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1.
J Phys Chem Lett ; 11(4): 1215-1221, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31978303

RESUMO

Site-selective dissociation induced by core photoexcitation of biomolecules is of key importance for the understanding of radiation damage processes and dynamics and for its promising use as "chemical scissors" in various applications. However, identifying products of site-selective dissociation in large molecules is challenging at the carbon, nitrogen, and oxygen edges because of the high recurrence of these atoms and related chemical groups. In this paper, we present the observation of site-selective dissociation at the sulfur L-edge in the gas-phase peptide methionine enkephalin, which contains only a single sulfur atom. Near-edge X-ray absorption mass spectrometry has revealed that the resonant S 2p → σ*C-S excitation of the sulfur contained in the methionine side chain leads to site-selective dissociation, which is not the case after core ionization above the sulfur L-edge. The prospects of such results for the study of charge dynamics in biomolecular systems are discussed.


Assuntos
Gases/química , Peptídeos/química , Enxofre/química , Espectroscopia por Absorção de Raios X , Encefalinas/química , Metionina/química , Prótons , Teoria Quântica
2.
Chemistry ; 26(1): 259-268, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31614021

RESUMO

In the effort to overcome issues of toxicity and resistance inherent to treatment by the approved platinum anticancer agents, a large number of cisplatin variants continues today to be prepared and tested. One of the applied strategies is to use monofunctional platinum complexes that, unlike traditional bifunctional compounds, are able to form only a single covalent bond with nuclear DNA. Chirality, aquation reaction, interaction with guanine and N-acetyl methionine as well as, intercalation into, binding to and distortion of DNA have been investigated by using both quantum mechanical DFT and molecular dynamics computations aiming at contributing to the elucidation of the molecular mechanism underlying the significantly enhanced spectrum of activity of the monofunctional PtII drug phenanthriplatin. Analogous calculations have been performed in parallel for other two less potent monofunctional PtII drugs, pyriplatin and enpyriplatin, which show very different cytotoxic effects.


Assuntos
Antineoplásicos/química , Teoria da Densidade Funcional , Simulação de Dinâmica Molecular , Compostos Organoplatínicos/química , Fenantridinas/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Humanos , Substâncias Intercalantes/química , Metionina/química , Conformação de Ácido Nucleico , Compostos Organoplatínicos/farmacologia , Fenantridinas/farmacologia , Termodinâmica , Transcrição Genética/efeitos dos fármacos
3.
Phys Chem Chem Phys ; 22(1): 107-113, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31807746

RESUMO

The non-enzymatic cleavage rates of amide bonds located in peptides in aqueous solution is pH-dependent and involves two distinct mechanisms: direct hydrolysis (herein termed "scission") and intramolecular aminolysis by the N-terminal amine (herein termed "backbiting"). While amide bond cleavage has been previously characterized using a variety of peptides, no systematic study has yet been reported addressing the effect of the pH on the interplay between the two amide bond cleavage pathways. In this study, the cleavage rates of the glycine dimer (GG), the glycine trimer (GGG), and the cyclic dimer (cGG), as well as the alanine trimer (AAA), were measured at pH 3, 5, 7, and 10 at 95 °C employing quantification based on 1H NMR. The distinct rate constants for scission and backbiting processes were obtained by solving the differential rate equations associated with the proposed kinetic model. Generalizations concerning the relative importance of the various amide bond cleavage pathways at pH 3, 5, 7, and 10 are presented. In particular, scission dominates at pH 10, while backbiting dominates at neutral pH. At the acidic pH of 3, both backbiting and scission are significant. The model of the reaction network, used in this work, enables the quantification of these multiple competing mechanisms and can be applied to longer peptides and to similar types of reaction networks.


