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1.
Molecules ; 26(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34577101

RESUMO

A fully mechanized multicommutated flow analysis (MCFA) system dedicated to determining horseradish peroxidase (HRP) activity was developed. Detection was conducted using a flow-through optoelectronic detector-constructed of paired LEDs operating according to the paired emitter-detector diode (PEDD) principle. The PEDD-MCFA system is dedicated to monitoring the enzyme-catalyzed oxidation of p-phenylenediamine (pPD) by a hydrogen peroxide. Under optimized conditions, the presented bioanalytical system was characterized by a linear response range (33.47-200 U/L) with a detection limit at 10.54 U/L HRP activity and 1.66 mV·L/U sensitivity, relatively high throughput (12 signals recordings per hour), and acceptable precision (RSD below 6%). Additionally, the utility of the developed PEDD-MCFA system for the determination of HRP inhibitors allowing the detection of selected thiols at micromolar levels, is demonstrated. The practical utility of the flow system was illustrated by the analysis of some dietary supplements containing L-cysteine, N-acetylcysteine, and L-glutathione.


Assuntos
Técnicas Eletroquímicas/métodos , Ensaios Enzimáticos/métodos , Análise de Injeção de Fluxo/métodos , Peroxidase do Rábano Silvestre/antagonistas & inibidores , Peroxidase do Rábano Silvestre/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Nefelometria e Turbidimetria/métodos , Calibragem , Peróxido de Hidrogênio/metabolismo , Limite de Detecção , Fenilenodiaminas/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
2.
Exp Eye Res ; 211: 108707, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34332989

RESUMO

The nuclear region of the lens is metabolically quiescent, but it is far from inert chemically. Without cellular renewal and with decades of environmental exposures, the lens proteome, lipidome, and metabolome change. The lens crystallins have evolved exquisite mechanisms for resisting, slowing, adapting to, and perhaps even harnessing the effects of these cumulative chemical modifications to minimize the amount of light-scattering aggregation in the lens over a lifetime. Redox chemistry is a major factor in these damages and mitigating adaptations, and as such, it is likely to be a key component of any successful therapeutic strategy for preserving or rescuing lens transparency, and perhaps flexibility, during aging. Protein redox chemistry is typically mediated by Cys residues. This review will therefore focus primarily on the Cys-rich γ-crystallins of the human lens, taking care to extend these findings to the ß- and α-crystallins where pertinent.


Assuntos
Cisteína/metabolismo , Cristalino/metabolismo , gama-Cristalinas/metabolismo , Envelhecimento/fisiologia , Dissulfetos/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Oxirredução , Compostos de Sulfidrila/metabolismo
3.
Pestic Biochem Physiol ; 178: 104915, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34446191

RESUMO

Pesticides are extensively employed worldwide, especially in agriculture to control weeds, insect infestation and diseases. Besides their targets, pesticides can also affect the health of non-target organisms, including humans The present study was conducted to study the effect of oral exposure of thiram, a dithiocarbamate fungicide, on the intestine of rats. Male rats were administered thiram at doses of 100, 250, 500 and 750 mg/kg body weight for 4 days. This treatment reduced cellular glutathione, total sulfhydryl groups but enhanced protein carbonyl content and hydrogen peroxide levels. In addition, the activities of all major antioxidant enzymes (catalase, thioredoxin reductase, glutathione peroxidase and glutathione-S-transferase) except superoxide dismutase were decreased. The antioxidant power of the intestine was impaired lowering the metal-reducing and free radical quenching ability. Administration of thiram also led to inhibition of intestinal brush border membrane enzymes, alkaline phosphatase, γ-glutamyl transferase, leucine aminopeptidase and sucrase. Activities of enzymes of pentose phosphate pathway, citric acid cycle, glycolysis and gluconeogenesis were also inhibited. Histopathology showed extensive damage in the intestine of thiram-treated rats at higher doses. All the observed effects were in a thiram dose-dependent manner. The results of this study show that thiram causes significant oxidative damage in the rat intestine which is associated with the marked impairment in the antioxidant defense system.


