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1.
J Agric Food Chem ; 67(41): 11444-11453, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31592644

RESUMO

Innovative approaches to develop flavors with high sensory appeal are critical in encouraging increased consumer preference and adoption of low sodium foods. Gas chromatography-olfactometry, coupled with stable isotope dilution assays and sensory experiments, led to the identification of the odorants responsible for an enhancement in saltiness perception of chicken broth prepared with thermally treated enzymatically hydrolyzed mushroom protein and cysteine, then reacted under kitchen-like cooking conditions. Comparative aroma extract dilution analysis revealed 36 odorants with flavor dilution factors between a range of 1 and 256. Sixteen odorants were quantitated and odor activity values (OAVs) calculated. Important odorants included 2-furfurylthiol (coffee, OAV 610), 1-(2-furyl)ethanethiol (meaty, OAV 78), 3-sulfanylpentan-2-one (catty, OAV 42), sotolon (maple, OAV 20), indole (animal, OAV 8), 2-methyl-3-(methyldithio)furan (meaty, OAV 3), and p-cresol (barnyard, OAV 1). An odor simulation model was evaluated in two consumer sensory studies. These studies confirmed that the addition of the aroma model increased the perceived saltiness of low sodium chicken broth (p < 0.05).


Assuntos
Agaricus/química , Cisteína/química , Proteínas Fúngicas/química , Odorantes/análise , Percepção Gustatória , Agaricus/metabolismo , Cloretos/análise , Cloretos/metabolismo , Cromatografia Gasosa , Culinária , Cisteína/metabolismo , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Temperatura Alta , Humanos , Hidrólise , Olfatometria , Olfato , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo
2.
Nihon Yakurigaku Zasshi ; 154(3): 133-137, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527363

RESUMO

Hydrogen sulfide (H2S) has been focused as a biological mediator, which modulates signal transduction and protects cells and tissues from oxidative stress. H2S is also expected as a neuroprotectant because it has a neuroprotective activity. Endogenous H2S is mainly generated from L-cysteine. However, it is difficult to use L-cysteine as a neuroprotectant because of its neurotoxicity. In 2013, a novel biogenesis pathway of H2S from D-cysteine has been identified. In this pathway, D-amino acid oxidase (DAO) converts D-cysteine to 3-mercaptopyruvate (3MP), followed by the generation of H2S from 3MP by 3-mercaptopyrvate sulfurtransferase. DAO is especially abundant in cerebellum among various brain regions and mediates efficient generation of H2S from D-cysteine in the cerebellar tissues. In addition, D-cysteine has more potent neuroprotective activity in cerebellar primary neurons than L-cysteine. Cerebella Purkinje cells (PCs) are characterized by the highly-branched dendrites and are important for cerebellar functions. The dendritic shrinkage and degeneration of PCs are frequently observed in patients and model mice of cerebellar ataxias. We revealed that D-cysteine enhanced dendritic development of primary cultured PCs, but L-cysteine impaired the dendritic development. This effect of D-cysteine was inhibited by DAO inhibitors and reproduced by 3MP and a H2S donor, suggesting that this enhancement of dendritic development is caused by the production of H2S from D-cysteine. Taken together, D-cysteine would be available as a neuroprotectant against cerebellar ataxias, which are accompanied with dendritic shrinkage of cerebellar PCs.


Assuntos
Cisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Neurônios/citologia , Fármacos Neuroprotetores/metabolismo , Animais , Células Cultivadas , D-Aminoácido Oxidase/antagonistas & inibidores , D-Aminoácido Oxidase/metabolismo , Humanos , Camundongos , Neurogênese , Estresse Oxidativo , Células de Purkinje/citologia
3.
Adv Exp Med Biol ; 1158: 197-216, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452142

