RESUMO
Quantitative monitoring of the concentrations of epigallocatechin gallate (EGCG) and cysteine (Cys) is of great significance for promoting human health. In this study, iron/aluminum bimetallic MOF material MIL-53 (Fe, Al) was rapidly prepared under room temperature using a co-precipitation method, followed by investigating the peroxidase-like (POD-like) activity of MIL-53(Fe, Al) using 3,3',5,5'-tetramethylbenzidine (TMB) as a chromogenic substrate. The results showed that the Michaelis -Menten constants of TMB and H2O2 as substrates were 0.167 mM and 0.108 mM, respectively. A colorimetric sensing platform for detecting EGCG and Cys was developed and successfully applied for analysis and quantitative detection using a smartphone. The linear detection range for EGCG was 15â¼80 µM (R2=0.994) and for Cys was 7â¼95 µM (R2=0.998). The limits of detection (LOD) were 0.719 µM and 0.363 µM for EGCG and Cys, respectively. This work provides a new and cost-effective approach for the real-time analysis of catechins and amino acids.
Assuntos
Antioxidantes , Técnicas Biossensoriais , Catequina , Colorimetria , Smartphone , Colorimetria/métodos , Colorimetria/instrumentação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Antioxidantes/análise , Antioxidantes/química , Catequina/análise , Catequina/análogos & derivados , Catequina/química , Cisteína/análise , Cisteína/análogos & derivados , Limite de Detecção , Análise de Alimentos/métodos , Análise de Alimentos/instrumentaçãoRESUMO
Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.
Assuntos
Cisteína , Corantes Fluorescentes , Análise de Alimentos , Glutationa , Lisossomos , Espectrometria de Fluorescência , Peixe-Zebra , Humanos , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Lisossomos/química , Lisossomos/metabolismo , Células HeLa , Cisteína/análise , Animais , Análise de Alimentos/métodos , Glutationa/análise , Espectrometria de Fluorescência/métodos , Homocisteína/análise , Arabidopsis/química , Limite de Detecção , Microscopia ConfocalRESUMO
Alcoholic liver disease (ALD) is a chronic toxic liver injury caused by long-term heavy drinking. Due to the increasing incidence, ALD is becoming one of important medical tasks. Many studies have shown that the main mechanism of liver damage caused by large amounts of alcohol may be related to antioxidant stress. As an important antioxidant, cysteine (Cys) is involved in maintaining the normal redox balance and detoxifying metabolic function of the liver, which may be closely related to the pathogenesis of ALD. Therefore, it is necessary to develop a simple non-invasive method for rapid monitoring of Cys in liver. Thus, a near-infrared (NIR) fluorescent probe DCI-Ac-Cys which undergoes Cys triggered cascade reaction to form coumarin fluorophore is developed. Using the DCI-Ac-Cys, decreased Cys was observed in the liver of ALD mice. Importantly, different levels of Cys were monitored in the livers of ALD mice taking silybin and curcumin with the antioxidant effects, indicating the excellent therapeutic effect on ALD. This study provides the important references for the accurate diagnosis of ALD and the pharmacodynamic evaluation of silybin and curcumin in the treatment of ALD, and support new ideas for the pathogenesis of ALD.