Assuntos
Concentração de Íons de Hidrogênio , Peptídeos/química , Alanina/química , Amidas/química , Aminas/química , Glicina/química , Hidrólise , Cinética , Metionina/química , Estabilidade Proteica , Termodinâmica
4.
J Anim Sci ; 97(10): 4242-4247, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581297

RESUMO

Two performance studies were conducted to investigate the effects of 3 different sources of Cu on production parameters of piglets. A total of 256 piglets weaned at 24 ± 2 d were randomly allocated into 4 treatments with 10 or 8 replicates per treatment of 4 or 3 piglets per pen in Exp. 1 and 2, respectively. The experimental period was divided into 3 feeding phases: Phase 1 (24 to 35 d), Phase 2 (36 to 49 d), and Phase 3 (50 to 70 d). Treatments included a Control group (fed 10 mg/kg of Cu from CuSO4), a group fed 160 mg/kg of either CuSO4 (CuSO4-160) or tri-basic copper chloride (TBCC), and a group fed Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1, 2, and 3, respectively. The methionine value of Cu-MHAC was accounted during diet formulation to achieve the same levels of methionine across treatments. Phases 1 and 2 diets contained 2,200 and 1,500 ppm of ZnO, respectively; and antibiotics were used as growth promoters. Performance parameters were analyzed as completely randomized block design, in which each experiment was considered as a block. In trial 2, blood serum and mucosal samples, from the fundic region of the stomach, were collected from 1 piglet per replicate at day 70 and tested for serum growth hormone levels (GH) and ghrelin mRNA expression, respectively. The contrast between Cu-MHAC vs. CuSO4-160 + TBCC showed that piglets fed Cu-MHAC exhibited better feed conversion ratio (FCR) in all feeding phases compared with feeding inorganic Cu (P < 0.05). Overall, feeding Cu-MHAC improved body weight (BW), BW gain, feed intake (FI), and FCR vs. Control diet fed piglets; yet, it improved BW and FCR vs. TBCC fed piglets, and improved BW, BW gain, and FI vs. CuSO4-160 fed piglets (P < 0.05). Feeding TBCC promoted similar performance than feeding CuSO4-160, regardless of age (P > 0.05). Both ghrelin expression and growth hormone serum levels were significantly increased by feeding Cu-MHAC vs. Control diet fed animals (P < 0.01). Feeding CuSO4-160 upregulated ghrelin expression vs. Control (P < 0.01) while GH serum levels and ghrelin expression did no change by feeding TBCC compared with Control diet fed animals (P > 0.05). It was concluded that feeding Cu-MHAC at the levels tested herein can improve growth performance of piglets beyond feeding 160 ppm of either CuSO4 or TBCC, which may be partially explained by the increased expression of ghrelin and GH serum levels.


Assuntos
Ração Animal/análise , Cobre/administração & dosagem , Suplementos Nutricionais/análise , Grelina/genética , Hormônio do Crescimento/sangue , Suínos/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Dieta/veterinária , Feminino , Masculino , Metionina/análogos & derivados , Metionina/química , RNA Mensageiro/genética , Distribuição Aleatória , Estômago/fisiologia , Suínos/genética , Suínos/crescimento & desenvolvimento , Desmame , Ganho de Peso/efeitos dos fármacos
5.
Inorg Chem ; 58(20): 14085-14106, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31589413

RESUMO

Ligand-switch reactions at the heme iron are common in biological systems, but their mechanisms and the features of the polypeptide fold that support dual ligation are not well understood. In cytochrome c (cyt c), two low-stability loops (Ω-loop C and Ω-loop D) are connected by the heme propionate HP6. At alkaline pH, the native Met80 ligand from Ω-loop D switches to a Lys residue from the same loop. Deprotonation of an as yet unknown group triggers the alkaline transition. We have created the two cyt c variants T49V/K79G and T78V/K79G with altered connections of these two loops to HP6. Electronic absorption, NMR, and EPR studies demonstrate that at pH 7.4 ferric forms of these variants are Lys-ligated, whereas ferrous forms maintain the native Met80 ligation. Measurements of protein stability, cyclic voltammetry, pH-jump and gated electron-transfer kinetics have revealed that these Thr to Val substitutions greatly affect the alkaline transition in both ferric and ferrous proteins. The substitutions modify the stability of the Met-ligated species and reduction potentials of the heme iron. The kinetics of ligand-switch processes are also altered, and analyses of these effects implicate redox-dependent differences in metal-ligand interactions and the role of the protein dynamics, including cross-talk between the two Ω-loops. With the two destabilized variants, it is possible to map energy levels for the Met- and Lys-ligated species in both ferric and ferrous proteins and assess the role of the protein scaffold in redox-dependent preferences for these two ligands. The estimated shift in the heme iron reduction potential upon deprotonation of the "trigger" group is consistent with those associated with deprotonation of an HP, suggesting that HP6, on its own or as a part of a hydrogen-bonded cluster, is a likely "trigger" for the Met to Lys ligand switch.