RESUMO

Mitochondria are dynamic organelles that perform a number of interconnected tasks that are elegantly intertwined with the regulation of cell functions. This includes the provision of ATP, reactive oxygen species (ROS), and building blocks for the biosynthesis of macromolecules while also serving as signaling platforms for the cell. Although the functions executed by mitochondria are complex, at its core these roles are, to a certain degree, fulfilled by electron transfer reactions and the establishment of a protonmotive force (PMF). Indeed, mitochondria are energy conserving organelles that extract electrons from nutrients to establish a PMF, which is then used to drive ATP and NADPH production, solute import, and many other functions including the propagation of cell signals. These same electrons extracted from nutrients are also used to produce ROS, pro-oxidants that can have potentially damaging effects at high levels, but also serve as secondary messengers at low amounts. Mitochondria are also enriched with antioxidant defenses, which are required to buffer cellular ROS. These same redox buffering networks also fulfill another important role; regulation of proteins through the reversible oxidation of cysteine switches. The modification of cysteine switches with the antioxidant glutathione, a process called protein S-glutathionylation, has been found to play an integral role in controlling various mitochondrial functions. In addition, recent findings have demonstrated that disrupting mitochondrial protein S-glutathionylation reactions can have some dire pathological consequences. Accordingly, this chapter focuses on the role of mitochondrial cysteine switches in the modulation of different physiological functions and how defects in these pathways contribute to the development of disease.


Assuntos
Cisteína , Metabolismo Energético , Mitocôndrias , Espécies Reativas de Oxigênio , Animais , Cisteína/metabolismo , Humanos , Mitocôndrias/metabolismo , Oxirredução
4.
Chem Commun (Camb) ; 55(69): 10214-10217, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31380528

RESUMO

The l,d-transpeptidases (Ldts) are promising antibiotic targets for treating tuberculosis. We report screening of cysteine-reactive inhibitors against LdtMt2 from Mycobacterium tuberculosis. Structural studies on LdtMt2 with potent inhibitor ebselen reveal opening of the benzisoselenazolone ring by a nucleophilic cysteine, forming a complex involving extensive hydrophobic interactions with a substrate-binding loop.


Assuntos
Azóis/química , Azóis/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/enzimologia , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Peptidil Transferases/antagonistas & inibidores , Antituberculosos/química , Antituberculosos/farmacologia , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Cisteína/metabolismo , Humanos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Peptidil Transferases/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
5.
Chem Commun (Camb) ; 55(65): 9629-9632, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31353368

RESUMO

Excessive accumulation of reducing agents in the ER leads to a constitutively high UPR. And the co-function of GSH, Cys and HOCl in biological processes is not well understood. To address this, a TP probe, NPCC, was developed for monitoring reductive stress in the ER. It can also distinguish cancer cells from normal cells.


Assuntos
Cumarínicos/química , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Pirazóis/química , Animais , Cumarínicos/síntese química , Cisteína/química , Cisteína/metabolismo , Corantes Fluorescentes/síntese química , Glutationa/química , Glutationa/metabolismo , Cabras , Células HeLa , Humanos , Ácido Hipocloroso/química , Ácido Hipocloroso/metabolismo , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Oxirredução , Pirazóis/síntese química , Peixe-Zebra
6.
J Agric Food Chem ; 67(31): 8559-8572, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298518

RESUMO

Avenin-like b protein is rich in cysteine residues, providing the possibility to form intermolecular disulfide bonds and then participate in glutenin polymerization. Site-directed mutagenesis was adopted to produce mutant avenin-like b gene encoding mutant avenin-like b protein, in which one tyrosine codon at the C-terminal is substituted by a cysteine codon. Compared with the control lines, both transgenic lines with wild-type and mutant avenin-like b genes demonstrated superior dough properties. While compared within the transgenic lines, the mutant lines showed relative weaker dough strength and decreased sodium-dodecyl-sulfate sedimentation volumes (from 69.7 mL in line WT alb-1 to 41.0 mL in line Mut alb-4). These inferior dough properties were accompanied by the lower contents of large-sized glutenin polymers, the decreased particle diameters of glutenin macropolymer (GMP), due to the lower content of intermolecular ß-sheets (from 39.48% for line WT alb-2 to 30.21% for line Mut alb-3) and the varied contents of disulfide bonds (from 137.37 µmol/g for line WT alb-1 to 105.49 µmol/g for line Mut alb-4) in wheat dough. The extra cysteine might alter the original disulfide bond structure, allowing cysteine residue usually involved in an intermolecular disulfide bond to become available for an intrachain disulfide bond. Avenin-like b proteins were detected in glutenin macropolymers, providing further evidence for this protein to participate in the polymerization of glutenin. This is the first time to investigate the effect of a specific cysteine residue in the avenin-like b protein on flour quality.


