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1.
Plant Physiol ; 164(2): 951-65, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24344171

RESUMO

The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endosperma/metabolismo , Giberelinas/farmacologia , Glicoproteínas/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Hordeum/metabolismo , Proteoma/metabolismo , Tunicamicina/farmacologia , Morte Celular/efeitos dos fármacos , Extratos Celulares , Análise por Conglomerados , Endosperma/citologia , Endosperma/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fluorescência , Glicosilação/efeitos dos fármacos , Hordeum/citologia , Hordeum/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Espaço Intracelular/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Modelos Biológicos , Proteínas de Plantas/metabolismo , Análise de Componente Principal , Proteômica , Coloração e Rotulagem , alfa-Amilases/biossíntese
2.
J Proteome Res ; 13(5): 2696-703, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24654981

RESUMO

Hydrophilic liquid chromatography (HILIC) is used extensively as a sample preparation step for glycopeptide enrichment in proteome research. Here, we have applied cotton wool and a zwitterionic HILIC (ZIC-HILIC) resin in solid-phase extraction microcolumns to provide a higher loading capacity and broader specificity for glycopeptide enrichment. This strategy was applied to tryptic digests of wheat flour albumin extracts followed by simulataneous site-specific (18)O labeling and deglycosylation using peptide-N-glycosidase A (PNGase A) in H(2)(18)O. Subsequent LC-MS/MS analysis allowed for assignment of 78 N-glycosylation sites in 67 albumin proteins. Bioinformatic analysis revealed that several of the identified glycoproteins show sequence similarity to known food allergens. In addition, the potential impact of some of the identified glycoproteins on wheat beer quality is discussed.


Assuntos
Albuminas/metabolismo , Cromatografia Líquida/métodos , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Triticum/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida/instrumentação , Fibra de Algodão , Farinha/análise , Glicopeptídeos/química , Glicosilação , Dados de Sequência Molecular , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Extração em Fase Sólida , Espectrometria de Massas em Tandem
3.
Heliyon ; 8(9): e10740, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36185148

RESUMO

Diabetes mellitus is the most common non-infective disease characterized by hyperglycemia (high level of blood glucose). Formation of advanced glycation end products (AGEs) in long termed-hyperglycemia and oxidative stress are the key factors to accelerate diabetic complications. To screen potential candidates for treating diabetes, total phenolic content, total flavonoid content, antioxidant activity from crude extracts of some Thai edible plants were primarily assessed, and the inhibiting potential of diabetes and its complications provided from some of these plants were evaluated in terms of their inhibitory activities of α-amylase, α-glycosidase, and AGEs formation. The highest amounts of phenolic and flavonoid compounds were found in the ethanolic extract of Caesalpinia mimosoides (S20, 12.63 ± 1.70 mg GAE/g DW) and Glochidion hirsutum (S8, 3.02 ± 0.25 mg CE/g DW), respectively. The highest antioxidant activity was found in Schinus terebinthifolius Raddi (S26, 217.94 ± 32.30 µg AAE/g DW) whereas the highest inhibitory activities of α-amylase and α-glycosidase were obtained from Basella alba L. (S11, IC50 = 0.21 ± 0.01 mg/ml) and S. terebinthifolius (S26, IC50 = 0.05 ± 0.02 mg/ml) respectively. The inhibitory effects of AGEs formation were studied in vitro using two model systems: BSA-glucose and BSA-methylglycoxal (MGO). The extracts of Glochidion hirsutum (Roxb.) Voigt (S8, IC50 = 0.20 ± 0.01 mg/ml) and Polygonum odoratum Lour. (S13, IC50 = 0.03 ± 0.01 mg/ml) exhibited the inhibitory activity of AGEs formation derived from glucose (BSA-glucose system) stronger than aminoguanidine (AG) (0.26 ± 0.00 mg/ml), which is a common AGEs formation inhibitory drug. By BSA-MGO assay, the inhibition of some selected extracts in this study (G. hirsutum, G. sphaerogynum, and S. terebinthifolius with IC50 = 0.11 ± 0.01, 0.11 ± 0.01, and 0.10 ± 0.00 mg/ml, respectively) were slightly less efficient than AG (the IC50 = 0.06 ± 0.00 mg/ml). These results indicated that some selected Thai edible plants in this present study provided potential applications towards the prevention of diabetes and their complications via the inhibitory of α-amylase, α-glycosidase, AGEs formation, and oxidative stress. This fundamental information would be important for alternative drug discovery and nutritional recommendations for diabetic patients.

5.
Biochem Biophys Res Commun ; 407(4): 708-13, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21439264

RESUMO

Bacillus thuringiensis Cry4Ba toxin selectively kills Aedes aegypti mosquito larvae as it is in part due to the presence of specific membrane-bound protein receptors. In this study, using data mining approach, we initially identified three potential glycosylphosphatidylinositol-linked aminopeptidase N (GPI-APN) isoforms, APN2778, APN2783 and APN5808, which are believed to act as Cry4Ba toxin receptors. These three isoforms that are functionally expressed in the larval midgut can be sequence-specific knocked down (ranging from ∼80 % to 95 %) by soaking the Aedes aegypti larvae in buffer of long double-stranded GPI-APN RNAs (∼300-680 bp). Finally, to see the physiological effect of APN knockdowns, the larvae were fed with Escherichia coli expressing Cry4Ba toxin. The results revealed that all the three identified GPI-APN isoforms may possibly function as a Cry4Ba receptor, particularly for APN2783 as those larvae with this transcript knockdown showed a dramatic increase in resistance to Cry4Ba toxicity.


Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Proteínas de Bactérias/toxicidade , Antígenos CD13/genética , Resistência a Medicamentos/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Inseticidas/toxicidade , Aedes/enzimologia , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Técnicas de Silenciamento de Genes , Glicosilfosfatidilinositóis/metabolismo , Isoenzimas/genética , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Dados de Sequência Molecular , Interferência de RNA , RNA Mensageiro/antagonistas & inibidores , Análise de Sequência de Proteína , Transcrição Gênica
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