Assuntos
Complexos de Coordenação/química , Citocromos c/química , Heme/química , Ferro/química , Metionina/química , Propionatos/química , Complexos de Coordenação/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Heme/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Cinética , Ligantes , Metionina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Propionatos/metabolismo
6.
Biochemistry (Mosc) ; 84(8): 923-930, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522674

RESUMO

Apoptosis, as the major type of programmed cell death, plays an important role in the organism renewal and removal of defective and transformed cells, including cancer cells. One of the earliest apoptotic events is lipid peroxidation in the inner mitochondrial membrane catalyzed by a complex of cytochrome c (CytC) with the mitochondrial phospholipid cardiolipin (CL). It was shown that mixing CytC and CL solutions results in the formation of CytC/CL complexes (Cyt-CL nanospheres) with a diameter of 11-12 nm composed of the molten globule protein molecule and a CL monolayer. Using the methods of dynamic light scattering for the Cyt-CL chloroform solution and small-angle X-ray scattering for the Cyt-CL sediment, it was found that in both cases, Cyt-CL formed nanospheres with a diameter of 8 and 11 nm, which corresponded to the earlier determined lipid/protein ratios of 13-14 and 35-50, respectively. These results showed that the Cyt-CL nanospheres can form not only during crystallization but also in a hydrophobic medium. CytC in the complex exists as a molten globule, as evidenced by the emergence of tryptophan and tyrosine fluorescence (absent in the native protein) due to the Förster resonance transfer of the electron excitation energy onto the heme. At the same time, the coordinate bond between the heme iron and the sulfur atom of methionine 80 in Cyt-CL is disrupted (the absorption band at ~700 nm disappears). Similar disruption of the iron-sulfur bond in Cyt-CL was observed in 50% methanol. These changes were reversible, which corroborates the conclusion on the CytC transition to the molten globule conformation in methanol-containing solutions.


Assuntos
Cardiolipinas/química , Citocromos c/química , Nanosferas/química , Animais , Apoptose/fisiologia , Cristalização , Difusão Dinâmica da Luz , Transferência Ressonante de Energia de Fluorescência , Heme/química , Cavalos , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Peroxidação de Lipídeos/fisiologia , Metanol/química , Metionina/química , Mitocôndrias Cardíacas/metabolismo , Membranas Mitocondriais/metabolismo , Conformação Proteica , Desdobramento de Proteína
7.
Chemistry ; 25(59): 13531-13536, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31393030

RESUMO

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein-protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metionina/análogos & derivados , Proteínas Serina-Treonina Quinases/química , Proteínas ras/química , Metionina/química , Metionina/farmacologia , Peptidomiméticos , Prenilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas ras/metabolismo
8.
J Zhejiang Univ Sci B ; 20(9): 699-712, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379141

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and metabolism in response to various environmental inputs, especially amino acids. In fact, the activity of mTORC1 is highly sensitive to changes in amino acid levels. Over past decades, a variety of proteins have been identified as participating in the mTORC1 pathway regulated by amino acids. Classically, the Rag guanosine triphosphatases (GTPases), which reside on the lysosome, transmit amino acid availability to the mTORC1 pathway and recruit mTORC1 to the lysosome upon amino acid sufficiency. Recently, several sensors of leucine, arginine, and S-adenosylmethionine for the amino acid-stimulated mTORC1 pathway have been coming to light. Characterization of these sensors is requisite for understanding how cells adjust amino acid sensing pathways to their different needs. In this review, we summarize recent advances in amino acid sensing mechanisms that regulate mTORC1 activity and highlight these identified sensors that accurately transmit specific amino acid signals to the mTORC1 pathway.