Assuntos
Compostos de Sulfidrila , Tiram , Administração Oral , Animais , Antioxidantes/metabolismo , Intestinos , Microvilosidades , Oxirredução , Estresse Oxidativo , Carbonilação Proteica , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo
4.
ACS Chem Biol ; 16(9): 1737-1744, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34423966

RESUMO

Nonribosomal peptide synthetase and polyketide synthase systems are home to complex enzymology and produce compounds of great therapeutic value. Despite this, they have continued to be difficult to characterize due to their substrates remaining enzyme-bound by a thioester bond. Here, we have developed a strategy to directly trap and characterize the thioester-bound enzyme intermediates and applied the strategy to the azinomycin biosynthetic pathway. The approach was initially applied in vitro to evaluate its efficacy and subsequently moved to an in situ system, where a protein of interest was isolated from the native organism to avoid needing to supply substrates. When the nonribosomal peptide synthetase AziA3 was isolated from Streptomyces sahachiroi, the capture strategy revealed AziA3 functions in the late stages of epoxide moiety formation of the azinomycins. The strategy was further validated in vitro with a nonribosomal peptide synthetase involved in colibactin biosynthesis. In the long term, this method will be utilized to characterize thioester-bound metabolites within not only the azinomycin biosynthetic pathway but also other cryptic metabolite pathways.


Assuntos
Compostos de Epóxi/metabolismo , Naftalenos/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Compostos de Sulfidrila/metabolismo , Proteínas de Bactérias , Vias Biossintéticas , Compostos de Epóxi/análise , Genes Bacterianos , Metabolômica , Naftalenos/análise , Peptídeo Sintases/genética , Peptídeos/análise , Policetídeo Sintases/genética , Policetídeos/análise , Streptomyces , Espectrometria de Massas em Tandem
5.
Sci Rep ; 11(1): 15134, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34302052

RESUMO

The epidemiological studies confirm that the overproduction of free radical is an important factor of cancer induction as well as development, and loss of antioxidant systems efficiency is associated with an increased risk of carcinogenesis. While bladder cancer is the fourth most common type of cancer all over the world, there is little evidence of the advancing changes in oxidative/nitrative stress during the progression of bladder cancer. Our study aimed to investigate the plasma levels of typical markers of oxidative/nitrative stress depending on the clinical classification of bladder cancer differentiation and infiltration degree. We examined 40 patients with newly diagnosed bladder cancer and 20 healthy volunteers as a control group. We analysed the plasma levels of protein carbonyls, thiol groups, 3-nitrotyrosine, lipid peroxidation, as well as non-enzymatic plasma antioxidant capacity using DPPH· and ABTS·+ radicals. We confirmed that all analysed biomarkers are higher in enrolled BC patients than in healthy subjects. Furthermore, our findings demonstrate a positive correlation between the degree of bladder cancer progression and the level of oxidative stress, but no correlation in the case of NT-3. Based on obtained results, we might conclude that during carcinogenesis of the bladder increased oxidative damage of biomolecules is manifested. This indicates the participation of oxidative stress in the development of bladder cancer, and it is important the ensure the proper antioxidant protection.


Assuntos
Biomarcadores/metabolismo , Estresse Oxidativo/fisiologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Adulto , Antioxidantes/metabolismo , Estudos de Casos e Controles , Progressão da Doença , Feminino , Radicais Livres/metabolismo , Humanos , Peroxidação de Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Compostos de Sulfidrila/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
6.
Toxicol Lett ; 350: 194-201, 2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34303790

RESUMO

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.


