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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360890

RESUMO

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membranas Intracelulares/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Proteoma , Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Filogenia , Processamento de Proteína Pós-Traducional , Proteômica/métodos
2.
Curr Protoc ; 1(8): e221, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34411463

RESUMO

This paper provides a guideline for optimizing and utilizing Mn2+ Phos-tag gel technology to separate phosphorylated proteins from their unphosphorylated counterparts. It provides key insights into methods for careful sample preparation and experimental directions for determining the appropriate Phos-tag gel compositions and electrophoresis and western blotting conditions. This protocol has been used to successfully resolve proteins extracted from cardiac and skeletal muscles. The guidelines can be extended for optimizing protocols to resolve proteins from other cells or tissue sources. With this, phosphoproteomics and the elucidation of underlying mechanisms of disease progression can be accelerated. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.


Assuntos
Fosfoproteínas , Resinas Acrílicas , Eletroforese em Gel de Poliacrilamida , Humanos , Fosfoproteínas/metabolismo , Fosforilação , Piridinas
3.
Methods Mol Biol ; 2341: 9-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264455

RESUMO

Zymography has been used to analyze enzymatic activity and processing of enzymes for many years. We have used bacterial cells copolymerized into the acrylamide gel to analyze specific activity of murein hydrolases of interest. In addition, this method has been widely used to examine and distinguish protease activities using different substrates. This chapter provides instruction for zymography of both extracellular murein hydrolases and proteases produced by Staphylococcus aureus.


Assuntos
N-Acetil-Muramil-L-Alanina Amidase/análise , Peptídeo Hidrolases/análise , Staphylococcus aureus/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Hidrolases/metabolismo , Staphylococcus aureus/enzimologia
4.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200462

RESUMO

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.


Assuntos
Gastrópodes/metabolismo , Muco/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Animais , Cromatografia Líquida/métodos , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Aprendizado de Máquina , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
5.
J Enzyme Inhib Med Chem ; 36(1): 1267-1281, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34210221

RESUMO

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.


Assuntos
Peptídeo Hidrolases/metabolismo , Periodontite/microbiologia , Inibidores de Proteases/farmacologia , Tannerella forsythia/enzimologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Descoberta de Drogas , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Acoplamento Molecular , Estrutura Molecular , Peptídeo Hidrolases/efeitos dos fármacos , Inibidores de Proteases/química , Tannerella forsythia/isolamento & purificação , Temperatura
6.
Electron. j. biotechnol ; 52: 85-92, July. 2021. graf, tab
Artigo em Inglês | LILACS | ID: biblio-1283600

RESUMO

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.


Assuntos
Peptídeo Sintases/metabolismo , Reatores Biológicos/microbiologia , Escherichia coli , Temperatura , Biotecnologia , Carbono/metabolismo , Modelos Estatísticos , Eletroforese em Gel de Poliacrilamida , Bioengenharia , Concentração de Íons de Hidrogênio
7.
Methods Mol Biol ; 2323: 67-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086274

RESUMO

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.


Assuntos
Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/química , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Ampicilina/farmacologia , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Capsídeo , Cloranfenicol/farmacologia , Simulação por Computador , Resistência Microbiana a Medicamentos/genética , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Levivirus/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Operadoras Genéticas , Plasmídeos/genética , RNA/biossíntese , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , RNA Viral/genética , RNA Viral/isolamento & purificação , Proteínas de Ligação a RNA/biossíntese
8.
Methods Mol Biol ; 2323: 75-97, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086275

RESUMO

Preparative synthesis of RNA is a challenging task that is usually accomplished by either chemical or enzymatic polymerization of ribonucleotides in vitro. Herein, we describe an alternative approach in which RNAs of interest are expressed as a fusion with a 5S rRNA-derived scaffold. The scaffold provides protection against cellular ribonucleases resulting in cellular accumulations comparable to those of regular ribosomal RNAs. After isolation of the chimeric RNA from the cells, the scaffold can be removed, if necessary, by deoxyribozyme-catalyzed cleavage followed by preparative electrophoretic separation of the reaction products. The protocol is designed for sustained production of high quality RNA on the milligram scale.