Assuntos
Cumarínicos , Cisteína , Corantes Fluorescentes , Hepatopatias Alcoólicas , Animais , Cisteína/análise , Cisteína/metabolismo , Cumarínicos/química , Corantes Fluorescentes/química , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Masculino , Fígado/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Curcumina/farmacologia , Espectrometria de Fluorescência , Silibina/farmacologia , Silibina/químicaRESUMO
Epilepsy is one of the most commonly-seen neurological disorders, and both endoplasmic reticulum stress (ERS) and oxidative stress (OS) have been demonstrated to be associated with epileptic seizures. As one of the three endogenous thiol-containing amino acids, cysteine (Cys) is recognized not only as an important biomarker of various biological processes but also widely used as a significant additive in the food industry. However, the exact role that Cys plays in ERS has not been well answered up to now. In this paper, we reported the first flavone-based fluorescent probe (namely BFC) with nice endoplasmic reticulum (ER)-targeting ability, which was capable of monitoring Cys in a fast response (3.0 min), large stokes shift (130 nm) and low detection limit (10.4 nM). The recognition mechanism of Cys could be attributed to the addition-cyclization reaction involving a Cys residue and an acrylate group, resulting in the release of the strong excited-state intramolecular proton transfer (ESIPT) emission molecule of benzoflavonol (BF). The low cytotoxicity and good biocompatibility of the probe BFC allowed for monitoring the fluctuation of endogenous Cys levels under both ERS and OS processes, as well as in zebrafish models of epilepsy. Quantitative determination of Cys with the probe BFC was also achieved in three different food samples. Additionally, a probe-immersed test strips integrated with a smartphone device was successfully constructed for on-site colorimetric detection of Cys. Undoubtedly, our work provided a valuable tool for tracking Cys levels in both an epilepsy model and real food samples.
Assuntos
Cisteína , Retículo Endoplasmático , Epilepsia , Flavonas , Corantes Fluorescentes , Análise de Alimentos , Peixe-Zebra , Corantes Fluorescentes/química , Cisteína/análise , Animais , Epilepsia/diagnóstico , Flavonas/análise , Flavonas/química , Retículo Endoplasmático/metabolismo , Análise de Alimentos/métodos , Espectrometria de Fluorescência/métodos , Humanos , Modelos Animais de Doenças , Limite de Detecção , Estresse do Retículo EndoplasmáticoRESUMO
BACKGROUND: Biothiols are important for numerous cellular processes, such as resisting oxidative stress and protecting cell health. Their abnormal levels and molecular configurations have been associated with various diseases. So, establishing an effective and reliable method for the specific detection and enantiomeric discrimination of diverse biothiols is highly meaningful. RESULTS: We have developed a new NMR and CD probe using 1,4-dinitroimidazole, specifically targeting the thiol group. This probe allows for the specific detection and enantiomeric recognition of biothiols in complex mixtures. We achieved this by identifying the distinguishable 1H NMR signals of 2nd in imidazole-ring of the resulting 4NI-biothiols in the downfield region at 7-8 ppm and newly discovered induced CD signals within 290-430 nm. Using this probe, the limits of detection of Cys, GSH, and Hcy, the recovery rates, and the concentration of GSH extracted from HEK293T cells were determined by measuring the unique downfield 1H NMR signals. Moreover, Cys, GSH, and Hcy can be discriminated simultaneously in complicated samples at a pH range of 2-3.5. Furthermore, this probe can also be utilized to sense chiral thiol-drugs. SIGNIFICANCE: This method offers a cost-effective and accurate sensing solution for the specific detection of biothiols in complex mixtures, with stereochemical recognition.
Assuntos
Imidazóis , Compostos de Sulfidrila , Humanos , Estereoisomerismo , Imidazóis/química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/análise , Células HEK293 , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Cisteína/análise , Glutationa/análise , Glutationa/química , Homocisteína/análise , Limite de Detecção , Estrutura MolecularRESUMO
Peptides are valuable for therapeutic development, with multicyclic peptides showing promise in mimicking antigen-binding potency of antibodies. However, our capability to engineer multicyclic peptide scaffolds, particularly for the construction of large combinatorial libraries, is still limited. Here, we study the interplay of disulfide pairing between three biscysteine motifs, and designed a range of triscysteine motifs with unique disulfide-directing capability for regulating the oxidative folding of multicyclic peptides. We demonstrate that incorporating these motifs into random sequences allows the design of disulfide-directed multicyclic peptide (DDMP) libraries with up to four disulfide bonds, which have been applied for the successful discovery of peptide binders with nanomolar affinity to several challenging targets. This study encourages the use of more diverse disulfide-directing motifs for creating multicyclic peptide libraries and opens an avenue for discovering functional peptides in sequence and structural space beyond existing peptide scaffolds, potentially advancing the field of peptide drug discovery.
