 1523-32, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23667907

RESUMO

Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49, involved in protein metabolism and oxidative stress response. DPPIII crystal structure shows the two lobe-like domains separated by a wide cleft. The human enzyme has a total of six cysteines, three in the lower (Cys19, Cys147,and Cys176) and three in the upper (Cys509, Cys519,and Cys654), catalytic, domain containing the active site zinc ion. To elucidate the molecular basis of this enzyme ' s susceptibility to sulfhydryl reagents, biochemical analysis of a set of Cys to Ala mutants was used, supported by mass spectrometry. Cys176, a residue 44 A apart from the catalytic center of the ligand-free enzyme, was found responsible for the inactivation with the submicromolar concentration of an organomercurial compound, and three additional cysteines contributed to sensitivity to aromatic disulfides. Upon treatment with oxidized glutathione [glutathione disulfide(GSSG)], cysteine residues at positions 147, 176, and 654 were found glutathionylated. The mutational analysis confirmed the involvement of Cys176 and Cys654 inhuman DPP III inactivation by GSSG. Observation that Cys176, a residue quite distant from the active center,contributes to enzyme inactivation, indicates that the substrate-binding site of human DPP III comprises both lower and upper protein domain.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Dissulfeto de Glutationa/metabolismo , Reagentes de Sulfidrila/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Cisteína/química , Cisteína/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína
10.
Hum Mol Genet ; 20(16): 3256-65, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21628316

RESUMO

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is the most common monogenic cause of stroke and vascular dementia. Accumulation and deposition of the NOTCH3 (N3) extracellular domain in small blood vessels has been recognized as a central pathological feature of the disease. Recent experiments suggested enhanced formation of higher order multimers for mutant N3 compared with wild-type (WT). However, the mechanisms and consequences of N3 multimerization are still poorly understood, in part because of the lack of an appropriate in vitro aggregation assay. We therefore developed and validated a robust assay based on recombinant N3 fragments purified from cell culture supernatants. Using single-molecule analysis techniques such as scanning for intensely fluorescent targets and single-particle fluorescence resonance energy transfer, we show that spontaneous aggregation is limited to CADASIL-mutant N3, recapitulating a central aspect of CADASIL pathology in vitro. N3 aggregation requires no co-factor and is facilitated by sulfhydryl crosslinking. Although WT N3 does not exhibit multimerization itself, it can participate in aggregates of mutant N3. Furthermore, we demonstrate that thrombospondin-2, a known interaction partner of N3, co-aggregates with mutant N3. Sequestration of WT N3 and other proteins into aggregates represents a potentially important disease mechanism. These findings in combination with a new assay for single-molecule aggregation analysis provide novel opportunities for the development of therapeutic strategies.


Assuntos
CADASIL/genética , Mutação/genética , Receptores Notch/química , Receptores Notch/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Humanos , Maleimidas/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Receptor Notch3 , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Reagentes de Sulfidrila/metabolismo , Trombospondinas/metabolismo
11.
Biochim Biophys Acta ; 1807(3): 302-10, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21167128

RESUMO

The mitochondrial oxoglutarate carrier belongs to the mitochondrial carrier family and exchanges oxoglutarate for malate and other dicarboxylates across the mitochondrial inner membrane. Here, single-cysteine mutant carriers were engineered for every residue in the amino- and carboxy-terminus, cytoplasmic loops, and matrix alpha-helices and their transport activity was measured in the presence and absence of sulfhydryl reagents. The analysis of the cytoplasmic side of the oxoglutarate carrier showed that the conserved and symmetric residues of the mitochondrial carrier motif [DE]XX[RK] localized at the C-terminal end of the even-numbered transmembrane alpha-helices are important for the function of the carrier, but the non-conserved cytoplasmic loops and termini are not. On the mitochondrial matrix side of the carrier most residues of the three matrix alpha-helices that are in the interface with the transmembrane alpha-helical bundle are important for function. Among these are the residues of the symmetric [ED]G motif present at the C-terminus of the matrix alpha-helices; the tyrosines of the symmetric YK motif at the N-terminus of the matrix alpha-helices; and the hydrophobic residues M147, I171 and I247. The functional role of these residues was assessed in the structural context of the homology model of OGC. Furthermore, in this study no evidence was found for the presence of a specific homo-dimerisation interface on the surface of the carrier consisting of conserved, asymmetric and transport-critical residues.