Assuntos
Clonagem Molecular/métodos , RNA Ribossômico 5S , RNA/biossíntese , Sequência de Bases , DNA Catalítico/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Pennisetum/genética , Plasmídeos/genética , Plasmídeos/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA de Plantas/genética , RNA Ribossômico 5S/genética , Transformação Bacteriana , Vibrio/genética
9.
Methods Mol Biol ; 2323: 109-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086277

RESUMO

RNA motifs guide the interaction with specific proteins leading to the assembly of ribonucleoprotein complexes that perform key functions in cellular processes. Internal ribosome entry site (IRES) elements are organized in structural domains that determine internal initiation of translation. In this chapter we describe a pull-down assay using streptavidin-aptamer tagged RNAs that combines RNA structure-dependent protein isolation with proteomic analysis to identify novel interactors recognizing RNA structural domains. This approach takes advantage of tRNA-scaffold guided expression, allowing the identification of factors belonging to networks involved in RNA and protein metabolism.


Assuntos
Motivos de Nucleotídeos , Proteínas de Ligação a RNA/isolamento & purificação , Aptâmeros de Nucleotídeos , Eletroforese em Gel de Poliacrilamida , Humanos , Sítios Internos de Entrada Ribossomal , Espectrometria de Massas , Motivos de Nucleotídeos/genética , Biossíntese de Proteínas , Proteômica/métodos , RNA/isolamento & purificação , RNA/metabolismo , RNA de Transferência/biossíntese , RNA de Transferência/química , Proteínas de Ligação a RNA/metabolismo , Estreptavidina , Especificidade por Substrato
10.
Methods Mol Biol ; 2323: 249-265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34086286

RESUMO

Research on RNA function and therapeutic potential is dominated by the use of chemoengineered RNA mimics. Recent efforts have led to the establishment of novel technologies for the production of recombinant or bioengineered RNA molecules, which should better recapitulate the structures, functions and safety profiles of natural RNAs because both are produced and folded in living cells. Herein, we describe a robust approach for reproducible fermentation production of bioengineered RNA agents (BERAs) carrying warhead miRNAs, siRNAs, aptamers, or other forms of small RNAs, based upon an optimal hybrid tRNA/pre-miRNA carrier. Target BERA/sRNAs are readily purified by fast protein liquid chromatography (FPLC) to a high degree of homogeneity (>97%). This approach offers a consistent high-level expression (>30% of total bacterial RNAs) and large-scale production of ready-to-use BERAs (multiple to tens milligrams from 1 L bacterial culture).


Assuntos
Bioengenharia/métodos , MicroRNAs/isolamento & purificação , RNA Bacteriano/isolamento & purificação , RNA de Transferência/isolamento & purificação , RNA não Traduzido/isolamento & purificação , RNA/isolamento & purificação , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Clonagem Molecular/métodos , Contaminação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Endotoxinas/análise , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , MicroRNAs/biossíntese , MicroRNAs/genética , Desnaturação de Ácido Nucleico , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , RNA/biossíntese , RNA/genética , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA de Transferência/biossíntese , RNA de Transferência/genética , RNA não Traduzido/genética
11.
Methods Mol Biol ; 2348: 243-253, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34160812

RESUMO

Viruses, like their metazoan hosts, have evolved to utilize intricate transcriptional mechanisms to generate a vast array of both coding and noncoding RNA transcripts. The resolution of specific noncoding RNA transcripts produced by viruses, particularly herpesviruses, presents a particularly difficult challenge due to their highly dense dsDNA genomes and their complex, overlapping, and context-dependent network of transcripts. While new long read sequencing platforms have facilitated the resolution of some noncoding transcripts from virus genomes, empirical molecular validation of transcripts from individual regions is essential. Herein, we demonstrate that the use of strand specific northern blots is essential for true validation of specific viral noncoding RNAs, and provide here a detailed molecular method for such an approach.


Assuntos
Northern Blotting , Homologia de Genes , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Viral/genética , Northern Blotting/métodos , Eletroforese em Gel de Poliacrilamida , Regulação Viral da Expressão Gênica , Genoma Viral , Herpesviridae/genética , Fases de Leitura Aberta , Vírus/genética
12.
Anal Chem ; 93(26): 9267-9276, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34165952

RESUMO

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.