Assuntos
Aminoácidos/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Arginina/química , Membrana Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Leucina/química , Lisossomos/metabolismo , Metionina/química , S-Adenosilmetionina/química , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
9.
Org Biomol Chem ; 17(32): 7493-7496, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31364664

RESUMO

The fluorinase enzyme from Streptomyces cattleya is shown to catalyse a direct displacement of bromide and iodide by fluoride ion from 5'-bromodeoxyadenosine (5'-BrDA) and 5'-iododeoxyadenosine (5'-IDA) respectively to form 5'-fluorodeoxyadenosine (5'-FDA) in the absence of l-methionine (l-Met) or S-adenosyl-l-methionine (SAM). 5'-BrDA is the most efficient substrate for this enzyme catalysed Finkelstein reaction.


Assuntos
Proteínas de Bactérias/química , Desoxiadenosinas/química , Halogênios/química , Oxirredutases/química , Catálise , Cinética , Metionina/química , Conformação Molecular , S-Adenosilmetionina/química , Streptomyces/enzimologia , Termodinâmica
10.
Food Chem ; 298: 124952, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260994

RESUMO

The riboflavin-mediated photo-degradation of methionine in white wine has been related to onset of undesired light-struck taste. This research investigated the effects of different concentrations of riboflavin and methionine, hydrolysable tannins from various sources (nut galls, chestnut and oak woods) and sulfur dioxide on methionine degradation in a model wine exposed to light. Increased methionine concentration resulted in its increased degradation with the consequent formation of volatile sulfur compounds, namely methanethiol, dimethyl disulphide and dimethyl trisulphide. Tannins, especially nut gall tannin, were effective in limiting both methionine degradation and the production of volatile sulfur compounds. Sulfur dioxide enhanced the methionine degradation although the light-struck taste was not perceived when sulfur dioxide concentration was higher than 50 mg/L. In conclusion, the use of hydrolysable tannins can represent a promising tool for protecting white wine against the light-struck taste also limiting the use of sulfur dioxide.


Assuntos
Taninos Hidrolisáveis/química , Metionina/química , Riboflavina/química , Dióxido de Enxofre/química , Vinho , Dissulfetos/química , Luz , Metionina/metabolismo , Processos Fotoquímicos , Compostos de Sulfidrila/química , Paladar/efeitos dos fármacos , Compostos Orgânicos Voláteis/química , Compostos Orgânicos Voláteis/metabolismo , Vinho/análise
11.
Int J Mol Sci ; 20(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261895

RESUMO

Preventive approaches for age-related memory decline and dementia have become a high priority in the aging society because of the lack of therapeutic approaches. Recent epidemiological studies have reported that fermented dairy products can help prevent dementia. Previously, we identified tryptophan-tyrosine (WY) and tryptophan-methionine (WM) peptides as the suppressants of activation of the primary microglia and showed that WY peptide consumption suppresses inflammation in the brains of Alzheimer's disease model mice. However, the effects of the WM peptide on inflammation in the brain and Alzheimer's pathology have not been investigated. Here, we evaluated the effect of WM peptide consumption on Alzheimer's disease model (5×FAD) mice. In 5×FAD mice, intake of WM peptide suppressed the production of inflammatory cytokines, activation of microglia, and infiltration of activated microglia around ß amyloid (Aß) depositions. WM peptide intake reduced Aß deposition in the cortex and hippocampus and then improved the object recognition memory. Taken together with previous reports, the current findings indicate that ingestion of tryptophan-related peptides or food material rich in tryptophan-related peptides, thereby regulating microglial activity, represents a potential preventive approach for cognitive decline and dementia related to inflammation.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Dipeptídeos/farmacologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Citocinas/metabolismo , Suplementos Nutricionais , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Dipeptídeos/uso terapêutico , Feminino , Metionina/química , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas do Leite/química , Triptofano/química
12.
Mater Sci Eng C Mater Biol Appl ; 102: 646-652, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147036