Assuntos
Cisteína , Dissulfetos , Biblioteca de Peptídeos , Dissulfetos/química , Cisteína/química , Motivos de Aminoácidos , Descoberta de Drogas/métodos , Sequência de Aminoácidos , Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Humanos , Oxirredução , Dobramento de ProteínaRESUMO
Glutathione transferase P1 (GSTP1) is a multi-functional protein that protects cells from electrophiles by catalyzing their conjugation with glutathione, and contributes to the regulation of cell proliferation, apoptosis, and signalling. GSTP1, usually described as a cytosolic enzyme, can localize to other cell compartments and we have reported its strong association with the plasma membrane. In the current study, the hypothesis that GSTP1 is palmitoylated and this modification facilitates its dynamic localization and function was investigated. Palmitoylation is the reversible post-translational addition of a 16-C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. GSTP1 in MCF7 cells was modified by palmitate, however, GSTP1 Cys to Ser mutants (individual and Cys-less) retained palmitoylation. Treatment of palmitoylated GSTP1 with 0.1 N NaOH, which cleaves ester bonds, did not remove palmitate. Purified GSTP1 was spontaneously palmitoylated in vitro and peptide sequencing revealed that Cys48 and Cys102 undergo S-palmitoylation, while Lys103 undergoes the rare N-palmitoylation. N-palmitoylation occurs via a stable NaOH-resistant amide bond. Analysis of subcellular fractions of MCF7-GSTP1 cells and a modified proximity ligation assay revealed that palmitoylated GSTP1 was present not only in the membrane fraction but also in the cytosol. GSTP1 isolated from E. coli, and MCF7 cells (grown under fatty acid free or regular conditions), associated with plasma membrane-enriched fractions and this association was not altered by palmitoyl CoA. Overall, GSTP1 is modified by palmitate, at multiple sites, including at least one non-Cys residue. These modifications could contribute to regulating the diverse functions of GSTP1.
Assuntos
Glutationa S-Transferase pi , Lipoilação , Palmitatos , Humanos , Glutationa S-Transferase pi/metabolismo , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/química , Células MCF-7 , Palmitatos/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Cisteína/metabolismo , Processamento de Proteína Pós-Traducional , Ácido Palmítico/metabolismoRESUMO
Fibroblast growth factor 2 (FGF2) is an attractive biomaterial for pharmaceuticals and functional cosmetics. To improve the thermo-stability of FGF2, we designed two mutants harboring four-point mutations: FGF2-M1 (D28E/C78L/C96I/S137P) and FGF2-M2 (D28E/C78I/C96I/S137P) through bioinformatics, molecular thermodynamics, and molecular modeling. The D28E mutation reduced fragmentation of the FGF2 wild type during preparation, and the substitution of a whale-specific amino acid, S137P, enhanced the thermal stability of FGF2. Surface-exposed cysteines that participate in oligomerization through intermolecular disulfide bond formation were substituted with hydrophobic residues (C78L/C78I and C96I) using the in silico method. High-resolution crystal structures revealed at the atomic level that the introduction of mutations stabilizes each local region by forming more favorable interactions with neighboring residues. In particular, P137 forms CH-π interactions with the side chain indole ring of W123, which seems to stabilize a ß-hairpin structure, containing a heparin-binding site of FGF2. Compared to the wild type, both FGF2-M1 and FGF2-M2 maintained greater solubility after a week at 45 °C, with their Tm values rising by ~ 5 °C. Furthermore, the duration for FGF2-M1 and FGF2-M2 to reach 50% residual activity at 45 °C extended to 8.8- and 8.2-fold longer, respectively, than that of the wild type. Interestingly, the hydrophobic substitution of surface-exposed cysteine in both FGF2 mutants makes them more resistant to proteolytic cleavage by trypsin, subtilisin, proteinase K, and actinase than the wild type and the Cys â Ser substitution. The hydrophobic replacements can influence protease resistance as well as oligomerization and thermal stability. It is notable that hydrophobic substitutions of surface-exposed cysteines, as well as D28E and S137P of the FGF2 mutants, were designed through various approaches with structural implications. Therefore, the engineering strategies and structural insights adopted in this study could be applied to improve the stability of other proteins.