Assuntos
Boratos , Dextranos , Eletroforese Capilar , Eletroforese em Gel de Poliacrilamida , Géis , Peso Molecular , Dodecilsulfato de Sódio
13.
Methods Mol Biol ; 2276: 103-112, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060035

RESUMO

Native electrophoresis is a powerful tool to analyze the mitochondrial electron transport chain complexes (Cx) I-V and their assembly into supercomplexes. Valuable information regarding the composition and bioenergetic regulation in physiological and pathological conditions can be obtained. This chapter compares different types of native electrophoresis to analyze mitochondrial supercomplexes.


Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Immunoblotting/métodos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Humanos , Proteínas Mitocondriais/química
14.
Food Chem ; 361: 130071, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34091398

RESUMO

In this study, conjugates of whey protein isolate (WPI) and four polyphenols (epigallocatechin gallate [EGCG], quercetin [QC], apigenin [AG], and naringenin [NG]) were prepared through free-radical grafting. The results for polyphenol binding equivalents and content of free amino and sulfhydryl groups as well as those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the covalent interaction between WPI and the polyphenols. Fourier transform infrared spectroscopy and fluorescence spectrum analysis identified the potential binding sites of the complexes and determined changes in the protein structure. The particle size distribution and scanning electron microscopy data demonstrated increases in conjugate particle sizes and surface changes in the complexes. The conjugation process significantly increased the polyphenols' antioxidant properties and thermal stabilities, whereas surface hydrophobicity was substantially reduced. WPI-EGCG had the best functional properties, followed by WPI-QC, WPI-AG, and WPI-NG.


Assuntos
Antioxidantes/química , Polifenóis/química , Proteínas do Soro do Leite/química , Apigenina/química , Catequina/análogos & derivados , Catequina/química , Eletroforese em Gel de Poliacrilamida , Flavanonas/química , Radicais Livres/química , Alimento Funcional , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Quercetina/química , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Expert Rev Mol Diagn ; 21(8): 767-787, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34115952

RESUMO

Introduction: Human blood and saliva are increasingly under investigation for the detection of biomarkers for early diagnosis of non-communicable (e.g.cancers) and communicable diseases like COVID-19. Exploring the potential application of human tears, an easily accessible body fluid, for the diagnosis of various diseases is the need of the hour.Areas covered: This review deals with a comprehensive account of applications of tear analysis using different techniques, their comparison and overall progress achieved till now. The techniques used for tear fluid analysis are HPLC/UPLC/SDS-PAGE, CE, etc., together with ELISA, Mass Spectrometry, etc. But, with advances in instrumentation and data processing methods, it has become easy to couple the various separation methods with highly sensitive optical techniques for the analysis of body fluids.Expert opinion: Tear analysis can provide valuable information about the health condition of the eyes since it contains several molecular constituents, and their relative concentrations may alter under abnormal conditions. Tear analysis has the advantage that it is totally non-invasive. This study recommends tear fluid as a reliable clinical sample to be probed by highly sensitive optical techniques to diagnose different health conditions, with special emphasis on eye diseases.


Assuntos
Biomarcadores/análise , Oftalmopatias/diagnóstico , Lágrimas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Espectrometria de Massas , Neoplasias/diagnóstico , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Análise Espectral Raman , Lágrimas/química
16.
Methods Mol Biol ; 2310: 247-258, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096006

RESUMO

We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.


Assuntos
Adipócitos Marrons/efeitos dos fármacos , Tecido Adiposo Marrom/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ácido Succínico/farmacologia , Adipócitos Marrons/enzimologia , Tecido Adiposo Marrom/enzimologia , Animais , Biomarcadores/metabolismo , Western Blotting , Fracionamento Celular , Complexo I de Transporte de Elétrons/genética , Eletroforese em Gel de Poliacrilamida , Fluorometria , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/genética , Ratos , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
17.
Molecules ; 26(10)2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065427