Assuntos
Albuminas/farmacologia , Proteínas de Bactérias/farmacologia , Inibidores Enzimáticos/farmacologia , Intoxicação por Organofosfatos/tratamento farmacológico , Hidrolases de Triester Fosfórico/metabolismo , Hidrolases de Triester Fosfórico/uso terapêutico , Compostos de Sulfidrila/metabolismo , Albuminas/metabolismo , Proteínas de Bactérias/metabolismo , Caulobacteraceae/química , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Inibidores Enzimáticos/metabolismo , Modelos Animais , Compostos Organofosforados/metabolismo , Compostos de Sulfidrila/sangue
7.
Molecules ; 26(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299536

RESUMO

The volatile thiol compound 3-sulfanylhexan-1-ol (3SH) is a key impact odorant of white wines such as Sauvignon Blanc. 3SH is produced during fermentation by metabolism of non-volatile precursors such as 3-S-gluthathionylhexanal (glut-3SH-al). The biogenesis of 3SH is not fully understood, and the role of glut-3SH-al in this pathway is yet to be elucidated. The aldehyde functional group of glut-3SH-al is known to make this compound more reactive than other precursors to 3SH, and we are reporting for the first time that glut-3SH-al can exist in both keto and enol forms in aqueous solutions. At wine typical pH (~3.5), glut-3SH-al exists predominantly as the enol form. The dominance of the enol form over the keto form has implications in terms of potential consumption/conversion of glut-3SH-al by previously unidentified pathways. Therefore, this work will aid in the further elucidation of the role of glut-3SH-al towards 3SH formation in wine, with significant implications for the study and analysis of analogous compounds.


Assuntos
Compostos de Enxofre/metabolismo , Aldeídos/metabolismo , Fermentação/fisiologia , Hexanóis/metabolismo , Odorantes/análise , Compostos de Sulfidrila/metabolismo , Vitis/metabolismo , Vinho/análise
8.
Nat Commun ; 12(1): 4336, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267196

RESUMO

Glutathione (GSH) is the most abundant cellular antioxidant. As reactive oxygen species (ROS) are widely believed to promote aging and age-related diseases, and antioxidants can neutralize ROS, it follows that GSH and its precursor, N-acetyl cysteine (NAC), are among the most popular dietary supplements. However, the long- term effects of GSH or NAC on healthy animals have not been thoroughly investigated. We employed C. elegans to demonstrate that chronic administration of GSH or NAC to young or aged animals perturbs global gene expression, inhibits skn-1-mediated transcription, and accelerates aging. In contrast, limiting the consumption of dietary thiols, including those naturally derived from the microbiota, extended lifespan. Pharmacological GSH restriction activates the unfolded protein response and increases proteotoxic stress resistance in worms and human cells. It is thus advantageous for healthy individuals to avoid excessive dietary antioxidants and, instead, rely on intrinsic GSH biosynthesis, which is fine-tuned to match the cellular redox status and to promote homeostatic ROS signaling.


Assuntos
Acetilcisteína/farmacologia , Envelhecimento/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Glutationa/farmacologia , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Suplementos Nutricionais , Escherichia coli , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Masculino , Paraquat/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/metabolismo , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas/fisiologia
9.
Geriatr Gerontol Int ; 21(7): 584-589, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34080286

RESUMO

AIM: Sarcopenia is characterized by progressive and generalized loss of skeletal muscle mass and strength. Chronic inflammatory conditions and increased oxidative stress are in the pathogenesis of sarcopenia. Our aim was to evaluate the relationship between sarcopenia and thiol-disulfide homeostasis and ischemia-modified albumin levels as an oxidative stress marker. METHODS: Patients aged ≥65 years were recruited in this study. Sarcopenia was diagnosed according to the European Working Group on Sarcopenia in Older People criterion. Total thiol, native thiol, disulfide and ischemia-modified albumin levels were measures according to clinical and laboratory features. Patients were divided into two groups according to their sarcopenia presence; thiol-disulfide homeostasis and ischemia-modified albumin levels were evaluated between these groups. RESULTS: Overall, 94 patients were analyzed. The mean age was 75.0 ± 6.71 years. A total of 39% of the patients were diagnosed as probable sarcopenia, 3.2% had sarcopenia, 6.4% had severe sarcopenia and 51.1% were diagnosed as normal. The levels of native thiol, total thiol, disulfide level and disulfide-native thiol, native thiol-total thiol and disulfide-total thiol ratios were similar in patients with sarcopenia when compared with the control group. In addition, there were no differences between albumin and ischemia-modified albumin levels. In univariate regression analysis, handgrip strength was found to be an independent predictor of native thiol and total thiol, and disulfide levels. CONCLUSION: This is the first study in the literature that evaluates the thiol-disulfide homeostasis and ischemia-modified albumin levels in sarcopenic older patients. Long-term studies are warranted to confirm the relationship between oxidative stress markers and sarcopenia. Geriatr Gerontol Int 2021; 21: 584-589.