RESUMO

In this study we examined the influence of ultra-small gold and magnetite­gold nanoparticles (NPs) stabilized with d,l-methionine, Fe3O4@Au@Met, on their antibacterial efficacy against three of twelve the worst bacterial family members included in the World Health Organization (WHO) list. In particular, gram-negative Acinetobacter baumannii, Salmonella enterica and gram-positive methicillin-resistant Staphylococcus aureus and Micrococcus luteus were tested. Apart from the synthesis, gold species reduction and NP stabilization, an excess of methionine has been used herein to detach ultra-small gold NPs from the Fe3O4@Au@Met surface, collect them and investigate. The antimicrobial efficiency of the ultra-small (Ø ~ 1.8 nm) Au@Met NPs and Fe3O4@Au@Met NPs was evaluated through the quantitative analysis by comparing with that of naked magnetite NPs, d,l-Met and BSA. It has been determined that compared with the control sample, 70 mg L-1 probe of Au@Met NPs exhibited the killing efficiency of 84.4-58.5% against gram-negative bacteria and 89.1-75.7% against gram-positive bacteria. The composition, structure, and morphology of the synthesized and tested herein NPs were investigated by inductively coupled plasma optical emission spectrometry, magnetic measurements, FTIR, XRD, XPS, AFM and HRTEM.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/química , Metionina/química , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de Fourier
13.
MAbs ; 11(6): 1101-1112, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31161859

RESUMO

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.


Assuntos
Anticorpos Biespecíficos/química , Anticorpos Monoclonais/química , Imunoglobulina G/química , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Asparagina/química , Asparagina/imunologia , Humanos , Imunoglobulina G/imunologia , Espectrometria de Massas , Metionina/química , Metionina/imunologia , Oxirredução , Receptores de IgG/química , Receptores de IgG/imunologia , Relação Estrutura-Atividade
14.
Analyst ; 144(13): 3988-3998, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31169288

RESUMO

Methylation of components involved in one-carbon metabolism is extremely important in cancer; comprehensive studies on methylation are essential and may provide us with a better understanding of tumorigenesis, and lead to the discovery of potential biomarkers. Here, we present an improved methodology for methylated metabolite profiling and its relative quantification in breast cancer cell lines by isotope dilution mass spectrometry based on 13CD3-methionine metabolic labeling using ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UPLC-HRMS/MS). First, all the methylated metabolites related to methionine were first screened and profiled by introducing 13CD3-methionine as the only medium into breast cancer cell growth cultures for both cellular polar metabolites and lipids. In total, we successfully found 20 labeled methylated metabolites and most of them were identified, some of which have not been reported before. We also developed a relative quantification method for all identified methylated metabolites based on isotope dilution mass spectrometry assays. Finally, the developed method was used for different breast cancer cells and mammary epithelial cells. Most methylated metabolites were disrupted in cancer cells. 1-Methyl-nicotinamide was decreased significantly, while trimethylglycine-glutamic acid-lysine and trimethyl-lysine were increased more than five times. This method offers a new insight into the methylation process, with several key pathways and important new metabolites being identified. Further investigation with biological assays should help to reveal the overall methylation metabolic network.


Assuntos
Metaboloma , Metabolômica/métodos , Metionina/metabolismo , Isótopos de Carbono/química , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Deutério/química , Humanos , Marcação por Isótopo , Metionina/química , Metilação , Espectrometria de Massas em Tandem
15.
PLoS One ; 14(6): e0217862, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31170222