RESUMO

Early plants began colonizing earth about 450 million years ago. During the process of coevolution, their metabolic cellular pathways produced a myriad of natural chemicals, many of which remain uncharacterized biologically. Popular preparations containing some of these molecules have been used medicinally for thousands of years. In Brazilian folk medicine, plant extracts from the bamboo plant Guadua paniculata Munro have been used for the treatment of infections and pain. However, the chemical basis of these therapeutic effects has not yet been identified. Here, we performed protein biochemistry and downstream pharmacological assays to determine the mechanisms underlying the anti-inflammatory and antinociceptive effects of an aqueous extract of the G. paniculata rhizome, which we termed AqGP. The anti-inflammatory and antinociceptive effects of AqGP were assessed in mice. We identified and purified a protein (AgGP), with an amino acid sequence similar to that of thaumatins (~20 kDa), capable of repressing inflammation through downregulation of neutrophil recruitment and of decreasing hyperalgesia in mice. In conclusion, we have identified the molecule and the molecular mechanism responsible for the anti-inflammatory and antinociceptive properties of a plant commonly used in Brazilian folk medicine.


Assuntos
Analgésicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Bambusa/química , Extratos Vegetais/uso terapêutico , Sequência de Aminoácidos , Analgésicos/administração & dosagem , Animais , Anti-Inflamatórios/administração & dosagem , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Hiperalgesia/tratamento farmacológico , Inflamação/tratamento farmacológico , Células MCF-7 , Masculino , Camundongos , Células NIH 3T3 , Extratos Vegetais/administração & dosagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Molecules ; 26(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066679

RESUMO

Microalgae are known to be rich in protein. In this study, we aim to investigate methods of producing and purifying proteins of 98 microalgae including Chlorella vulgaris, Arthrospira platensis, Nostoc sp., Dunaliella salina, and Pleurochrysis carterae (Baltic Sea). Therefore, we studied their amino acid composition and developed a two-stage protein concentrate purification method from the microalgae biomass. After an additional stage of purification, the mass fraction of protein substances with a molecular weight greater than 50 kDa in the protein concentrate isolated from the biomass of the microalga Dunaliella salina increased by 2.58 times as compared with the mass fraction before filtration. In the protein concentrate isolated from the biomass of the microalga Pleurochrysis cartera, the relative content of the fraction with a molecular weight greater than 50.0 kDa reached 82.4%, which was 2.43 times higher than the relative content of the same fractions in the protein concentrate isolated from this culture before the two-stage purification. The possibilities of large-scale industrial production of microalgae biomass and an expanded range of uses determine the need to search for highly productive protein strains of microalgae and to optimize the conditions for isolating amino acids from them.


Assuntos
Proteínas de Algas/química , Aminoácidos/química , Aminoácidos/isolamento & purificação , Chlorella vulgaris/química , Haptófitas/química , Microalgas/química , Nostoc/química , Spirulina/química , Biomassa , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Ultrafiltração
19.
Food Chem ; 362: 130170, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34091164

RESUMO

Impact of globulin addition on the functional and protein structural properties of dough and cooked noodles were investigated. The underlying mechanism was explored through analyzing the interaction between globulin and gluten by using SDS-PAGE, size exclusion chromatography, free sulfhydryl/disulfide bond analysis, laser scanning confocal microscopy and Fourier transform infrared spectroscopy. Results showed that the stiffness/hardness and maximum resistance of dough and cooked noodles were both increased when globulin addition was 1.5% or higher. Besides, extensibility of cooked noodles was also improved when the addition up to 3.0%. The addition of globulin facilitated weakening the S-S bonds in the gluten network and cross-linked with SDS-soluble gluten mainly through non-covalent interactions, especially hydrophobic interactions. Meanwhile, a more rigid protein network structure was observed. Additionally, following cooking, globulin addition accelerated the aggregation of protein molecules. When the addition reached 3%, the protein conformation was transformed from ß-sheets and random coils to ß-turns.


Assuntos
Farinha , Globulinas/química , Triticum/química , Cromatografia em Gel , Culinária , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Farinha/análise , Qualidade dos Alimentos , Glutens/química , Dureza , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal , Proteínas de Vegetais Comestíveis/química , Reologia , Espectroscopia de Infravermelho com Transformada de Fourier
20.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067755

RESUMO

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.


Assuntos
Cromatografia em Gel/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Isomerases de Dissulfetos de Proteínas/metabolismo , Diferenciação Celular , Eletroforese em Gel de Poliacrilamida/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/fisiologia , Transporte Proteico , Solubilidade
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