Assuntos
Biomarcadores/metabolismo , Dissulfetos/metabolismo , Sarcopenia/diagnóstico , Albumina Sérica Humana/metabolismo , Compostos de Sulfidrila/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos Transversais , Dissulfetos/sangue , Feminino , Avaliação Geriátrica , Força da Mão , Homeostase , Humanos , Inflamação , Masculino , Estresse Oxidativo/fisiologia , Compostos de Sulfidrila/sangue , Turquia
11.
Angew Chem Int Ed Engl ; 60(35): 19102-19106, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34173696

RESUMO

Oligonucleotide phosphorothioates (OPS) are DNA or RNA mimics where one phosphate oxygen is replaced by a sulfur atom. They have been shown to enter mammalian cells much more efficiently than non-modified DNA. Thus, solving one of the key challenges with oligonucleotide technology, OPS became very useful in practice, with several FDA-approved drugs on the market or in late clinical trials. However, the mechanism accounting for this facile cellular uptake is unknown. Here, we show that OPS enter cells by thiol-mediated uptake. The transient adaptive network produced by dynamic covalent pseudo-disulfide exchange is characterized in action. Inhibitors with nanomolar efficiency are provided, together with activators that reduce endosomal capture for efficient delivery of OPS into the cytosol, the site of action.


Assuntos
Transporte Biológico/fisiologia , Oligonucleotídeos Fosforotioatos/metabolismo , Compostos de Sulfidrila/metabolismo , Endocitose/fisiologia , Células HeLa , Humanos , Oxirredução , Oligonucleotídeos Fosforotioatos/química , Compostos de Sulfidrila/química
12.
Nat Commun ; 12(1): 3124, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035275

RESUMO

Linear nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) template the modular biosynthesis of numerous nonribosomal peptides, polyketides and their hybrids through assembly line chemistry. This chemistry can be complex and highly varied, and thus challenges our understanding in NRPS and PKS-programmed, diverse biosynthetic processes using amino acid and carboxylate building blocks. Here, we report that caerulomycin and collismycin peptide-polyketide hybrid antibiotics share an assembly line that involves unusual NRPS activity to engage a trans-acting flavoprotein in C-C bond formation and heterocyclization during 2,2'-bipyridine formation. Simultaneously, this assembly line provides dethiolated and thiolated 2,2'-bipyridine intermediates through differential treatment of the sulfhydryl group arising from L-cysteine incorporation. Subsequent L-leucine extension, which does not contribute any atoms to either caerulomycins or collismycins, plays a key role in sulfur fate determination by selectively advancing one of the two 2,2'-bipyridine intermediates down a path to the final products with or without sulfur decoration. These findings further the appreciation of assembly line chemistry and will facilitate the development of related molecules using synthetic biology approaches.


Assuntos
2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Flavoproteínas/química , 2,2'-Dipiridil/síntese química , 2,2'-Dipiridil/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Cisteína/química , Cisteína/metabolismo , Flavoproteínas/metabolismo , Compostos Heterocíclicos/química , Compostos Heterocíclicos/metabolismo , Modelos Químicos , Estrutura Molecular , Peptídeo Sintases/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/química , Policetídeos/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
13.
ACS Appl Mater Interfaces ; 13(19): 22955-22969, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-33969998