Assuntos
Cisteína , Fator 2 de Crescimento de Fibroblastos , Interações Hidrofóbicas e Hidrofílicas , Estabilidade Proteica , Cisteína/química , Cisteína/genética , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Mutação , Modelos Moleculares , Cristalografia por Raios X , Substituição de Aminoácidos , Humanos , TermodinâmicaRESUMO
In plants, a local infection can lead to systemic acquired resistance (SAR) through increased production of salicylic acid (SA). For many years, the identity of the mobile signal and its direct transduction mechanism for systemic SA synthesis in initiating SAR have been debated. We found that in Arabidopsis thaliana, after a local infection, the conserved cysteine residue of the transcription factor CCA1 HIKING EXPEDITION (CHE) undergoes sulfenylation in systemic tissues, which enhances its binding to the promoter of the SA-synthesis gene ISOCHORISMATE SYNTHASE1 (ICS1) and increases SA production. Furthermore, hydrogen peroxide (H2O2) produced through NADPH oxidases is the mobile signal that sulfenylates CHE in a concentration-dependent manner. Accumulation of SA and the previously reported signal molecules, such as N-hydroxypipecolic acid (NHP), then form a signal amplification loop to establish SAR.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Peróxido de Hidrogênio , Doenças das Plantas , Ácido Salicílico , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/microbiologia , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Peróxido de Hidrogênio/metabolismo , Ácido Salicílico/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genética , Regiões Promotoras Genéticas , Transferases Intramoleculares/metabolismo , Transferases Intramoleculares/genética , NADPH Oxidases/metabolismo , NADPH Oxidases/genética , Regulação da Expressão Gênica de Plantas , Cisteína/metabolismo , Transdução de Sinais , Pseudomonas syringaeRESUMO
Hydrogen sulfide (H2S), mainly produced from L-cysteine (Cys), renders bacteria highly resistant to oxidative stress and potentially increases antimicrobial resistance (AMR). CyuR is a Cys-dependent transcription regulator, responsible for the activation of the cyuPA operon and generation of H2S. Despite its potential importance, its regulatory network remains poorly understood. In this study, we investigate the roles of the CyuR regulon in a Cys-dependent AMR mechanism in E. coli strains. We show: (1) Generation of H2S from Cys affects the sensitivities to growth inhibitors; (2) Cys supplementation decreases stress responses; (3) CyuR negatively controls the expression of mdlAB encoding a potential transporter for antibiotics; (4) CyuR binds to a DNA sequence motif 'GAAwAAATTGTxGxxATTTsyCC' in the absence of Cys; and (5) CyuR may regulate 25 additional genes which were not reported previously. Collectively, our findings expand the understanding of the biological roles of CyuR relevant to antibiotic resistance associated with Cys.
Assuntos
Cisteína , Farmacorresistência Bacteriana , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Farmacorresistência Bacteriana/genética , Antibacterianos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Óperon , RegulonRESUMO
RAF kinases, consisting of ARAF, BRAF and CRAF, are direct effectors of RAS GTPases and critical for signal transduction through the RAS-MAPK pathway. Driver mutations in BRAF are commonplace in human cancer, while germline mutations in BRAF and CRAF cause RASopathy development syndromes. However, there remains a lack of effective drugs that target RAF function, which is partially due to the complexity of the RAF activation cycle. Therefore, greater understanding of RAF regulation is required to identify new approaches that target its function in disease. A key piece of this puzzle is the RAF zinc finger, often referred to as the cysteine-rich domain (CRD). The CRD is a lipid and protein binding domain which plays complex and opposing roles in the RAF activation cycle. Firstly, it supports the RAS-RAF interaction during RAF activation by binding to phosphatidylserine (PS) in the plasma membrane and by making direct RAS contacts. Conversely, under quiescent conditions the CRD also plays a critical role in maintaining RAF in a closed, autoinhibited state. However, the interplay between these activities and their relative importance for RAF activation were not well understood. Recent structural and biochemical studies have contributed greatly to our understanding of these roles and identified functional differences between BRAF CRD and that of CRAF. This chapter provides an in-depth review of the CRDs roles in RAF regulation and how they may inform novel approaches to target RAF function.
