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1.
Nucleic Acids Res ; 49(7): 3661-3671, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33772594

RESUMO

Among the many in vitro-selected aptamers derived from SELEX protocols, only a small fraction has the potential to be applied for synthetic riboswitch engineering. Here, we present a comparative study of the binding properties of three different aptamers that bind to ciprofloxacin with similar KD values, yet only two of them can be applied as riboswitches. We used the inherent ligand fluorescence that is quenched upon binding as the reporter signal in fluorescence titration and in time-resolved stopped-flow experiments. Thus, we were able to demonstrate differences in the binding kinetics of regulating and non-regulating aptamers. All aptamers studied underwent a two-step binding mechanism that suggests an initial association step followed by a reorganization of the aptamer to accommodate the ligand. We show that increasing regulatory potential is correlated with a decreasing back-reaction rate of the second binding step, thus resulting in a virtually irreversible last binding step of regulating aptamers. We suggest that a highly favoured structural adaption of the RNA to the ligand during the final binding step is essential for turning an aptamer into a riboswitch. In addition, our results provide an explanation for the fact that so few aptamers with regulating capacity have been found to date. Based on our data, we propose an adjustment of the selection protocol for efficient riboswitch detection.


Assuntos
Aptâmeros de Nucleotídeos/química , Ciprofloxacina/química , RNA/química , Riboswitch , Técnica de Seleção de Aptâmeros/métodos , Ligantes , Conformação de Ácido Nucleico
2.
Plant Physiol Biochem ; 92: 39-47, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25900423

RESUMO

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human ß1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.


Assuntos
Arabidopsis/genética , N-Acetil-Lactosamina Sintase/genética , Nicotiana/genética , Fenótipo , Plantas Geneticamente Modificadas , Polissacarídeos/biossíntese , Arabidopsis/metabolismo , Parede Celular/metabolismo , Epitopos , Galactosiltransferases/metabolismo , Engenharia Genética , Glicosilação , Humanos , Peróxido de Hidrogênio/metabolismo , Mucoproteínas/metabolismo , N-Acetil-Lactosamina Sintase/metabolismo , Pectinas/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismo
4.
Plant Biotechnol J ; 12(7): 832-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24618259

RESUMO

Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co-expression of BChE with a novel gene-stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N-glycans. The N-glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma-derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER-typical oligomannosidic structures. Biochemical analyses and live-cell imaging experiments indicated that impaired N-glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic-reticulum-derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in-depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.


Assuntos
Butirilcolinesterase/metabolismo , Nicotiana/metabolismo , Butirilcolinesterase/genética , Butirilcolinesterase/isolamento & purificação , Vetores Genéticos/metabolismo , Glicosilação , Humanos , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Recombinantes/metabolismo , Nicotiana/genética
5.
Biotechnol J ; 9(4): 501-10, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24130173

RESUMO

Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre- and post-exposure treatment. However, the presence and functional necessity of complex N-glycans (i.e. sialylated structures) is a challenging issue in respect to its recombinant expression. Here we transiently co-expressed BChE cDNA in the model plant Nicotiana benthamiana with vectors carrying the genes necessary for in planta protein sialylation. Site-specific sugar profiling of secreted recombinant BChE (rBChE) collected from the intercellular fluid revealed the presence of mono- and di-sialylated N-glycans, which largely resembles to the plasma-derived orthologue. Attempts to increase that sialylation content of rBChE by the over-expression of an additional glycosylation enzyme that generates branched N-glycans (i.e. ß1,4-N-acetylglucosaminyl-transferase IV), allowed the production of rBChE decorated with tri-sialylated structures (up to 70%). Sialylated and non-sialylated plant-derived rBChE exhibited functional in vitro activity comparable to that of its commercially available equine-derived counterpart. These results demonstrate the ability of plants to generate valuable proteins with designed sialylated glycosylation profiles optimized for therapeutic efficacy. Moreover, the efficient synthesis of carbohydrates present only in minute amounts on the native protein (tri-sialylated N-glycans) facilitates the generation of a product with superior efficacies and/or new therapeutic functions.


Assuntos
Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Butirilcolinesterase/genética , Butiriltiocolina/análise , Butiriltiocolina/metabolismo , Glicosilação , Humanos , Ácido N-Acetilneuramínico , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Nicotiana/genética , Nicotiana/metabolismo
6.
Biochimie ; 95(12): 2445-53, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24047540