RESUMO

Amalgamation of the reactive oxygen species (ROS)-responsive stimulus with nanoparticles has gained considerable interest owing to their high tumor specificity. Hypoxia plays a pivotal role in the acceleration of intracellular ROS production. Herein, we report the construction of a cancer cell (PD-L1)- and ROS-responsive, dual-targeted, temozolomide (TMZ)-laden nanosystem which offers a better anticancer effect in a hypoxic tumor microenvironment. A dual-targeted system boosted permeation in the cancer cells. Hypoxic conditions elevating the high ROS level accelerated the in situ release of TMZ from anti-PD-L1-TKNPs. Hyperaccumulated ROS engendered from TMZ caused oxidative damage leading to mitochondria-mediated apoptosis. TMZ fabricated in the multifunctional nanosystem (anti-PD-L1-TMZ-TKNPs) provided excellent tumor accumulation and retarded tumor growth under in vivo conditions. The elevated apoptosis effect with the activation of an apoptotic marker, DNA double-strand breakage marker, and downregulation of the angiogenesis marker in the tumor tissue following treatment with anti-PD-L1-TMZ-TKNPs exerts robust anticancer effect. Collectively, the nanoconstruct offers deep tumor permeation and high drug release and broadens the application of the ROS-responsive nanosystem for a successful anticancer effect.


Assuntos
Apoptose , Antígeno B7-H1/metabolismo , Mitocôndrias/metabolismo , Nanopartículas , Espécies Reativas de Oxigênio/metabolismo , Animais , Biomarcadores/metabolismo , Humanos , Compostos de Sulfidrila/metabolismo
14.
Plant Cell Rep ; 40(8): 1585-1602, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34003317

RESUMO

KEY MESSAGE: Melatonin enhanced arsenic (As) tolerance by inhibiting As bioaccumulation, modulating the expression of As transporters and phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid. The present study was aimed at investigating the influence of exogenous melatonin on the regulation of endogenous plant growth regulators and their cumulative effects on metal(loid)-binding ligands in two contrasting indica rice cultivars, viz., Khitish (arsenic sensitive) and Muktashri (arsenic tolerant) under arsenic stress. Melatonin supplementation ameliorated arsenic-induced perturbations by triggering endogenous levels of gibberellic acid and melatonin, via up-regulating the expression of key biosynthetic genes like GA3ox, TDC, SNAT and ASMT. The endogenous abscisic acid content was also enhanced upon melatonin treatment by induced expression of the key anabolic gene, NCED3 and concomitant suppression of ABA8ox1. Enhanced melatonin content induced accumulation of higher polyamines (spermidine and spermine), together with up-regulation of SPDS and SPMS in Khitish, thereby modulating stress condition. On the contrary, melatonin escalated putrescine and spermidine levels in Muktashri, via enhanced expression of ADC and SAMDC. The role of melatonin appeared to be more prominent in Khitish, as evident from better utilization of thiol components like cysteine, GSH, non-protein thiols and phytochelatins, with higher GSH/GSSG ratio, despite down-regulated expression of corresponding thiol-metabolic genes (OsMT2 and OsPCS1) to deal with arsenic toxicity. The extent of arsenic bioaccumulation, which was magnified several folds, particularly in Khitish, was decreased upon melatonin application. Overall, our observation highlighted the fact that melatonin enhanced arsenic tolerance by inhibiting arsenic bioaccumulation, via modulating the expression levels of selected arsenic transporters (OsNramp1, OsPT2, OsPT8, OsLsi1) and controlling endogenous phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid.