Assuntos
Cisteína/genética , Farinha/análise , Plantas Geneticamente Modificadas/genética , Prolaminas/genética , Triticum/genética , Pão/análise , Cisteína/metabolismo , Dissulfetos/química , Manipulação de Alimentos , Mutagênese Sítio-Dirigida , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/metabolismo , Prolaminas/metabolismo , Triticum/química , Triticum/metabolismo
7.
Aquat Toxicol ; 213: 105228, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31229888

RESUMO

The present work was conducted to study how restoration of perturbed oxidant and antioxidant homeostasis is achieved in the UV-C radiation exposed cells of cyanobacterium Nostoc muscorum Meg1. Exposure to varying doses of UV-C radiation (6, 12, 18 and 24 mJ/cm2) showed damage to ultrastructures especially cytoplasmic membrane, cell wall and organisation of thylakoid membranes of the cyanobacterium under transmission electron microscope (TEM). All doses of UV-C exposure significantly induced most of the enzymatic antioxidant {catalase, superoxide dismutase (SOD) and glutathione reductase (GR)} activities, their protein levels (western blot analysis) and mRNA levels (real time PCR analysis) within the first hour of post UV-C radiation incubation period. In the same way, contents of many non-enzymatic antioxidants such as ascorbic acid, reduced glutathione, proline, phenol and flavonoids were also augmented in response to such UV-C radiation exposure. Although notable increase in ROS level was only seen in cultures treated with 24 mJ/cm2 UV-C exposure which also registered increase in protein oxidation (22%) and lipid peroxidation (20%), this boost in both enzymatic and non-enzymatic antioxidants was significant in all radiation exposed cells indicating cell's preparation to combat rise in oxidants. Further, albeit all antioxidants increased considerably, their levels were restored back to control values by day seventh re-establishing physiological redox state for normal metabolic function. The combined efficiency of the enzymatic and non-enzymatic antioxidants were so effective that they were able to bring down the increase levels of ROS, lipid peroxidation and protein oxidation to the physiological levels within 1 h of radiation exposure signifying their importance in the defensive roles in protecting the organism from oxidative toxicity induced by UV-C radiation exposure.


Assuntos
Antioxidantes/metabolismo , Homeostase , Nostoc muscorum/fisiologia , Nostoc muscorum/efeitos da radiação , Oxidantes/metabolismo , Estresse Fisiológico/efeitos da radiação , Raios Ultravioleta , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Cisteína/metabolismo , Flavonoides/metabolismo , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Nostoc muscorum/ultraestrutura , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Prolina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/toxicidade
8.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 33(6): 689-697, 2019 Jun 15.
Artigo em Chinês | MEDLINE | ID: mdl-31197995