Assuntos
Aminoácidos/química , Aminoácidos/fisiologia , Citosol/metabolismo , Proteínas de Membrana Transportadoras/química , Mitocôndrias/fisiologia , Aminoácidos/genética , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Bovinos , Ácidos Cetoglutáricos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Reagentes de Sulfidrila/metabolismo
12.
Biochemistry ; 50(1): 82-92, 2011 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-21117647

RESUMO

Apurinic/apyrimidinic endonuclease (APE1) is an essential base excision repair protein that also functions as a reduction and oxidation (redox) factor in mammals. Through a thiol-based mechanism, APE1 reduces a number of important transcription factors, including AP-1, p53, NF-κB, and HIF-1α. What is known about the mechanism to date is that the buried residues Cys 65 and Cys 93 are critical for APE1's redox activity. To further detail the redox mechanism, we developed a chemical footprinting-mass spectrometric assay using N-ethylmaleimide (NEM), an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When APE1 was incubated with E3330, two NEM-modified products were observed, one with two and a second with seven added NEMs; this latter product corresponds to a fully modified APE1. In a similar control reaction without E3330, only the +2NEM product was observed in which the two solvent-accessible Cys residues, C99 and C138, were modified by NEM. Through hydrogen-deuterium amide exchange with analysis by mass spectrometry, we found that the +7NEM-modified species incorporates approximately 40 more deuterium atoms than the native protein, which exchanges nearly identically as the +2NEM product, suggesting that APE1 can be trapped in a partially unfolded state. E3330 was also found to increase the extent of disulfide bond formation involving redox critical Cys residues in APE1 as assessed by liquid chromatography and tandem mass spectrometry, suggesting a basis for its inhibitory effects on APE1's redox activity. Collectively, our results suggest that APE1 adopts a partially unfolded state, which we propose is the redox active form of the enzyme.


Assuntos
Benzoquinonas/farmacologia , Cisteína/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Etilmaleimida/metabolismo , Oxirredução/efeitos dos fármacos , Propionatos/farmacologia , Reagentes de Sulfidrila/metabolismo , Animais , Cisteína/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Humanos , Conformação Proteica/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Temperatura
13.
Toxicol Sci ; 117(2): 249-52, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20660472

RESUMO

Conversion of arsenate to arsenite is a critical event in the pathway that leads from inorganic arsenic to a variety of methylated metabolites. The formation of methylated metabolites influences distribution and retention of arsenic and affects the reactivity and toxicity of these intermediates. Indeed, some of the toxic and carcinogenic effects associated with exposure to arsenate or arsenite are probably mediated by methylated arsenicals. Recent work has demonstrated a biologically plausible role for phosphorolytic-arsenolytic enzymes in a reaction scheme in which an "activated" arsenate ester is readily reduced by thiols to arsenite. Thiol-dependent reduction of arsenate esters formed by arsenolysis may be one of several functionally reductant processes that control the flux of arsenic into the cellular pathway for arsenic methylation. Integrating these reductive processes into a conceptual model for arsenic metabolism may provide new insights into the cellular machinery for handling this toxic metalloid.


Assuntos
Arseniatos/metabolismo , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/metabolismo , Animais , Arseniatos/química , Arseniatos/toxicidade , Glutationa/metabolismo , Oxirredução , Purina-Núcleosídeo Fosforilase/metabolismo , Compostos de Sulfidrila/química , Reagentes de Sulfidrila/química , Reagentes de Sulfidrila/toxicidade
14.
Antioxid Redox Signal ; 12(10): 1167-78, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19769462

RESUMO

Hydrogen sulfide (H(2)S) is an endogenous opener of K(ATP) channels in many different types of cells. However, the molecular mechanism for an interaction between H(2)S and K(ATP) channel proteins remains unclear. The whole-cell patch-clamp technique and mutagenesis approach were used to examine the effects of H(2)S on different K(ATP) channel subunits, rvKir6.1 and rvSUR1, heterologously expressed in HEK-293 cells. H(2)S stimulated coexpressed rvKir6.1/rvSUR1 K(ATP) channels, but had no effect on K(ATP) currents generated by rvKir6.1 expression alone. Intracellularly applied sulfhydryl alkylating agent (N-ethylmaleimide, NEM), oxidizing agent (chloramine T, CLT), and a disulfide bond-oxidizing enzyme (protein disulfide isomerase) did not alter H(2)S effects on this recombinant channels. CLT, but not NEM, inhibited basal rvKir6.1/rvSUR1 currents, and both abolished the stimulatory effects of H(2)S on K(ATP) currents, when applied extracellularly. After selective cysteine residues (C6S and C26S but not C1051S and C1057S) in the extracellular loop of rvSUR1 subunits were point-mutated, H(2)S lost its stimulatory effects on rvKir6.1/rvSUR1 currents. Our results demonstrate that H(2)S interacts with Cys6 and Cys26 residues of the extracellular N terminal of rvSUR1 subunit of K(ATP) channel complex. Direct chemical modification of rvSUR1 subunit protein constitutes a molecular mechanism for the activation of K(ATP) channels by H(2)S.