Assuntos
Neoplasias , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/genética , Animais , Quinases raf/metabolismo , Domínios Proteicos , Cisteína/metabolismo , Mutação , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The ER is the organelle of nucleated cells that produces lipids, sugars, and proteins. More than 20 ER-resident members of the protein disulfide isomerase (PDI) family regulate formation, isomerization, and disassembly of covalent bonds in newly synthesized polypeptides. The PDI family includes few membrane-bound members. Among these, TMX1, TMX2, TMX3, TMX4, and TMX5 belong to the thioredoxin-related transmembrane (TMX) protein family. TMX5 is the least-known member of the family. Here, we establish that TMX5 covalently engages via its active site cysteine residue at position 220 a subset of secretory proteins, mainly single- and multipass Golgi-resident polypeptides. TMX5 also interacts non-covalently, and covalently, via non-catalytic cysteine residues, with the PDI family members PDI, ERp57, and ERp44. The association between TMX5 and ERp44 requires formation of a mixed disulfide between the catalytic cysteine residue 29 of ERp44 and the non-catalytic cysteine residues 114 and/or 124 of TMX5 and controls the ER localization of TMX5 in pre-Golgi compartments. Thus, TMX5 belongs to the family of proteins including Ero1α, Ero1ß, Prx4, ERAP1, and SUMF1 that operate in pre-Golgi compartments but lack localization sequences required to position themselves and rely on ERp44 engagement for proper intercompartmental distribution.
Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Humanos , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Complexo de Golgi/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Cisteína/metabolismo , Células HeLa , Mutação , Domínio Catalítico/genética , Ligação Proteica , Chaperonas MolecularesRESUMO
L-cysteine is an essential component in pharmaceutical and agricultural industries, and synthetic biology has made strides in developing new metabolic pathways for its production, particularly in archaea with unique O-phosphoserine sulfhydrylases (OPSS) as key enzymes. In this study, we employed database mining to identify a highly catalytic activity OPSS from Acetobacterium sp. (AsOPSS). However, it was observed that the enzymatic activity of AsOPSS suffered significant feedback inhibition from the product L-cysteine, exhibiting an IC50 value of merely 1.2 mM. A semi-rational design combined with tunnel analysis strategy was conducted to engineer AsOPSS. The best variant, AsOPSSA218R was achieved, totally eliminating product inhibition without sacrificing catalytic efficiency. Molecular docking and molecular dynamic simulations indicated that the binding conformation of AsOPSSA218R with L-cys was altered, leading to a reduced affinity between L-cysteine and the active pocket. Tunnel analysis revealed that the AsOPSSA218R variant reshaped the landscape of the tunnel, resulting in the construction of a new tunnel. Furthermore, random acceleration molecular dynamics simulation and umbrella sampling simulation demonstrated that the novel tunnel improved the suitability for product release and effectively separated the interference between the product release and substrate binding processes. Finally, more than 45 mM of L-cysteine was produced in vitro within 2 h using the AsOPSSA218R variant. Our findings emphasize the potential for relieving feedback inhibition by artificially generating new product release channels, while also laying an enzymatic foundation for efficient L-cysteine production.