RESUMO

Objective: To observe the change of stromal cell-derived factor 1α/cysteine X cysteine receptor 4 (SDF-1α/CXCR4) signaling pathway during the process of axial stress stimulation promoting bone regeneration, and to further explore its mechanism. Methods: A total of 72 male New Zealand white rabbits were selected to prepare the single cortical bone defect in diameter of 8 mm at the proximal end of the right tibia that repaired with deproteinized cancellous bone. All models were randomly divided into 3 groups ( n=24). Group A was treated with intraperitoneally injection of PBS; Group B was treated with stress stimulation and intraperitoneally injection of PBS; Group C was treated with stress stimulation and intraperitoneally injection of AMD3100 solution. The X-ray films were taken and Lane-Sandhu scores of bone healing were scored at 2, 4, 8, and 12 weeks after operation, while specimens were harvested for HE staining, immunohistochemical staining of vascular endothelial growth factor (VEGF) and CXCR4, and Western blot (SDF-1α and CXCR4). The bone healing area was scanned by Micro-CT at 12 weeks after operation, and the volume and density of new bone were calculated. Results: X-ray film showed that the Lane-Sandhu scores of bone healing in group B were significantly higher than those in groups A and C at 4, 8, and 12 weeks after operation ( P<0.05). Micro-CT scan showed that the bone defect was repaired in group B and the pulp cavity was re-passed at 12 weeks after operation. The volume and density of new bone were higher in group B than in groups A and C ( P<0.05). HE staining showed that the new bone growth in bone defect area and the degradation of scaffolds were faster in group B than in groups A and C after 4 weeks. The immunohistochemical staining showed that the expressions of VEGF and CXCR4 in 3 groups reached the peak at 4 weeks, and group B was higher than groups A and C ( P<0.05). Western blot analysis showed that the expressions of SDF-1α and CXCR4 in group B were significantly higher than those in groups A and C at 4 and 8 weeks after operation ( P<0.05). Conclusion: Axial stress stimulation can promote the expression of SDF-1α in bone defect tissue, activate and regulate the CXCR4 signal collected by marrow mesenchymal stem cells, and accelerate bone regeneration in bone defect area.


Assuntos
Regeneração Óssea , Quimiocina CXCL12 , Cisteína , Animais , Fenômenos Biomecânicos , Quimiocina CXCL12/metabolismo , Cisteína/metabolismo , Masculino , Coelhos , Receptores CXCR4/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Arch Virol ; 164(9): 2309-2314, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31172288

RESUMO

The surface (SU) and transmembrane (TM) glycoproteins of many retroviruses are linked by disulphide bonds, and the interaction of SU with a cellular receptor results in disulphide bond isomerisation triggered by the CXXC motif in SU. This reaction leads to the fusion of viral and host cell membranes. In this work, we show that the cysteine at amino acid position 212 in the CAIC motif of the SU glycoprotein of bovine leukaemia virus has a free thiol group. A C-to-A mutation at position 212, either individually or in combination with a C-to-A mutation at position 215, was found to inhibit the maturation process, suggesting its involvement in the formation of the covalent bond with TM.


Assuntos
Cisteína/metabolismo , Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Cisteína/genética , Vírus da Leucemia Bovina/química , Vírus da Leucemia Bovina/isolamento & purificação , Vírus da Leucemia Bovina/fisiologia , Glicoproteínas de Membrana/genética , Mutação
10.
Photochem Photobiol Sci ; 18(7): 1761-1772, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31111854

RESUMO

Biomolecules like cysteine and cytosine play a significant role in many physiological processes, and their unusual level in biological systems can lead to many diseases including cancer. Indeed, the need for selective detection of these moieties by a fluorescence probe is imperative. Thus, thiophene based Schiff N,N'-bis(thiophene-2-ylmethylene)thiophenemethane (BMTM) was synthesized and then characterized using several analytical techniques before converting it into organic nanoparticles (ONPs). Then, fluorescent organic inorganic nanohybrids (FONs) were obtained after decorating ONPs with AuNPs to yield BMTM-Au-ONPs (FONPs). The morphology of the particles, analyzed using a Transmission Electron Microscope (TEM), shows that AuNPs were embedded with low density organic matter (ONPs). FONPs were employed to recognize cysteine and cytosine simultaneously. No interference was observed from other moieties such as guanine, uracyl, NADH, NAD, ATP, and adenine during the detection. It means that the intensity of the fluorescence signal was significantly changed (enhanced for cytosine and quenched for cysteine). So, FONPs were used to detect cysteine and cytosine in real samples, like Saccharomyces cerevisiae cells. As expected, no considerable fluorescence signal for cysteine was observed, while for cytosine, strong fluorescence signals were detected in the cells. DFT was used to explain the interaction of FONPs with cysteine or cytosine.