Assuntos
Arsênio/toxicidade , Melatonina/farmacologia , Oryza/efeitos dos fármacos , Reguladores de Crescimento de Plantas/metabolismo , Compostos de Sulfidrila/metabolismo , Ácido Abscísico/metabolismo , Arsênio/farmacocinética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/metabolismo , Glutationa/metabolismo , Homeostase/efeitos dos fármacos , Inativação Metabólica , Melatonina/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poliaminas/metabolismo , Plântula/efeitos dos fármacos , Plântula/metabolismo , Estresse Fisiológico/efeitos dos fármacos
15.
Biochim Biophys Acta Bioenerg ; 1862(8): 148434, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33932368

RESUMO

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain that contains a cysteine couple which interacts with the torque-transmitting ßDELSEED-loop. This thiol modulation equips CF1Fo with an important environmental fine-tuning mechanism. In vitro, disulfide formation in the γ-redox domain slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient ( [Formula: see text] ), which agrees with its proposed role as chock based on recently solved structure. The γ-dithiol formation at the onset of light is crucial to maximize photosynthetic efficiency since it lowers the [Formula: see text] activation level for ATP synthesis in vitro. Here, we validate these findings in vivo by utilizing absorption spectroscopy in Arabidopsis thaliana. To do so, we monitored the [Formula: see text] present in darkness and identified its mitochondrial sources. By following the fate and components of light-induced extra [Formula: see text] , we estimated the ATP lifetime that lasted up to tens of minutes after long illuminations. Based on the relationship between [Formula: see text] and CF1Fo activity, we conclude that the dithiol configuration in vivo facilitates photosynthesis by driving the same ATP synthesis rate at a significative lower [Formula: see text] than in the γ-disulfide state. The presented in vivo findings are an additional proof of the importance of CF1Fo thiol modulation, reconciling biochemical in vitro studies and structural insights.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Força Próton-Motriz , Compostos de Sulfidrila/metabolismo , Arabidopsis/crescimento & desenvolvimento , Oxirredução , Folhas de Planta/crescimento & desenvolvimento
16.
Angew Chem Int Ed Engl ; 60(31): 16906-16910, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34057803

RESUMO

Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow-up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin-dependent methyltransferases exploiting thiols like ethyl 3-mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90 %. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one-gram scale in 97 % isolated yield.


Assuntos
Guaiacol/metabolismo , Oxigenases de Função Mista/metabolismo , Compostos de Sulfidrila/metabolismo , Biocatálise , Desmetilação , Guaiacol/química , Oxigenases de Função Mista/química , Estrutura Molecular , Compostos de Sulfidrila/química
17.
J Photochem Photobiol B ; 220: 112212, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34049180

RESUMO

Red light (670 nm) promotes ex vivo dilation of blood vessels in a nitric oxide (NO) dependent, but eNOS independent manner by secreting a quasi-stable and transferable vasoactive substance with the characteristics of S-nitrosothiols (RSNO) from the endothelium. In the present work we establish that 670 nm light mediated vasodilation occurs in vivo and is physiologically stable. Light exposure depletes intracellular S-nitroso protein while concomitantly increasing extracellular RNSO, suggesting vesicular pathways are involved. Furthermore, we demonstrate this RSNO vasodilator is embedded in extracellular vesicles (EV). The action of red light on vesicular trafficking appears to increase expression of endosome associated membrane protein CD63 in bovine aortic endothelial cells, enhance endosome localization in the endothelium, and induce exit of RSNO containing EVs from murine facialis arteries. We suggest a mechanism by which the concerted actions of 670 nm light initiate formation of RSNO containing EVs which exit the endothelium and trigger relaxation of smooth muscle cells.


Assuntos
Vesículas Extracelulares/metabolismo , Luz , Vasodilatação/efeitos da radiação , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Compostos Nitrosos/metabolismo , Compostos de Sulfidrila/metabolismo
18.
Angew Chem Int Ed Engl ; 60(31): 17171-17177, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34021957

RESUMO

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.