Assuntos
Cisteína Sintase , Cisteína , Simulação de Dinâmica Molecular , Cisteína/química , Cisteína/metabolismo , Cisteína Sintase/química , Cisteína Sintase/metabolismo , Cisteína Sintase/genética , Simulação de Acoplamento Molecular , Engenharia de Proteínas/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genéticaRESUMO
Owing to the on-going emission of Hg into the global environment, new insight into their bioinorganic chemistry in mammals is urgently required to better understand their adverse health effects and analytical methods to quantify Hg2+ and MeHg+ in environmental samples are needed. Analytical separations can help to address both of these needs. While Hg2+ and MeHg+ have been most frequently separated by cation and reversed-phase (RP) HPLC, we here report on using anion-exchange (AEX) HPLC in conjunction with a flame atomic absorption spectrometer (FAAS) to observe the retention behavior of these mercury species in the pH range 5.0-8.0 using mobile phases comprised of 10 mM l-cysteine (Cys) in 100 mM phosphate buffer. The results obtained for pH 5.0 served as a starting point to develop a rapid HPLC separation for these mercurials. The addition of 5-20 % methanol (MeOH) to this mobile phase revealed that MeOH did not appreciably change the retention of Hg2+, but significantly reduced the retention of MeHg+. A 15 % MeOH-containing mobile phase offered the best compromise between achieving a rapid baseline separation in <400 s at affordable costs. To assess the suitability and robustness of the developed AEX-HPLC separation method for the analysis of environmental samples an inductively coupled plasma atomic emission spectrometer (ICP-AES) was employed as the mercury-specific detector. The developed AEX-HPLC-ICP-AES method allowed to achieve detection limits of 1.5 ppm for Hg2+ and 2.9 ppm for MeHg+ and was successfully applied to analyze wastewater that had been spiked with Hg2+ and MeHg+.
Assuntos
Cisteína , Mercúrio , Compostos de Metilmercúrio , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/química , Mercúrio/análise , Mercúrio/química , Mercúrio/isolamento & purificação , Compostos de Metilmercúrio/análise , Compostos de Metilmercúrio/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Limite de Detecção , Espectrofotometria Atômica/métodos , Concentração de Íons de Hidrogênio , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química , Poluentes Químicos da Água/isolamento & purificaçãoRESUMO
1,3-Butadiene (BD) is a carcinogenic air pollutant. N-acetyl-S-(4-hydroxy-2-buten-1-yl)-L-cysteine (MHBMA3 or 4HBeMA), an urinary BD metabolite with unspecified configuration, is considered the most sensitive BD biomarker and has been used in routine biomonitoring since 2012. However, two issues remain unaddressed: why its concentrations are unusually high relative to other urinary BD biomarkers and why some authors reported no detection of the biomarker whereas other authors readily quantitated it. To address the issues, we synthesized and structurally characterized the authentic trans- and cis-isomers of MHBMA3 (designated NE and NZ, respectively), developed an isotope-dilution LC-MS/MS method for their quantification, and examined 67 urine samples from barbecue restaurant personnel (n = 47) and hotel administrative staff (n = 20). The restaurant personnel were exposed to barbecue fumes, which contain relatively high concentrations of BD. The results showed that NE and NZ had highly similar NMR spectra, and were difficult to be well separated chromatographically. The NMR data showed that the MHBMA3 isomer investigated in most previous studies was NE. We did not detect NE and NZ in any samples; however, an interfering peak with varying heights was observed in most samples. Notably, under the chromatographic conditions used in the literature, the peak exhibited indistinguishable retention time from that of NE. Thus, it is highly likely that the interfering peak has been mis-identified as NE in previous studies, providing a reasonable explanation for the high MHBMA3 concentration in urine. The contradiction in the presence of MHBMA3 in urine was also caused by the mis-identification, because the researchers who reported the absence of MHBMA3 were actually detecting NZ. Thus, we clarified the confusion on MHBMA3 in previous studies through correctly identifying the two MHBMA3 isomers. The presence of NE and NZ in human urine warrants further investigations.