Assuntos
Cisteína/análise , Citosina/análise , Ouro/química , Nanopartículas Metálicas/química , Tiofenos/química , Cisteína/metabolismo , Citosina/metabolismo , Teoria da Densidade Funcional , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Microscopia Confocal , Microscopia Eletrônica de Transmissão , NAD/química , Saccharomyces cerevisiae/metabolismo
11.
Chemphyschem ; 20(13): 1719-1727, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31090243

RESUMO

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.


Assuntos
Luciferina de Vaga-Lumes/metabolismo , Animais , Cisteína/metabolismo , Vaga-Lumes/química , Vaga-Lumes/enzimologia , Luciferina de Vaga-Lumes/química , Hidrólise , Luciferases de Vaga-Lume/metabolismo , Modelos Químicos , Estereoisomerismo , Tioléster Hidrolases/metabolismo
12.
Am J Chin Med ; 47(4): 787-801, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31091973

RESUMO

American ginseng and Asian ginseng, which occupy prominent positions in the list of best-selling natural products in the West and East, are suitable for different indications in the traditional pharmacological uses. Currently, the effects of American ginseng and Asian ginseng in the protection against metabolic dysfunction and the differences between them are still unknown. Herein, an untargeted metabolomics based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was determined. The serum metabolomics and dynamic feces metabolomics revealed significant metabolic distinction between American ginseng and Asian ginseng in diet-induced obese (DIO) mice. The results show that American ginseng and Asian ginseng alleviate glucose and lipid metabolism disorder in DIO mice. A total of 45 differential metabolites were confirmed between the drug-naïve and American ginseng group, and 32 metabolites were confirmed between the drug-naïve and Asian ginseng group. Metabolic pathways analysis shows that these two ginsengs treatment dynamic rectifies metabolic disorder in DIO mice mainly via regulating linoleic acids metabolism, cysteine and methionine metabolism and biosynthesis of unsaturated fatty acid. Moreover, American ginseng's specific function in monitoring the carnitines and taurine/hypotaurine metabolism might make it more effective in meliorating lipids metabolism disorder than Asian ginseng.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolômica/métodos , Obesidade/etiologia , Obesidade/metabolismo , Panax/química , Panax/classificação , Extratos Vegetais/farmacologia , Animais , Carnitina/metabolismo , Cromatografia Líquida , Cisteína/metabolismo , Ácidos Graxos/biossíntese , Ácido Linoleico/metabolismo , Masculino , Espectrometria de Massas , Metionina/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Taurina/metabolismo
13.
Nat Commun ; 10(1): 2195, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097712

RESUMO

Cysteine modifications emerge as important players in cellular signaling and homeostasis. Here, we present a chemical proteomics strategy for quantitative analysis of reversibly modified Cysteines using bioorthogonal cleavable-linker and switch technique (Cys-BOOST). Compared to iodoTMT for total Cysteine analysis, Cys-BOOST shows a threefold higher sensitivity and considerably higher specificity and precision. Analyzing S-nitrosylation (SNO) in S-nitrosoglutathione (GSNO)-treated and non-treated HeLa extracts Cys-BOOST identifies 8,304 SNO sites on 3,632 proteins covering a wide dynamic range of the proteome. Consensus motifs of SNO sites with differential GSNO reactivity confirm the relevance of both acid-base catalysis and local hydrophobicity for NO targeting to particular Cysteines. Applying Cys-BOOST to SH-SY5Y cells, we identify 2,151 SNO sites under basal conditions and reveal significantly changed SNO levels as response to early nitrosative stress, involving neuro(axono)genesis, glutamatergic synaptic transmission, protein folding/translation, and DNA replication. Our work suggests SNO as a global regulator of protein function akin to phosphorylation and ubiquitination.


Assuntos
Cisteína/análise , Proteoma/metabolismo , Proteômica/métodos , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/metabolismo , Células HeLa , Humanos , Nitrosação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/análise , Proteômica/instrumentação , S-Nitrosoglutationa/química , S-Nitrosoglutationa/metabolismo , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
14.
Chem Commun (Camb) ; 55(49): 7029-7032, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31140482

RESUMO

A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The hydrazinolysis followed by thioesterification affords cysteinyl prolyl thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide thioesters. The developed protocol was successfully applied to the conversion of recombinant proteins to thioesters.


