RESUMEN

Thiol dioxygenases (TDOs) are non­heme Fe(II)­dependent enzymes that catalyze the O2-dependent oxidation of thiol substrates to their corresponding sulfinic acids. Six classes of TDOs have thus far been identified and two, cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO), are found in eukaryotes. All TDOs belong to the cupin superfamily of enzymes, which share a common ß­barrel fold and two cupin motifs: G(X)5HXH(X)3-6E(X)6G and G(X)5-7PXG(X)2H(X)3N. Crystal structures of TDOs revealed that these enzymes contain a relatively rare, neutral 3­His iron­binding facial triad. Despite this shared metal-binding site, TDOs vary greatly in their secondary coordination spheres. Site­directed mutagenesis has been used extensively to explore the impact of changes in secondary sphere residues on substrate specificity and enzymatic efficiency. This chapter summarizes site-directed mutagenesis studies of eukaryotic TDOs, focusing on the tools and practicality of non­standard amino acid incorporation.

Asunto(s)

Aminoácidos , Dioxigenasas , Mutagénesis Sitio-Dirigida , Dioxigenasas/química , Dioxigenasas/metabolismo , Dioxigenasas/genética , Aminoácidos/metabolismo , Aminoácidos/química , Especificidad por Sustrato , Cisteína-Dioxigenasa/química , Cisteína-Dioxigenasa/metabolismo , Cisteína-Dioxigenasa/genética , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/química , Humanos , Animales , Modelos Moleculares

RESUMEN

The present study investigated the effect of adding allopathic doxorubicin (DOX 0.3 µg/mL), the vehicle of ultradiluted/dynamized doxorubicin (0.2 % ethanol), different dynamizations of ultradiluted/dynamized doxorubicin (DOX 6CH, DOX 12CH and DOX 30CH), both in the absence or presence of chemical stress induced by doxorubicin at 0.3 µg/mL on follicular survival and activation, antioxidant capacity of the medium, Catalase activity (CAT), production of reactive protein thiol, maintenance of type I and III collagen fibers and accumulation of lipofuscin in porcine ovarian tissue cultured in vitro for 48 hours. To do this, part of the ovarian tissue fragments was fixed for the uncultured control and the rest were cultured in: MEM (cultured control), DOX 0.3 µg/mL, Ethanol, DOX 6CH, DOX 12CH, DOX 30CH, DOX (0.3 µg/mL) + DOX 6CH, DOX (0.3 µg/mL) + DOX 12CH, DOX (0.3 µg/mL) + DOX 30CH treatments. The results showed that, in general, ultradiluted/dynamized doxorubicin (DOX 6CH, DOX 12CH and DOX 30CH) mitigated the toxic effect of allopathic doxorubicin (0.3 µg/mL) on the morphology of preantral follicles, the content of type I and III collagen fibers, and the production of lipofuscin in the tissue. However, only DOX (0.3 µg/mL) + DOX 6CH attenuated the oxidative stress induced by DOX (0.3 µg/mL), maintaining adequate CAT activity that was similar to the uncultured control. Additionally, when the three isolated ultradiluted/dynamized doxorubicin were considered, only DOX 12CH increased the reduced thiol levels compared to the uncultured control and MEM. In conclusion, supplementing the culture medium with ultradiluted/dynamized DOX (DOX 6CH, DOX 12CH and DOX 30CH) attenuated the toxicity induced by allopathic doxorubicin during the in vitro culture of pig preantral follicles enclosed in ovarian tissue.

Asunto(s)

Antibióticos Antineoplásicos , Doxorrubicina , Folículo Ovárico , Animales , Doxorrubicina/toxicidad , Femenino , Porcinos , Antibióticos Antineoplásicos/toxicidad , Folículo Ovárico/efectos de los fármacos , Catalasa/metabolismo , Técnicas de Cultivo de Tejidos , Lipofuscina/metabolismo , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/farmacología , Colágeno Tipo I/metabolismo , Ovario/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Colágeno Tipo III/metabolismo