RESUMO

Turnip (Brassica rapa spp. rapa) is an important vegetable species, with a unique physiology. Several plant parts, including both the turnip tubers and leaves, are important for human consumption. During the development of turnip plants, the leaves function as metabolic source tissues, while the tuber first functions as a sink, while later the tuber turns into a source for development of flowers and seeds. In the present study, chemical changes were determined for two genotypes with different genetic background, and included seedling, young leaves, mature leaves, tuber surface, tuber core, stalk, flower and seed tissues, at seven different time points during plant development. As a basis for understanding changes in glucosinolates during plant development, the profile of glucosinolates was analysed using liquid chromatography (LC) coupled to mass spectrometry (MS). This analysis was complemented by a gene expression analysis, focussed on GLS biosynthesis, which could explain part of the observed variation, pointing to important roles of specific gene orthologues for defining the chemical differences. Substantial differences in glucosinolate profiles were observed between above-ground tissues and turnip tuber, reflecting the differences in physiological role. In addition, differences between the two genotypes and between tissues that were harvested early or late during the plant lifecycle. The importance of the observed differences in glucosinolate profile for the ecophysiology of the turnip and for breeding turnips with optimal chemical profiles is discussed.


Assuntos
Brassica napus/crescimento & desenvolvimento , Brassica napus/metabolismo , Glucosinolatos/metabolismo , Organogênese , Brassica napus/genética , Cruzamentos Genéticos , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genótipo , Glucosinolatos/biossíntese , Glucosinolatos/química , Redes e Vias Metabólicas , Metionina/química , Metionina/metabolismo , Especificidade de Órgãos/genética , Organogênese/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Análise de Componente Principal
16.
J Am Soc Mass Spectrom ; 30(9): 1601-1608, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31222676

RESUMO

Mass spectrometry affords rapid and sensitive analysis of peptides and proteins. Coupling spectroscopy with mass spectrometry allows for the development of new methods to enhance biomolecular structure determination. Herein, we demonstrate two new energy acceptors that can be utilized for action-excitation energy transfer experiments. In the first system, C-S bonds in methionine act as energy acceptors from native chromophores, including tyrosine, tryptophan, and phenylalanine. Comparison among chromophores reveals that tyrosine transfers energy most efficiently at 266 nm, but phenylalanine and tryptophan also transfer energy with comparable efficiencies. Overall, the C-S bond dissociation yields following energy transfer are low for methionine, which led to an investigation of selenomethionine, a common analog that is found in many naturally occurring proteins. Sulfur and selenium are chemically similar, but C-Se bonds are weaker than C-S bonds and have lower lying σ* anti-bonding orbitals. Excitation of peptides containing tyrosine and tryptophan results in efficient energy transfer to selenomethionine and abundant C-Se bond dissociation. A series of helical peptides were examined where the positions of the donor or acceptor were systematically scanned to explore the influence of distance and helix orientation on energy transfer. The distance was found to be the primary factor affecting energy transfer efficiency, suggesting that selenomethionine may be a useful acceptor for probing protein structure in the gas phase.


Assuntos
Metionina/química , Peptídeos/química , Selenometionina/química , Carbono/química , Transferência de Energia , Gases/química , Espectrometria de Massas , Modelos Moleculares , Simulação de Dinâmica Molecular , Fenilalanina/química , Enxofre/química , Triptofano/química , Tirosina/química
17.
Pharm Res ; 36(7): 103, 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101998

RESUMO

PURPOSE: A rapid and broadly applicable method to assess relevant oxidative damage in biopharmaceuticals is important for lifecycle management of product quality. Multiple methods are currently employed as stress tests to induce oxidative damage for assessment of stability, safety, and efficacy. We compared two common methods for inducing oxidative damage to assess differences in impact on bioactivity and structure of the biopharmaceuticals. METHODS: Biopharmaceuticals were treated with either metal-catalyzed oxidation (MCO) conditions or the reactive-oxygen species (ROS) inducer 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), then analyzed for changes in structure and bioactivity. RESULTS: We demonstrate that commonly used chemical methods for assessing oxidation yield distinct oxidation profiles for each of the biotechnology products analyzed, including monoclonal antibodies. We further report oxidant- and product-specific changes in bioactivity under oxidizing conditions, along with differential oxidation on the molecular subunits of monoclonal antibodies. CONCLUSION: Our results highlight the need for product-specific optimization and selection of orthogonal, relevant oxidizers when characterizing stress responses in biopharmaceuticals.