Assuntos
Sulfeto de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Canais KATP/metabolismo , Poluentes Atmosféricos/metabolismo , Poluentes Atmosféricos/farmacologia , Linhagem Celular , Cloraminas/metabolismo , Cisteína/metabolismo , Etilmaleimida/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Canais KATP/genética , Mutagênese Sítio-Dirigida , Oxidantes/metabolismo , Técnicas de Patch-Clamp , Mutação Puntual , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , Subunidades Proteicas/metabolismo , Reagentes de Sulfidrila/metabolismo , Compostos de Tosil/metabolismo
15.
Virol J ; 4: 110, 2007 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-17961263

RESUMO

BACKGROUND: A human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity. RESULTS: HPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region. CONCLUSION: HPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.


Assuntos
Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Reagentes de Sulfidrila/metabolismo , Transporte Biológico/efeitos dos fármacos , Capsídeo , Proteínas do Capsídeo/química , Linhagem Celular Transformada , Cisteína/metabolismo , Humanos , Proteínas Oncogênicas Virais/química , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologia , Vírion/metabolismo
16.
J Mol Biol ; 369(2): 400-12, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17442340

RESUMO

The mitochondrial oxoglutarate carrier (OGC) plays an important role in the malate-aspartate shuttle, the oxoglutarate-isocitrate shuttle and gluconeogenesis. To establish amino acid residues that are important for function, each residue in the transmembrane alpha-helices H1, H3 and H5 was replaced systematically by a cysteine in a fully functional mutant carrier that was devoid of cysteine residues. The transport activity of the mutant carriers was measured in the presence and absence of sulfhydryl reagents. The observed effects were rationalized by using a comparative structural model of the OGC. Most of the residues that are critical for function are found at the bottom of the cavity and they belong to the signature motifs P-X-[DE]-X-X-[KR] that form a network of three inter-helical salt bridges that close the carrier at the matrix side. The OGC deviates from most other carriers, because it has a conserved leucine (L144) rather than a positively charged residue in the signature motif of the second repeat and thus the salt bridge network is lacking one salt bridge. Incomplete salt-bridge networks due to hydrophobic, aromatic or polar substitutions are observed in other dicarboxylate, phosphate and adenine nucleotide transporters. The interaction between the carrier and the substrate has to provide the activation energy to trigger the re-arrangement of the salt-bridge network and other structural changes required for substrate translocation. For substrates such as malate, which has only two carboxylic and one hydroxyl group, a reduction in the number of salt bridges in the network may be required to lower the energy barrier for translocation. Another group of key residues, consisting of T36, A134, and T233, is close to the putative substrate binding site and substitutions or modifications of these residues may interfere with substrate binding and ion coupling. Residues G32, A35, Q40, G130, G133, A134, G230, and S237 are potentially engaged in inter-helical interactions and they may be involved in the movements of the alpha-helices during translocation.


Assuntos
Ácidos Cetoglutáricos/metabolismo , Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Estrutura Secundária de Proteína , Animais , Transporte Biológico/fisiologia , Bovinos , Cisteína/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Reagentes de Sulfidrila/metabolismo
17.
J Toxicol Environ Health A ; 70(8): 715-21, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17365626

RESUMO

This study examined the difference between sulfhydryl-reactive metals (mercury, lead, arsenic, and cadmium) in the hair of 45 children with autism (1-6 yr of age) as compared to 45 gender-, age-, and race-matched typical children. Hair samples were measured with inductively coupled mass spectrometry. Some studies, such as Holmes et al. (2003), suggested that children with autism may be poor detoxifiers relative to normally developing children. Metals that are not eliminated sequester in the brain. Our study found that arsenic, cadmium, and lead were significantly lower in the hair of children with autism than in matched controls. Mercury was in the same direction (lower in autism) following the same pattern, but did not achieve statistical significance. The evidence from our study supports the notion that children with autism may have trouble excreting these metals, resulting in a higher body burden that may contribute to symptoms of autism.


Assuntos
Transtorno Autístico/metabolismo , Cabelo/química , Metais Pesados/metabolismo , Reagentes de Sulfidrila/metabolismo , Arsênio/análise , Arsênio/metabolismo , Carga Corporal (Radioterapia) , Cádmio/metabolismo , Criança , Pré-Escolar , Monitoramento Ambiental , Feminino , Humanos , Lactente , Chumbo/metabolismo , Masculino , Mercúrio/metabolismo , Metais Pesados/análise , Índice de Gravidade de Doença
18.
FEMS Yeast Res ; 7(3): 391-403, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17253982

RESUMO

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-cysteine synthetase) and, particularly, GLR1 (glutathione reductase) are required for survival on DPS. DPS is uniquely thiol-specific, and we found that the cellular mechanisms for DPS detoxification differ substantially from that of the commonly used thiol oxidant diamide. In contrast to this oxidant, the full antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS sensitivity. DPS seems to induce a specific disulfide stress, where an increase in the cytoplasmic/nuclear GSSG/GSH ratio results in putative DPS target(s) becoming sensitive to DPS.