Assuntos
Carboxipeptidases/metabolismo , Cisteína/metabolismo , Ésteres/metabolismo , Hidrazinas/metabolismo , Compostos de Sulfidrila/metabolismo , Cisteína/química , Ésteres/química , Hidrazinas/química , Conformação Molecular , Compostos de Sulfidrila/química
15.
Prep Biochem Biotechnol ; 49(6): 567-577, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929621

RESUMO

Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage reserve material stored by gram-negative bacteria under nutrient limitation. PHAs are best alternative biodegradable plastics (bio-plastics) due to their resemblance to conventional synthetic plastic. The present study investigated the synergistic effect of nutritional supplements (amino acid and vitamin) on the PHA production by Alcaligenes sp. NCIM 5085 utilizing a sugar refinery waste (cane molasses) under submerged fermentation process. Initially, the effect of individual factor on PHA yield was studied by supplementing amino acids (cysteine, isoleucine, and methionine), vitamin (thiamin), and cane molasses at varying concentration in the production medium. Further, the cultivation medium was optimized by varying the levels of cane molasses, methionine and thiamin using response surface methodology to enhance the PHA yield. The maximum PHA yield of 70.89% was obtained under the optimized condition, which was then scaled up on 7.5 L-bioreactor. Batch cultivation in 7.5 L-bioreactor under the optimized condition gave a maximum PHA yield and productivity of 79.26% and 0.312 gL-1 h-1, respectively. The PHA produced was subsequently characterized as PHB by FTIR. PHB extracted was of relatively high molecular weight and crystallinity index. DSC analysis gave Tg, Tm, and Xc of 4.2, 179 °C and 66%, respectively. TGA analysis showed thermal stability with maximized degradation occurring at 302 °C, which is above the melting temperature (179 °C) of the purified polymer. The extracted polymer, therefore, possessed desirable material properties to be used in food packaging.


Assuntos
Aminoácidos/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Tiamina/metabolismo , Alcaligenes/metabolismo , Reatores Biológicos , Cisteína/metabolismo , Fermentação , Embalagem de Alimentos , Resíduos Industriais/prevenção & controle , Isoleucina/metabolismo , Metionina/metabolismo , Melaço , Peso Molecular , Poli-Hidroxialcanoatos/química , Temperatura de Transição , Gerenciamento de Resíduos/métodos
16.
Methods Mol Biol ; 1977: 83-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30980324

RESUMO

Reduction and alkylation are common processing steps in sample preparation for qualitative and quantitative proteomic analyses. In principle, these steps mitigate the limitations resulting from the presence of disulfide bridges. There has been recurring debate in the proteomics community around their use, with concern over negative impacts that result from overalkylation (off-target, non-thiol sites) or incomplete reduction and/or S-alkylation of cysteine. This chapter integrates findings from a number of studies on different reduction and alkylation strategies, to guide users in experimental design for their optimal use in proteomic workflows.


Assuntos
Cisteína/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteômica , Alquilantes/farmacologia , Alquilação/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Proteômica/métodos , Substâncias Redutoras/farmacologia , Fluxo de Trabalho
17.
MBio ; 10(2)2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940703

RESUMO

Mercury (Hg) is a widely distributed, toxic heavy metal with no known cellular role. Mercury toxicity has been linked to the production of reactive oxygen species (ROS), but Hg does not directly perform redox chemistry with oxygen. How exposure to the ionic form, Hg(II), generates ROS is unknown. Exposure of Thermus thermophilus to Hg(II) triggered ROS accumulation and increased transcription and activity of superoxide dismutase (Sod) and pseudocatalase (Pcat); however, Hg(II) inactivated Sod and Pcat. Strains lacking Sod or Pcat had increased oxidized bacillithiol (BSH) levels and were more sensitive to Hg(II) than the wild type. The ΔbshA Δsod and ΔbshA Δpcat double mutant strains were as sensitive to Hg(II) as the ΔbshA strain that lacks bacillithiol, suggesting that the increased sensitivity to Hg(II) in the Δsod and Δpcat mutant strains is due to a decrease of reduced BSH. Treatment of T. thermophilus with Hg(II) decreased aconitase activity and increased the intracellular concentration of free Fe, and these phenotypes were exacerbated in Δsod and Δpcat mutant strains. Treatment with Hg(II) also increased DNA damage. We conclude that sequestration of the redox buffering thiol BSH by Hg(II), in conjunction with direct inactivation of ROS-scavenging enzymes, impairs the ability of T. thermophilus to effectively metabolize ROS generated as a normal consequence of growth in aerobic environments.IMPORTANCE Thermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus-Thermus phylum that, together with the Aquificae, constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243-249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321-331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.