RESUMO

In search for yet uncharacterized proteins involved in lipid metabolism of the chicken, we have isolated a hitherto unknown protein from the serum lipoprotein fraction with a buoyant density of ≤1.063 g/ml. Data obtained by protein microsequencing and molecular cloning of cDNA defined a 537 bp cDNA encoding a precursor molecule of 178 residues. As determined by SDS-PAGE, the major circulating form of the protein, which we designate apolipoprotein-VLDL-IV (Apo-IV), has an apparent Mr of approximately 17 kDa. Northern Blot analysis of different tissues of laying hens revealed Apo-IV expression mainly in the liver and small intestine, compatible with an involvement of the protein in lipoprotein metabolism. To further investigate the biology of Apo-IV, we raised an antibody against a GST-Apo-IV fusion protein, which allowed the detection of the 17-kDa protein in rooster plasma, whereas in laying hens it was detectable only in the isolated ≤1.063 g/ml density lipoprotein fraction. Interestingly, estrogen treatment of roosters caused a reduction of Apo-IV in the liver and in the circulation to levels similar to those in mature hens. Furthermore, the antibody crossreacted with a 17-kDa protein in quail plasma, indicating conservation of Apo-IV in avian species. In search for mammalian counterparts of Apo-IV, alignment of the sequence of the novel chicken protein with those of different mammalian apolipoproteins revealed stretches with limited similarity to regions of ApoC-IV and possibly with ApoE from various mammalian species. These data suggest that Apo-IV is a newly identified avian apolipoprotein.


Assuntos
Apolipoproteínas/sangue , Etinilestradiol/farmacologia , Sequência de Aminoácidos , Animais , Apolipoproteínas/biossíntese , Apolipoproteínas/efeitos dos fármacos , Apolipoproteínas/imunologia , Sequência de Bases , Galinhas/sangue , Galinhas/genética , Feminino , Masculino , Dados de Sequência Molecular , Oviposição
7.
Eur J Clin Invest ; 43(2): 174-82, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23278361

RESUMO

BACKGROUND: Hypoxia precedes cardiomyocyte necrosis in acute myocardial infarction (AMI). We therefore hypothesized that uric acid - as a marker of oxidative stress and hypoxia - might be useful in the early diagnosis and risk stratification of patients with suspected AMI. MATERIALS AND METHODS: In this prospective observational study, uric acid was measured at presentation in 892 consecutive patients presenting to the emergency department with suspected AMI. The final diagnosis was adjudicated by two independent cardiologists. Patients were followed 24 months regarding mortality. Primary outcome was the diagnosis of AMI, secondary outcome was short- and long-term mortality. RESULTS: Uric acid at presentation was higher in patients with AMI than in patients without (372 µM vs. 336 µM; P < 0·001). The diagnostic accuracy of uric acid for AMI as quantified by the area under the receiver operating characteristic curve (AUC) was 0·60 (95%Cl 0·56-0·65). When added to cardiac troponin T (cTnT), uric acid significantly increased the AUC of cTnT from 0·89 (95%Cl 0·85-0·93) to 0·92 (95%Cl 0·89-0·95, P = 0·020 for comparison). Cumulative 24-month mortality rates were 2·2% in the first, 5·4% in the second and the third and 15·6% in the fourth quartile of uric acid (P < 0·001 for log-rank). Uric acid predicted 24-month mortality independently. Adding uric acid to TIMI and GRACE risk score improved their prognostic accuracy as shown by an integrated discrimination improvement of 0·04 (P = 0·007) respective 0·02 (P = 0·021). CONCLUSIONS: Uric acid, an inexpensive widely available biomarker, improves both the early diagnosis and risk stratification of patients with suspected AMI.


Assuntos
Biomarcadores/sangue , Infarto do Miocárdio/diagnóstico , Ácido Úrico/sangue , Idoso , Diagnóstico Precoce , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/mortalidade , Estresse Oxidativo , Valor Preditivo dos Testes , Fatores de Risco
8.
Mol Cell Biochem ; 359(1-2): 271-81, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21842374

RESUMO

Calnexin is an endoplasmic reticulum protein that has a role in folding newly synthesized glycoproteins. In this study, we used site-specific mutagenesis to disrupt cysteine and histidine amino acid residues in the N- and P-domains of calnexin and determined whether these mutations impact the structure and function of calnexin. We identified that disruption of the N-domain cysteines resulted in significant loss of the chaperone activity of calnexin toward the glycosylated substrate, IgY, while disruption of the P-domain cysteines only had a small impact toward IgY. We observed that wild-type calnexin as well as the P-domain double cysteine mutant contained an intramolecular disulfide bond which is lost when the N-domain cysteines are mutated. Mutation to the N-domain histidine and N-domain cysteines resulted in increased binding of ERp57. Mutations to the P-domain cysteines further enhanced ERp57 binding to calnexin. Taken together, these observations indicated that the cysteine residues within calnexin were important for the structure and function of calnexin.


Assuntos
Calnexina/química , Cisteína/fisiologia , Calnexina/genética , Calnexina/metabolismo , Dissulfetos , Histidina , Humanos , Imunoglobulinas , Chaperonas Moleculares , Mutagênese Sítio-Dirigida , Isomerases de Dissulfetos de Proteínas/metabolismo , Transporte Proteico
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