RESUMEN

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic in vitro organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping. The shelf life of the HLM chips in the wetted state is ca. 2-3 weeks only and requires cold storage at 4 °C. In this study, we developed a freeze-drying method for lyophilization of HLMs that are readily immobilized inside microfluidic pillar arrays made from off-stoichiometric thiol-ene polymer. The success of lyophilization was evaluated by monitoring the cytochrome P450 and UDP-glucuronosyltransferase enzyme activities of rehydrated HLMs for several months post-freeze-drying. By adapting the freeze-drying protocol, the HLM chips could be stored at room temperature (protected from light and moisture) for at least 9 months (n = 2 independent batches) and up to 16 months at best, with recovered enzyme activities within 60-120% of the non-freeze-dried control chips. This is a major improvement over the cold-storage requirement and the limited shelf life of the non-freeze-dried HLM chips, which can significantly ease the design of experiments, decrease energy consumption during storage, and reduce the shipping costs with a view to commercial adaptation.

Asunto(s)

Liofilización , Microsomas Hepáticos , Humanos , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/química , Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Técnicas Analíticas Microfluídicas/instrumentación

RESUMEN

The aromatic profile of wine determines its overall final quality, and among the volatile molecules that define it, varietal thiols are responsible for shaping the distinctive character of certain wine varieties. In grape must, these thiols are conjugated to amino acids or small peptides in a non-volatile form. During wine fermentation, yeasts play a principal role in expressing these aromatic compounds as they internalise and cleavage these precursors, releasing the corresponding free and aroma-impacting fraction. Here, we investigate the impact of three wine yeasts (Saccharomyces cerevisiae, Torulaspora delbrueckii and Lachancea thermotolerans) on thiol releasing in synthetic grape must fermentations supplemented with different cysteinylated (Cys-4MSP and Cys-3SH) and glutathionylated (GSH-4MSP and GSH-3SH) precursors. We demonstrate higher consumption levels of cysteinylated precursors, and consequently, higher amounts of thiols are released from them compared to glutathionylated ones. We also report a significant impact of yeast inoculated on the final thiols released. Meanwhile T. delkbrueckii exhibits a great 3SHA releasing capacity, L. thermotolerans stands out because of its high 3SH release. We also highlight the synergic effect of the co-inoculation strategy, especially relevant in the case of S. cerevisiae and L. thermotolerans mixed fermentation, that has an outstanding release of 4MSP thiol. Although our results stem from a specific experimental approach that differs from real winemaking situations, these findings reveal the potential of unravelling the specific role of different yeast species, thiol precursors and their interaction, to improve wine production processes in the context of wine aroma enhancement.

Asunto(s)

Fermentación , Saccharomyces cerevisiae , Compuestos de Sulfhidrilo , Torulaspora , Vino , Vino/microbiología , Vino/análisis , Compuestos de Sulfhidrilo/metabolismo , Saccharomyces cerevisiae/metabolismo , Torulaspora/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/crecimiento & desarrollo , Vitis/microbiología , Odorantes/análisis

RESUMEN

Post-transcriptional modification of nucleosides in transfer RNAs (tRNAs) is an important process for accurate and efficient translation of the genetic information during protein synthesis in all domains of life. In particular, specific enzymes catalyze the biosynthesis of sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A), within tRNAs. Whereas the mechanism that has prevailed for decades involved persulfide chemistry, more and more tRNA thiolation enzymes have now been shown to contain a [4Fe-4S] cluster. This review summarizes the information over the last ten years concerning the biochemical, spectroscopic and structural characterization of [4Fe-4S]-dependent non-redox tRNA thiolation enzymes.