Assuntos
Produtos Biológicos/química , Estresse Oxidativo , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Indicadores e Reagentes/química , Metionina/química , Oxidantes/química , Oxirredução , Espécies Reativas de Oxigênio/química , Rituximab/química , Rituximab/farmacologia , Trastuzumab/química , Trastuzumab/farmacologia
18.
Cell ; 177(3): 711-721.e8, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30982603

RESUMO

Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-ß polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.


Assuntos
Proteínas de Transporte/metabolismo , Metionina/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Peróxido de Hidrogênio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Transição de Fase , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
19.
J Dairy Sci ; 102(6): 5530-5541, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954259

RESUMO

Enhancing the supply of rumen-protected Met (RPM) during the peripartum period alleviates inflammation and oxidative stress status in dairy cows. We tested the hypothesis that RPM could increase abundance of genes and proteins related to glutathione (GSH) metabolism and the antioxidant transcription factor nuclear factor erythroid 2-like 2 (NFE2L2) in subcutaneous adipose tissue. Multiparous Holstein cows were fed a basal diet [control prepartum diet = 1.47 Mcal/kg of dry matter (DM) and 15.3% crude protein; control postpartum diet = 1.67 Mcal/kg of DM and 17.7% crude protein] or the control plus ethyl-cellulose RPM at a rate of 0.09 and 0.10% of DM intake before expected calving and after calving, respectively. Sixty cows were assigned to treatments based on parity, previous 305-d milk yield, and body condition score at 28 d from parturition. Diets were fed from -28 to 30 d. Biopsies of subcutaneous adipose tissue collected on d -10, 10, and 30 relative to parturition from 7 cows in each group were used for measuring concentrations of GSH, reactive oxygen species, superoxide dismutase, malondialdehyde, and mRNA and protein abundance (Western blotting). A repeated-measures ANOVA was used for statistics. The statistical model included the random effect of block and fixed effects of treatment, time, and its interaction. There was a diet × time effect for reactive oxygen species due to lower concentrations in Met versus control cows specifically at d -10. Cows fed Met also had lower concentrations of malondialdehyde in subcutaneous adipose tissue. Compared with controls, overall mRNA abundance of the GSH metabolism-related genes cystathionine-ß-synthase (CBS), glutamate-cysteine ligase modifier subunit (GCLM), glutathione reductase (GSR), and glutathione peroxidase 1 (GPX1) was greater in cows fed Met. Furthermore, supply of Met resulted in an overall upregulation of protein abundance of glutathione peroxidase (GPX) 1, GPX3, glutathione S-transferase mu 1 (GSTM1), and glutathione S-transferase α 4 (GSTA4), all related to GSH metabolism. There was a diet × time effect for protein abundance of NFE2L2 and its repressor Kelch-like ECH associated protein 1 (KEAP1) due to lower values at 30 d in cows fed Met versus controls. The abundance of phosphorylated NFE2L2 was lower at 30 d in response to Met. Overall, the data suggest that exogenous Met may play a role in activating GSH metabolism and the antioxidant NFE2L2 pathways in subcutaneous adipose tissue.


Assuntos
Bovinos/fisiologia , Suplementos Nutricionais , Glutationa/metabolismo , Inflamação/veterinária , Metionina/administração & dosagem , Fator 2 Relacionado a NF-E2/metabolismo , Tecido Adiposo/metabolismo , Animais , Antioxidantes/metabolismo , Celulose/análogos & derivados , Celulose/química , Dieta/veterinária , Feminino , Inflamação/prevenção & controle , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Metionina/química , Leite/metabolismo , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Período Periparto , Fosforilação , Período Pós-Parto/efeitos dos fármacos , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Rúmen/metabolismo
20.
Methods Mol Biol ; 1947: 31-55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969410

RESUMO

Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete 13CH3-methionine labeling of a thermostable neurotensin receptor 1 (enNTS1) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6 mg enNTS1 per liter of expression culture containing 50 mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6 mg of functional Apo-state enNTS1.


Assuntos
Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Metionina/química , Receptores de Neurotensina/isolamento & purificação , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Isótopos de Carbono/química , Escherichia coli/genética , Humanos , Mutação , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusão/genética
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