Assuntos
Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Acetilcisteína/urina , Acetilcisteína/análogos & derivados , Acetilcisteína/química , Isomerismo , Limite de Detecção , Butadienos/química , Butadienos/urina , Reprodutibilidade dos Testes , Cisteína/urina , Cisteína/análogos & derivados , Cisteína/química , Biomarcadores/urina , MasculinoRESUMO
Leydig cells are the primary source of testosterone or androgen production in male mammals. The blood-testis barrier (BTB) maintains structural integrity and safeguards germ cells from harmful substances by blocking their entry into the seminiferous tubules. L-cysteine is essential to the production of glutathione, a powerful antioxidant crucial to protecting against oxidative stress-induced damage. Animal studies have demonstrated the protective effect of L-cysteine in preventing testicular damage caused by chemicals or radiation. This study examines whether L-cysteine enhances the expression of testosterone biosynthesis and the BTB genes in human Leydig cells and THP-1 monocytes. The Leydig cells and THP-1 monocytes were treated with L-cysteine for 24 h. RNA was extracted following treatment, and the gene expression was analyzed using quantitative RT-PCR. Testosterone levels in the cell supernatant were measured using an ELISA kit. L-cysteine treatment in Leydig cells significantly upregulated the expression of CYP11A1 (p = 0.03) and the BTB genes CLDN1 (p = 0.03), CLDN11 (p = 0.02), and TJP1 (p = 0.02). Similarly, L-cysteine significantly upregulated the expression of CYP11A1 (p = 0.03) and CYP19A1 (p < 0.01), and the BTB genes CLDN1 (p = 0.04), CLDN2 (p < 0.01), CLDN4 (p < 0.01), CLDN11 (p < 0.01), and TJP1 (p = 0.03) in THP-1 monocytes. Further, L-cysteine supplementation increased the testosterone secretion levels in human Leydig cells. The findings suggest that L-cysteine supplementation could be used as an adjuvant therapy to promote the integrity of the BTB genes, testosterone biosynthesis and secretion, and the maintenance of testicular functions, which in turn mitigates the risk of male infertility.
Assuntos
Barreira Hematotesticular , Cisteína , Células Intersticiais do Testículo , Monócitos , Testosterona , Humanos , Masculino , Células Intersticiais do Testículo/metabolismo , Células Intersticiais do Testículo/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Barreira Hematotesticular/efeitos dos fármacos , Cisteína/farmacologia , Cisteína/metabolismo , Monócitos/metabolismo , Monócitos/efeitos dos fármacos , Células THP-1 , Regulação para Cima/efeitos dos fármacos , Células CultivadasRESUMO
Blood-testis barrier (BTB) genes are crucial for the cellular mechanisms of spermatogenesis as they protect against detrimental cytotoxic agents, chemicals, and pathogens, thereby maintaining a sterile environment necessary for sperm development. BTB proteins predominantly consist of extensive tight and gap junctions formed between Sertoli cells. These junctions form a crucial immunological barrier restricting the intercellular movement of substances and molecules within the adluminal compartment. Epithelial tight junctions are complex membrane structures composed of various integral membrane proteins, including claudins, zonula occludens-1, and occludin. Inter-testicular cell junction proteins undergo a constant process of degradation and renewal. In addition, the downregulation of genes crucial to the development and preservation of cell junctions could disrupt the functionality of the BTB, potentially leading to male infertility. Oxidative stress and inflammation may contribute to disrupted spermatogenesis, resulting in male infertility. L-cysteine is a precursor to glutathione, a crucial antioxidant that helps mitigate damage and inflammation resulting from oxidative stress. Preclinical research indicates that L-cysteine may offer protective benefits against testicular injury and promote the expression of BTB genes. This review emphasizes various BTB genes essential for preserving its structural integrity and facilitating spermatogenesis and male fertility. Furthermore, it consolidates various research findings suggesting that L-cysteine may promote the expression of BTB-associated genes, thereby aiding in the maintenance of testicular functions.
Assuntos
Barreira Hematotesticular , Cisteína , Espermatogênese , Masculino , Barreira Hematotesticular/metabolismo , Humanos , Animais , Cisteína/metabolismo , Junções Íntimas/metabolismo , Estresse Oxidativo , Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Células de Sertoli/metabolismo , Células de Sertoli/efeitos dos fármacos , Testículo/metabolismoRESUMO
Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.