Asunto(s)

Proteínas Hierro-Azufre , ARN de Transferencia , ARN de Transferencia/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/química , Oxidación-Reducción , Procesamiento Postranscripcional del ARN , Humanos , Tiouridina/análogos & derivados , Tiouridina/metabolismo , Tiouridina/química

RESUMEN

Complexes with low-molecular-weight thiols are crucial species of methylmercury (MeHg) excreted by anaerobic Hg-methylating microbes, notably, MeHg-cysteine (MeHg-Cys). As MeHg-Cys diffuses into surface water, it would undergo a ligand exchange process with dissolved organic matter (DOM) under nonsulfidic conditions, inevitably altering MeHg speciation and bioavailability to phytoplankton. In this study, we investigated the competitive binding kinetics between MeHg-Cys and Suwannee River natural organic matter, and their influence on the adsorption and uptake of MeHg by the cyanobacterium, Synechocystis sp. PCC6803. Liquid chromatography-inductively coupled plasma mass spectrometry was employed to monitor the kinetics processes involving competition of DOM with Cys for MeHg binding, which revealed that competitive binding kinetics were dictated by the abundance of thiol moieties in DOM. Thiol concentrations of 0.97 and 49.34 µmol of thiol (g C)-1 resulted in competitive binding rate constant (k values) of 0.30 and 3.47 h-1, respectively. Furthermore, the time-dependent competitive binding of DOM toward MeHg-Cys significantly inhibited MeHg adsorption and uptake by cyanobacteria, an effect that was amplified by an increased thiol abundance in DOM. These findings offer valuable insights into the kinetic characteristics of MeHg's fate and transport, as well as their impact on bioconcentration in aquatic organisms within natural aquatic ecosystems.

Asunto(s)

Compuestos de Metilmercurio , Compuestos de Sulfhidrilo , Compuestos de Metilmercurio/metabolismo , Compuestos de Metilmercurio/química , Adsorción , Cinética , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/química , Cisteína/metabolismo , Cisteína/química , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/química

RESUMEN

Water buffalo horn (WBH), a traditional Chinese medicine, is known for its antipyretic, anti-inflammatory and antioxidant properties. This study aims to investigate the therapeutic potential of WBH keratin (WBHK) and its derived thiol-rich peptide fractions (SHPF) for oxidative stress and inflammation. WBHK and SHPF were prepared and tested using various models including LPS-induced fever in rabbits, H2O2-induced oxidative damage in bEnd.3 cells, TNF-α-induced inflammation in bEnd.3 cells and LPS-induced inflammation in RAW 264.7 cells. Expression of key markers, such as Nrf2, Hmox-1 and NF-κB, were analyzed using qRT-PCR, ELISA and Western blotting. Label-free quantitative proteomic analysis was used to identify key differential proteins associated with the efficacy of SHPF. Our results demonstrated that treatment with WBHK significantly reduced body temperature after 0.5 h of administration in the fever rabbit model. SHPF could alleviate cellular inflammatory injury and oxidative damage by activating the key transcription factor Nrf2 and increasing the expression level of Hmox-1. SHPF could inhibit the NF-κB pathway by reducing IκB phosphorylation. It was also found that SHPF could reduce pro-inflammatory cytokine (IL-6, COX-2 and PGE2) and inhibit the expression of VCAM-1, ICAM-1, IL-6 and MCP-1. Proteomics analysis showed that SHPF could inhibit HMGB1 expression and release. The results indicated that SHPF could significantly reduce inflammation and oxidative stress by regulating the Nrf2/Hmox-1 and NF-κB pathways. These findings suggest the potential therapeutic applications of WBH components in the treatment of oxidative stress and inflammation-related diseases.

Asunto(s)

Hemo-Oxigenasa 1 , Inflamación , Queratinas , Factor 2 Relacionado con NF-E2 , FN-kappa B , Estrés Oxidativo , Péptidos , Transducción de Señal , Animales , Estrés Oxidativo/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Conejos , FN-kappa B/metabolismo , Transducción de Señal/efectos de los fármacos , Ratones , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Inflamación/inducido químicamente , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Queratinas/metabolismo , Péptidos/farmacología , Búfalos , Células RAW 264.7 , Compuestos de Sulfhidrilo/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/química , Antioxidantes/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Cuernos/química , Lipopolisacáridos , Fiebre/tratamiento farmacológico , Fiebre/inducido químicamente , Fiebre/metabolismo , Peróxido de Hidrógeno/metabolismo , Masculino , Medicina Tradicional China

RESUMEN

A microbial metabolite influences myelination in the brain.