Assuntos
Compostos de Sulfidrila/metabolismo , Ubiquitina-Proteína Ligases/análise , Ubiquitina/química , Biocatálise , Humanos , Estrutura Molecular , Compostos de Sulfidrila/química , Ubiquitina/síntese química , Ubiquitina-Proteína Ligases/metabolismo
19.
J Alzheimers Dis ; 82(2): 527-540, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34024827

RESUMO

BACKGROUND: Serum homocysteine (Hcy) level is considered to be an important biomarker for Alzheimer's disease (AD); however, the status of Hcy in brain tissue, and the association between brain and serum levels of Hcy in AD patients remain unclear. OBJECTIVE: We aimed to examine whether the changes of three thiols are consistent in serum of AD patients and the brain of APP/PS1 mice, and to verify the effectiveness of Hcy as a biomarker for early AD detection. METHODS: The levels of Hcy, cysteine (Cys), and glutathione (GSH) in Aß1-42-treated PC12 cells, the brain and hippocampus of APP/PS1 mouse, and the serum of AD patients were evaluated using ethyl (E)-3-(9-chloro-11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f] pyrido [3,2,1 -ij] quinolin-10-yl)-2-cyanoacrylate (Probe 1) and ELISA assay or LC-MS. RESULTS: Measurement by Probe 1 revealed a significant increase in Hcy level, and a decrease in Cys and GSH levels in Aß1-42-treated PC12 cells and the serum of AD patients. The hippocampus and whole brain of APP/PS1 mice also showed a significant increase in Hcy level alongside the accumulation of age-related AD symptoms. The upregulation of Hcy and the downregulation of Cys and GSH were reversed in the Aß1-42-treated PC12 cells and the brain of APP/PS1 mice when supplemented with VB6. CONCLUSION: Changes in Hcy, Cys, and GSH levels in the brain of APP/PS1 mice and Aß1-42-treated PC12 cells were observed in situ with a new fluorescent probe, which were consistent with the abnormal changes in Hcy, Cys, and GSH levels in the serum of AD patients. VB6 supplementation was successful in ameliorating abnormal increases in Hcy levels.


Assuntos
Doença de Alzheimer , Encéfalo/metabolismo , Homocisteína , Compostos de Sulfidrila , Vitamina B 6/farmacologia , Doença de Alzheimer/sangue , Doença de Alzheimer/diagnóstico , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Cisteína/metabolismo , Regulação para Baixo , Diagnóstico Precoce , Feminino , Corantes Fluorescentes , Glutationa/metabolismo , Homocisteína/sangue , Homocisteína/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Células PC12 , Ratos , Espectrometria de Fluorescência/métodos , Compostos de Sulfidrila/classificação , Compostos de Sulfidrila/metabolismo , Regulação para Cima , Complexo Vitamínico B/farmacologia
20.
Mycoses ; 64(8): 947-953, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33964024

RESUMO

OBJECTIVES: Onychomycosis is the general term to define fungal nail infections that arise from dermatophytes, non-dermatophytic moulds and yeasts. Thiol/disulphide homeostasis is a new indicator of oxidative stress. In this study, we aimed to investigate the role of thiol/disulphide balance in the pathogenesis of onychomycosis. METHODS: This cross-sectional study included adult patients with onychomycosis who were admitted to the dermatology department and healthy adult volunteers without any dermatologic or systemic condition. The patients and controls were evaluated in terms of native thiol, total thiol, and disulphide levels, and disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios. The possible association between these parameters and clinical subtypes of onychomycosis and demographic characteristics was also investigated. RESULTS: A total of 52 patients with onychomycosis and 50 healthy subjects were enrolled in the study. The patient group showed lower levels of total thiol, native thiol and native thiol/total thiol ratio, and higher ratios of disulphide/native thiol and disulphide/total thiol. No statistically significant relationship was found between the parameters, clinical subtypes of onychomycosis and demographic characteristics (p > .05). CONCLUSION: Patients with onychomycosis showed a shifted thiol/disulphide homeostasis towards oxidative stress with a reduction in thiols and an increase in disulphide/native thiol, and disulphide/total thiol ratios. These findings may indicate the role of oxidative stress in the pathogenesis of onychomycosis.


Assuntos
Dissulfetos/metabolismo , Homeostase , Onicomicose/fisiopatologia , Estresse Oxidativo , Compostos de Sulfidrila/metabolismo , Adulto , Estudos de Casos e Controles , Estudos Transversais , Feminino , Humanos , Masculino , Onicomicose/microbiologia
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