Assuntos
Cisteína , Dissulfetos , Simulação de Dinâmica Molecular , Anticorpos de Domínio Único , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Animais , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Estabilidade Proteica , Receptores de Antígenos/química , Receptores de Antígenos/metabolismo , Receptores de Antígenos/genética , Receptores de Antígenos/imunologia , Afinidade de Anticorpos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Muramidase/química , Muramidase/metabolismo , Muramidase/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , MutaçãoRESUMO
Cys is one of the least abundant amino acids in proteins. However, it is often highly conserved and is usually found in important structural and functional regions of proteins. Its unique chemical properties allow it to undergo several post-translational modifications, many of which are mediated by reactive oxygen, nitrogen, sulfur, or carbonyl species. Thus, in addition to their role in catalysis, protein stability, and metal binding, Cys residues are crucial for the redox regulation of metabolism and signal transduction. In this review, we discuss Cys post-translational modifications (PTMs) and their role in plant metabolism and signal transduction. These modifications include the oxidation of the thiol group (S-sulfenylation, S-sulfinylation and S-sulfonylation), the formation of disulfide bridges, S-glutathionylation, persulfidation, S-cyanylation S-nitrosation, S-carbonylation, S-acylation, prenylation, CoAlation, and the formation of thiohemiacetal. For each of these PTMs, we discuss the origin of the modifier, the mechanisms involved in PTM, and their reversibility. Examples of the involvement of Cys PTMs in the modulation of protein structure, function, stability, and localization are presented to highlight their importance in the regulation of plant metabolic and signaling pathways.
Assuntos
Cisteína , Proteínas de Plantas , Plantas , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Cisteína/metabolismo , Cisteína/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Plantas/metabolismo , OxirreduçãoRESUMO
Biothiols, characterized by thiol groups, exhibit remarkable affinity for certain metals, playing pivotal roles in intracellular and extracellular biological processes. Fluctuations in their levels profoundly impact overall physiological health. Despite the development of various probes for biothiol detection and quantification, their inability to monitor thiol-to-disulfide state transitions persists as a limitation. Given their association with pathologies, early detection remains imperative. Gold nanorod (AuNR)-based colorimetric probes have garnered attention for their utility in visual diagnostic assays. Herein, we present a cost-effective, and sensitive multicolor ratio measuring probe enabling on-site simultaneous identification, discrimination, and quantification of essential biothiolsâcysteine (CYS), glutathione (GSH), cystine (CYSS), and glutathione disulfide (GSSG)âwhile also quantifying thiol-to-disulfide ratios. Our investigation clarifies the probe's functionality, elucidating etching and antietching mechanisms based on sulfhydryl group coordination with Hg2+. This coordination impedes gold amalgam formation, facilitating discriminative detection via AuNR size and aspect ratio modulation, validated by transmission electron microscopy. Notably, distinct rainbow-like fingerprint patterns were discernible both visually and spectroscopically for the aforementioned biothiols and their respective thiol-to-disulfide ratios. Subsequent qualitative and quantitative analyses via linear discriminant analysis (LDA) and partial least squares regression revealed linear correlations over broad concentration ranges (CYS: 1.9-40 µmol L-1, GSH: 3.2-200.0 µmol L-1, CYSS: 2.0-70.0 µmol L-1, GSSG: 3.7-100.0 µmol L-1), with detection limits of 0.66 µmol L-1 (CYS), 1.07 µmol L-1 (GSH), 0.69 µmol L-1 (CYSS), and 1.24 µmol L-1 (GSSG). Moreover, thiol-to-disulfide ratios exhibited linear patterns within 0.2-5 µmol L-1, with detection limits of 0.13 and 0.09 µmol L-1, and exceptional analytical sensitivities of 32.648 and 49.782 for (CYS/CYSS) and (GSH/GSSG), respectively. Lastly, we evaluated the probe's performance in complex matrices relative to aqueous media, both quantitatively and qualitatively.