Asunto(s)

Bacterias , Eje Cerebro-Intestino , Encéfalo , Microbioma Gastrointestinal , Vaina de Mielina , Compuestos de Sulfhidrilo , Animales , Humanos , Ratones , Bacterias/metabolismo , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Vaina de Mielina/metabolismo , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

Aquatic biota are exposed to toxic substances resulting from human activities, reducing environmental quality and can compromise the health of the organisms. This study aimed to employ Danio rerio as an environmental bioindicator, analyzing the effects of water from distinct urban aquatic environments. An active biomonitoring system was set up to compare the temporal dynamics of histological biomarkers for gill and liver and the patterns of non-protein thiols (NPSH) in muscle in specimens exposed for 3, 6, and 12 days. Three large urban basins in the city of Campo Grande (Midwest of Brazil) were selected. Two sites are in a very populous area (i.e Lagoa and Bandeira) and another on in an area with agricultural activities (i.e Anhanduí). All the streams displayed distinct qualitative characteristics. The presence of metals, including Mn, Zn, Fe, and Al, as well as pH, temperature, and dissolved oxygen, accounted for 38% of the variability (PC1), while total solids, conductivity, ammonia, nitrite, and explained 24 % (PC2). Degree tissue changes index (DTC) in gill and the concentration of NPSH increased in all streams during 3, 6 and 12 days of exposure. DTC in liver increases in all exposure times in most populous stream (i.e Lagoa and Bandeira). Histopathological evidence in the gill, including proliferation, desquamation, and necrosis of the primary lamellar epithelium; fusion and aneurysms in the secondary lamellar epithelium were observed after three days of exposure. Degenerative nuclear figures were noted in the liver after three days of exposure, followed by hepatocellular hypertrophy, lipidosis, and necrosis at twelve days. Our findings showing time-dependent effects of urban aquatic environments in histopathological (i.e DTC) and biochemical biomarkers in zebrafish. The biomonitoring model enabled a comparison of the temporal dynamics of various health markers, using zebrafish as bioindicator. Future studies might use this experimental model and biomarkers for environmental biomonitoring program.

Asunto(s)

Monitoreo Biológico , Monitoreo del Ambiente , Branquias , Hígado , Músculos , Ríos , Compuestos de Sulfhidrilo , Contaminantes Químicos del Agua , Pez Cebra , Animales , Branquias/patología , Branquias/metabolismo , Hígado/metabolismo , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/análisis , Monitoreo del Ambiente/métodos , Compuestos de Sulfhidrilo/metabolismo , Ríos/química , Músculos/química , Músculos/metabolismo , Brasil , Biomarcadores/metabolismo

RESUMEN

The process of harmless treatment of livestock manure produces a large amount of odor, which poses a potential threat to human and livestock health. A vertical fermentation tank system is commonly used for the environmentally sound treatment of chicken manure in China, but the composition and concentration of the odor produced and the factors affecting odor emissions remain unclear. In this study, we investigated the types and concentrations of odors produced in the mixing room (MR), vertical fermenter (VF), and aging room (AR) of the system, and analyzed the effects of bacterial communities and metabolic genes on odor production. The results revealed that 34, 26 and 26 odors were detected in the VF, MR and AR, respectively. The total odor concentration in the VF was 66613 ± 10097, which was significantly greater than that in the MR (1157 ± 675) and AR (1143 ± 1005) (P < 0.001), suggesting that the VF was the main source of odor in the vertical fermentation tank system. Methyl mercaptan had the greatest contribution to the odor produced by VF, reaching 47.82%, and the concentration was 0.6145 ± 0.2164 mg/m3. The abundance of metabolic genes did not correlate significantly with odor production, but PICRUSt analysis showed that cysteine and methionine metabolism involved in methyl mercaptan production was significantly more enriched in MR and VF than in AR. Bacillus was the most abundant genus in the VF, with a relative abundance significantly greater than that in the MR (P < 0.05). The RDA results revealed that Bacillus was significantly and positively correlated with methyl mercaptan. The use of large-scale aerobic fermentation systems to treat chicken manure needs to focused on the production of methyl mercaptan.