Assuntos
Dissulfetos/farmacologia , Dissulfeto de Glutationa/metabolismo , Glutationa/metabolismo , Piridinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Reagentes de Sulfidrila/farmacologia , Dissulfetos/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Proteínas de Membrana/metabolismo , Mutagênese Insercional , Oxirredução , Plasmídeos/genética , Piridinas/metabolismo , Receptores de Peptídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Reagentes de Sulfidrila/metabolismo , Superóxido Dismutase/metabolismo , Tiorredoxinas/metabolismo , Fatores de Transcrição/metabolismo
19.
Kidney Blood Press Res ; 29(5): 280-93, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17035713

RESUMO

Chloroacetaldehyde (CAA) is a metabolite of the alkylating agent ifosfamide (IFO) and putatively responsible for renal damage following anti-tumor therapy with IFO. Depletion of sulfhydryl (SH) groups has been reported from cell culture, animal and clinical studies. In this work the effect of CAA on human proximal tubule cells in primary culture (hRPTEC) was investigated. Toxicity of CAA was determined by protein content, cell number, LDH release, trypan blue exclusion assay and caspase-3 activity. Free thiols were measured by the method of Ellman. CAA reduced hRPTEC cell number and protein, induced a loss in free intracellular thiols and an increase in necrosis markers. CAA but not acrolein inhibited the cysteine proteases caspase-3, caspase-8 and cathepsin B. Caspase activation by cisplatin was inhibited by CAA. In cells stained with fluorescent dyes targeting lysosomes, CAA induced an increase in lysosomal size and lysosomal leakage. The effects of CAA on cysteine protease activities and thiols could be reproduced in cell lysate. Acidification, which slowed the reaction of CAA with thiol donors, could also attenuate effects of CAA on necrosis markers, thiol depletion and cysteine protease inhibition in living cells. Thus, CAA directly reacts with cellular protein and non-protein thiols, mediating its toxicity on hRPTEC. This effect can be reduced by acidification. Therefore, urinary acidification could be an option to prevent IFO nephropathy in patients.


Assuntos
Acetaldeído/análogos & derivados , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/toxicidade , Ifosfamida/metabolismo , Ifosfamida/toxicidade , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Compostos de Sulfidrila/toxicidade , Reagentes de Sulfidrila/metabolismo , Acetaldeído/química , Acetaldeído/metabolismo , Biomarcadores , Caspase 3/metabolismo , Caspase 8/metabolismo , Catepsina B/metabolismo , Contagem de Células , Morte Celular , Células Cultivadas , Corantes , Cisteína Endopeptidases/metabolismo , Humanos , Nefropatias/enzimologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , L-Lactato Desidrogenase/metabolismo , Microscopia de Fluorescência , Necrose , Compostos de Sulfidrila/química , Reagentes de Sulfidrila/química , Azul Tripano , gama-Glutamiltransferase/metabolismo
20.
J Biol Chem ; 281(36): 26382-90, 2006 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-16840784

RESUMO

Desulfitobacterium dehalogenans can use chlorinated aromatics including polychlorinated biphenyls as electron acceptors in a process called dehalorespiration. Expression of the cpr gene cluster involved in this process is regulated by CprK, which is a member of the CRP/FNR (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. High affinity interaction of the chlorinated aromatic compound with the effector domain of CprK triggers binding of CprK to an upstream target DNA sequence, which leads to transcriptional activation of the cpr gene cluster. When incubated with oxygen or diamide, CprK undergoes inactivation; subsequent treatment with dithiothreitol restores activity. Using mass spectrometry, this study identifies two classes of redox-active thiol groups that form disulfide bonds upon oxidation. Under oxidative conditions, Cys105, which is conserved in FNR and most other CprK homologs, forms an intramolecular disulfide bond with Cys111, whereas an intermolecular disulfide bond is formed between Cys11 and Cys200. SDS-PAGE and site-directed mutagenesis experiments indicate that the Cys11/Cys200 disulfide bond links two CprK subunits in an inactive dimer. Isothermal calorimetry and intrinsic fluorescence quenching studies show that oxidation does not change the affinity of CprK for the effector. Therefore, reversible redox inactivation is manifested at the level of DNA binding. Our studies reveal a strategy for limiting expression of a redox-sensitive pathway by using a thiol-based redox switch in the transcription factor.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Desulfitobacterium , Regulação Bacteriana da Expressão Gênica , Proteínas Ferro-Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desulfitobacterium/genética , Desulfitobacterium/metabolismo , Diamida/metabolismo , Dimerização , Dissulfetos/química , Dissulfetos/metabolismo , Ditiotreitol/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Oxirredução , Oxigênio/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Reagentes de Sulfidrila/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
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