Assuntos
Catalase/metabolismo , Cisteína/análogos & derivados , Tolerância a Medicamentos , Glucosamina/análogos & derivados , Compostos de Mercúrio/toxicidade , Superóxido Dismutase/metabolismo , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/enzimologia , Catalase/genética , Cisteína/metabolismo , Deleção de Genes , Glucosamina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Thermus thermophilus/genética , Thermus thermophilus/metabolismo
18.
Food Chem ; 289: 657-663, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30955661

RESUMO

Polyphenol oxidase from Granny Smith apples was purified and characterized in both its soluble form (sPPO) and its membrane-bound form (mPPO). Both forms were purified by temperature-induced phase partitioning, precipitation with ammonium sulfate, and ion exchange chromatography. The specific activity of mPPO was 19.17 times that of sPPO. The optimum pH and temperature for both forms were 7.0 and 35 °C when catechol was the substrate. The Michaelis constant and maximum reaction rate for sPPO were 34.1 mM and 500 U/mL/min, whereas those for mPPO were 53 mM and 10,000 U/mL/min, respectively. The enzymes exhibited diphenolase activity, and their affinity was highest for catechol (sPPO) and 4-methylcatechol (mPPO). Inhibitors of sPPO and mPPO included ascorbic acid, glutathione, and l-cysteine. However, ethylenediaminetetraacetic acid increased the activity of mPPO. Purified sPPO was dimeric with a molecular weight of 31 kDa, whereas mPPO was monomeric with an estimated molecular weight of 65 kDa.


Assuntos
Catecol Oxidase/metabolismo , Frutas/enzimologia , Malus/enzimologia , Ácido Ascórbico/metabolismo , Catecóis/química , Cisteína/metabolismo , Ácido Edético/metabolismo , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Temperatura Ambiente
19.
J Agric Food Chem ; 67(16): 4435-4443, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30945533

RESUMO

Aspergillus niger, which is a fungal pathogen, causes rot in a variety of fruits. In this study, the cystathionine ß-synthase cbsA gene was deleted by homologous recombination to study its role in sulfur metabolism and pathogenicity of A. niger. The results showed that Δ cbsA strain maintained normal mycelia growth and sporulation compared with the control strain A. niger MA 70.15, whereas the contents of cysteine and glutathione (GSH) increased significantly after cbsA deletion. However, Δ cbsA strain showed reduced endogenous H2S production. Further results showed that cbsA gene deletion induced higher resistance to cadmium stress and stronger infectivity to pears. It was also found that a stronger response of reactive oxygen species (ROS) production was induced in Δ cbsA mutant-infected pear compared with the control strain. In all, the present research suggested the important role of cbsA in sulfur metabolism and pathogenicity of A. niger in pear fruit.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/patogenicidade , Cistationina beta-Sintase/metabolismo , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Pyrus/microbiologia , Enxofre/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Cistationina beta-Sintase/genética , Cisteína/metabolismo , Frutas/microbiologia , Proteínas Fúngicas/genética , Glutationa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Virulência
20.
Proc Natl Acad Sci U S A ; 116(12): 5370-5375, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30824597

RESUMO

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Cα-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.


Assuntos
Cobre/metabolismo , Glicina/análogos & derivados , Oxigênio/metabolismo , Catálise , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Cisteína/metabolismo , Glicina/metabolismo , Oxirredução , Sulfatases/metabolismo
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