Asunto(s)

Pollos , Fermentación , Estiércol , Odorantes , Compuestos de Sulfhidrilo , Animales , Odorantes/análisis , Compuestos de Sulfhidrilo/metabolismo , Reactores Biológicos

RESUMEN

Nitrosyl iron complexes are remarkably multifactorial pharmacological agents. These compounds have been proven to be particularly effective in treating cardiovascular and oncological diseases. We evaluated and compared the antioxidant activity of tetranitrosyl iron complexes (TNICs) with thiosulfate ligands and dinitrosyl iron complexes (DNICs) with glutathione (DNIC-GS) or phosphate (DNIC-PO4-) ligands in hemoglobin-containing systems. The studied effects included the production of free radical intermediates during hemoglobin (Hb) oxidation by tert-butyl hydroperoxide, oxidative modification of Hb, and antioxidant properties of nitrosyl iron complexes. Measuring luminol chemiluminescence revealed that the antioxidant effect of TNICs was higher compared to DNIC-PO4-. DNIC-GS either did not exhibit antioxidant activity or exerted prooxidant effects at certain concentrations, which might have resulted from thiyl radical formation. TNICs and DNIC-PO4- efficiently protected the Hb heme group from decomposition by organic hydroperoxides. DNIC-GS did not exert any protective effects on the heme group; however, it abolished oxoferrylHb generation. TNICs inhibited the formation of Hb multimeric forms more efficiently than DNICs. Thus, TNICs had more pronounced antioxidant activity than DNICs in Hb-containing systems.

Asunto(s)

Antioxidantes , Hemoglobinas , Hierro , Fosfatos , Tiosulfatos , Tiosulfatos/farmacología , Tiosulfatos/química , Hemoglobinas/metabolismo , Hemoglobinas/química , Hierro/metabolismo , Hierro/química , Fosfatos/química , Fosfatos/metabolismo , Ligandos , Antioxidantes/farmacología , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Oxidación-Reducción/efectos de los fármacos , Óxidos de Nitrógeno/química , Óxidos de Nitrógeno/farmacología , Óxidos de Nitrógeno/metabolismo , Glutatión/metabolismo , Animales

RESUMEN

A small molecule disulfide unit technology platform based on dynamic thiol exchange chemistry at the cell membrane has the potential for drug delivery. However, the alteration of the CSSC dihedral angle of the disulfide unit caused by diverse substituents directly affects the effectiveness of this technology platform as well as its own chemical stability. The highly stable open-loop relaxed type disulfide unit plays a limited role in drug delivery due to its low dihedral angle. Here, we have built a novel disulfide unit starship based on the 3,4,5-trihydroxyphenyl skeleton through trigonometric bundling. The intracellular delivery results showed that the trigonometric bundling of the disulfide unit starship effectively promoted cellular uptake without any toxicity, which is far more than 100 times more active than that of equipment with a single disulfide unit in particular. Then, the significant reduction in cell uptake capacity (73-93%) using thiol erasers proves that the trigonometric bundling of the disulfide starship is an endocytosis-independent internalization mechanism via a dynamic covalent disulfide exchange mediated by thiols on the cell surface. Furthermore, analysis of the molecular dynamics simulations demonstrated that trigonometric bundling of the disulfide starship can significantly change the membrane curvature while pushing lipid molecules in multiple directions, resulting in a significant distortion in the membrane structure and excellent membrane permeation performance. In conclusion, the starship system we built fully compensates for the inefficiency deficiencies induced by poor dihedral angles.

Asunto(s)

Disulfuros , Disulfuros/química , Humanos , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Endocitosis , Membrana Celular/metabolismo , Simulación de Dinámica Molecular

RESUMEN

In mitochondria, the detoxification of molar excess H2S as polysulfide proceeded via an oxidation process promoted by Cu/Zn containing superoxide dismutase (SOD1) enzyme, which has been very recently reported as the alternative enzyme for cytosolic H2S oxidation. Herein, we present Ni(II) complexes bearing the terminal SH group as a synthetic functional analogue for the sulfide oxidase function of SOD1. Synthesis, crystal structure and complete spectroscopic characterization of two sets of complexes, [NiLOMe/tBu(PPh3)] (2OMe/tBu) and tetraethyl salt of [NiLOMe/tBu(SH)]-1 (3OMe/tBu), were described (LOMe = (E)-2-methoxy-6-(((2-sulfidophenyl)imino)methyl)phenolate and LtBu = (E)-2,4-di-tert-butyl-6-(((2-sulfidophenyl)imino)methyl)phenolate). Under anaerobic conditions, 3OMe/tBu responded to a catalytic sulfur atom transfer (SAT) reaction with PPh3 to produce SPPh3. The SAT reaction was analyzed using detailed studies of 1H and 31P NMR spectra. Finally, the SAT reactivity pattern was compared with the same in the native enzyme of SOD1.

Asunto(s)

Complejos de Coordinación , Níquel , Azufre , Níquel/química , Níquel/metabolismo , Complejos de Coordinación/química , Complejos de Coordinación/metabolismo , Complejos de Coordinación/síntesis química , Azufre/química , Azufre/metabolismo , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismo , Modelos Moleculares , Catálisis , Anaerobiosis , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

Melasma is a common acquired hyperpigmentation disorder that affects mostly women and individuals with darker skin types. Oxidative stress may play a role in the pathogenesis of melasma. Dynamic thiol/disulfide homeostasis is one of the most important indicators of oxidative stress. This study aimed to investigate the presence of oxidative stress in patients with melasma by evaluating thiol/disulfide homeostasis. Sixty-seven patients with melasma and 41 healthy age- and sex-matched controls were included in the study. Disease severity was evaluated using the modified melasma area and severity index (mMASI). Thiol/disulfide homeostasis parameters of the melasma and control groups were measured using a novel, fully automated spectrophotometric method. Our data indicated the presence of oxidative stress in melasma, which may be correlated with disease severity. Because research on the presence of oxidative stress in melasma is limited, further studies are needed to support these conclusions.

Asunto(s)

Disulfuros , Homeostasis , Melanosis , Estrés Oxidativo , Índice de Severidad de la Enfermedad , Compuestos de Sulfhidrilo , Humanos , Melanosis/metabolismo , Femenino , Compuestos de Sulfhidrilo/metabolismo , Adulto , Homeostasis/fisiología , Masculino , Estudios de Casos y Controles , Persona de Mediana Edad , Espectrofotometría

RESUMEN

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

Asunto(s)

Amiloide , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Cisteína , Oxidación-Reducción , Amiloide/metabolismo , Amiloide/química , Humanos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Cisteína/metabolismo , Cisteína/química , Disulfuros/metabolismo , Disulfuros/química , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/química , Mutación , Polimerizacion

RESUMEN

Thiol-disulfide interconversions are pivotal in the intricate chemistry of biological systems. They play a vital role in governing cellular redox potential and shielding against oxidative harm. These interconversions can also act as molecular switches within an expanding array of redox-regulated proteins, facilitating dynamic and responsive processes. Furthermore, metal-binding proteins often use thiols for coordination. Reverse thiol trapping is a valuable analytical tool to study the redox state of cysteines in biological systems. By selectively capturing and stabilizing free thiol species with an alkylating agent, reverse thiol trapping allows for their subsequent identification and quantification. Various methods can be employed to analyze the trapped thiol adducts, including electrophoresis-based methods, mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatographic techniques. In this chapter, we will focus on describing a simple and sensitive method to sequentially block thiols in their cellular state with a cell-permeant agent (iodoacetamide), and following reduction and denaturation of the samples, trap the native disulfides with a second blocker that shifts the apparent molecular weight of the protein. The oxidation status of proteins for which suitable antibodies are available can then be analyzed by immunoblotting. We present examples of mitochondrial proteins that use cysteine thiols to coordinate metal factors such as iron-sulfur clusters, zinc, and copper.

Asunto(s)

Proteínas Mitocondriales , Oxidación-Reducción , Compuestos de Sulfhidrilo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Humanos , Yodoacetamida/química , Disulfuros/química , Disulfuros/metabolismo , Metales/química , Metales/metabolismo , Cisteína/química , Cisteína/metabolismo

RESUMEN

Global warming, heat waves, and seasonal drought pose serious threats to crops, such as grapevine, that are valued for their secondary metabolites, which are of primary importance for the wine industry. Discriminating the effects of distinct environmental factors in the open field is challenging. In the present study, in vitro cultured berries of Sauvignon Blanc were exposed to individual and combined stress factors to investigate the effects on the biosynthesis of the thiol precursors. Our results confirm the complexity and extreme reactivity of the accumulation process in grapes. However, they also indicate that heat stress has a positive effect on the production of the Cys-3SH precursor. Moreover, we identified several candidate genes, such as VvGSTs and VvGGT that are potentially involved in biosynthesis and consistently modulated. Nonetheless, we were unable to conclusively determine the effects of stresses on the biosynthesis of other precursors nor could we formulate hypotheses regarding their regulation.

Asunto(s)

Ácido Abscísico , Frutas , Calor , Compuestos de Sulfhidrilo , Vitis , Vitis/metabolismo , Vitis/química , Vitis/genética , Frutas/metabolismo , Frutas/química , Frutas/genética , Compuestos de Sulfhidrilo/metabolismo , Ácido Abscísico/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Estrés Fisiológico

RESUMEN

Redox modulation is a common posttranslational modification to regulate protein activity. The targets of oxidizing agents are cysteine residues (Cys), which have to be exposed at the surface of the proteins and are characterized by an environment that favors redox modulation. This includes their protonation state and the neighboring amino acids. The Cys redox state can be assessed experimentally by redox titrations to determine the midpoint redox potential in the protein. Exposed cysteine residues and putative intramolecular disulfide bonds can be predicted by alignments with structural data using dedicated software tools and information on conserved cysteine residues. Labeling with light and heavy reagents, such as N-ethylmaleimide (NEM), followed by mass spectrometric analysis, allows for the experimental determination of redox-responsive cysteine residues. This type of thiol redox proteomics is a powerful approach to assessing the redox state of the cell, e.g., in dependence on environmental conditions and, in particular, under abiotic stress.

Asunto(s)

Cisteína , Oxidación-Reducción , Proteómica , Compuestos de Sulfhidrilo , Cisteína/metabolismo , Cisteína/química , Proteómica/métodos , Compuestos de Sulfhidrilo/metabolismo , Compuestos de Sulfhidrilo/química , Estrés Fisiológico , Procesamiento Proteico-Postraduccional , Espectrometría de Masas/métodos , Proteínas/química , Proteínas/metabolismo

RESUMEN

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Asunto(s)

Lisina , Compuestos de Sulfhidrilo , Lisina/metabolismo , Lisina/química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Acetilación , Cristalinas/metabolismo , Cristalinas/química , Cristalino/metabolismo , Procesamiento Proteico-Postraduccional , Humanos , Acetilcoenzima A/metabolismo , Acetilcoenzima A/química

RESUMEN

Unnatural product (uNP) nonribosomal peptides promise to be a valuable source of pharmacophores for drug discovery. However, the extremely large size and complexity of the nonribosomal peptide synthetase (NRPS) enzymes pose formidable challenges to the production of such uNPs by combinatorial biosynthesis and synthetic biology. Here we report a new NRPS dissection strategy that facilitates the engineering and heterologous production of these NRPSs. This strategy divides NRPSs into "splitting units", each forming an enzyme subunit that contains catalytically independent modules. Functional collaboration between the subunits is then facilitated by artificially duplicating, at the N-terminus of the downstream subunit, the linker - thiolation domain - linker fragment that is resident at the C-terminus of the upstream subunit. Using the suggested split site that follows a conserved motif in the linker connecting the adenylation and the thiolation domains allows cognate or chimeric splitting unit pairs to achieve productivities that match, and in many cases surpass those of hybrid chimeric enzymes, and even those of intact NRPSs, upon production in a heterologous chassis. Our strategy provides facile options for the rational engineering of fungal NRPSs and for the combinatorial reprogramming of nonribosomal peptide production.

Asunto(s)

Péptido Sintasas , Ingeniería de Proteínas , Péptido Sintasas/metabolismo , Péptido Sintasas/química , Péptido Sintasas/genética , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo