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1.
Nat Commun ; 12(1): 71, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397984

RESUMO

Signaling complexes are often organized in a spatiotemporal manner and on a minute timescale. Proximity labeling based on engineered ascorbate peroxidase APEX2 pioneered in situ capture of spatiotemporal membrane protein complexes in living cells, but its application to cytosolic proteins remains limited due to the high labeling background. Here, we develop proximity labeling probes with increased labeling selectivity. These probes, in combination with label-free quantitative proteomics, allow exploring cytosolic protein assemblies such as phosphotyrosine-mediated protein complexes formed in response to minute-scale EGF stimulation. As proof-of-concept, we systematically profile the spatiotemporal interactome of the EGFR signaling component STS1. For STS1 core complexes, our proximity proteomics approach shows comparable performance to affinity purification-mass spectrometry-based temporal interactome profiling, while also capturing additional-especially endosomally-located-protein complexes. In summary, we provide a generic approach for exploring the interactome of mobile cytosolic proteins in living cells at a temporal resolution of minutes.


Assuntos
Citosol/metabolismo , Proteômica , Transdução de Sinais , Biotina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Células HeLa , Humanos , Fenóis , Mapeamento de Interação de Proteínas , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de Tempo
2.
J Immunol Methods ; 490: 112952, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33358997

RESUMO

The ability to quantify protein-ligand interactions in an accurate and high-throughput manner is important in diverse areas of biology and medicine. Multiplex bead binding assays (MBBAs) are powerful methods that allow for simultaneous analysis of many protein-ligand interactions. Although there are a number of well-established MBBA platforms, there are few platforms suitable for research and development that offer rapid experimentation at low costs and without the need for specialized reagents or instruments dedicated for MBBA. Here, we describe a MBBA method that uses low-cost reagents and standard cytometers. The key innovation is the use of the essentially irreversible biotin-streptavidin interaction. We prepared a biotin-conjugated fluorescent dye and used it to produce streptavidin-coated magnetic beads that are labeled at distinct levels of fluorescence. We show the utility of our method in characterization of phage-displayed antibodies against multiple antigens of SARS-CoV-2, which substantially improves the throughput and dramatically reduces antigen consumption compared with conventional phage ELISA methods. This approach will make MBBAs more broadly accessible.


Assuntos
/métodos , /metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas de Bactérias/metabolismo , Biotina/análogos & derivados , Biotina/metabolismo , Técnicas de Visualização da Superfície Celular , Citometria de Fluxo , Corantes Fluorescentes , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Separação Imunomagnética , Microesferas , Mutação/genética , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/genética
3.
Nat Commun ; 11(1): 5598, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154364

RESUMO

Pimelic acid, a seven carbon α,ω-dicarboxylic acid (heptanedioic acid), is known to provide seven of the ten biotin carbon atoms including all those of the valeryl side chain. Distinct pimelate synthesis pathways were recently elucidated in Escherichia coli and Bacillus subtilis where fatty acid synthesis plus dedicated biotin enzymes produce the pimelate moiety. In contrast, the α-proteobacteria which include important plant and mammalian pathogens plus plant symbionts, lack all of the known pimelate synthesis genes and instead encode bioZ genes. Here we report a pathway in which BioZ proteins catalyze a 3-ketoacyl-acyl carrier protein (ACP) synthase III-like reaction to produce pimeloyl-ACP with five of the seven pimelate carbon atoms being derived from glutaryl-CoA, an intermediate in lysine degradation. Agrobacterium tumefaciens strains either deleted for bioZ or which encode a BioZ active site mutant are biotin auxotrophs, as are strains defective in CaiB which catalyzes glutaryl-CoA synthesis from glutarate and succinyl-CoA.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Alphaproteobacteria/metabolismo , Biotina/metabolismo , Lisina/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/metabolismo , Adipatos/metabolismo , Alphaproteobacteria/enzimologia , Alphaproteobacteria/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Glutaratos/metabolismo , Mutação , Ácidos Pimélicos/metabolismo
4.
Clin Dermatol ; 38(4): 477-483, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32972606

RESUMO

Biotinidase deficiency is a rare hereditary metabolic disease. Only a few cases have been reported in China, almost all of which have been in the pediatric population. We report a case of a girl with characteristic skin and hair findings with a negative family history, although her grandparents were consanguineous. The metabolites in the proband's blood and urine increased prominently, and the percentage of biotinase was 1.168%, much lower than normal. Genotyping identified two heterozygous mutations, which were C.1457T>A (p.L486Q) and C.1491dupT (p.L498Ffs*13) in the BTD gene. The diagnosis of biotinidase deficiency was established. No relevant reports about the missense mutation at the mutation site C.1457T>A (p.L486Q) of the BTD gene have been retrieved. Biotin replacement therapy was administered in the dose of 20 mg/d. The dermatitis subsided after 1 month, and the hair color was almost normal after 3 months. This reminds dermatologists to include biotinidase deficiency in their clinical differential when faced with children's intractable dermatitis, yellow hair, and alopecia.


Assuntos
Alopecia/etiologia , Biotina/administração & dosagem , Biotina/metabolismo , Deficiência de Biotinidase/complicações , Deficiência de Biotinidase/diagnóstico , Deficiência de Biotinidase/genética , Biotinidase/genética , Eczema/etiologia , Cor de Cabelo , Deficiência de Biotinidase/tratamento farmacológico , Criança , Feminino , Heterozigoto , Humanos , Mutação , Resultado do Tratamento
5.
J Vis Exp ; (159)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32478734

RESUMO

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.


Assuntos
Biotina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Ligação a RNA/metabolismo , Transcriptoma/genética , Humanos
6.
Nat Commun ; 11(1): 3244, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591520

RESUMO

Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.


Assuntos
Organofosfonatos/metabolismo , Biossíntese de Proteínas , Animais , Biotina/metabolismo , Bovinos , Células HeLa , Humanos , Interferon gama/farmacologia , Células Jurkat , Organofosfonatos/química , Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Soroalbumina Bovina/metabolismo , Coloração e Rotulagem , Estreptavidina/metabolismo
7.
J Biosci Bioeng ; 130(2): 195-199, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32370929

RESUMO

Ectoine production using inexpensive and renewable biomass resources has attracted great interest among the researchers due to the low yields of ectoine in current fermentation approaches that complicate the large-scale production of ectoine. In this study, ectoine was produced from corn steep liquor (CSL) and soybean hydrolysate (SH) in replacement to yeast extract as the nitrogen sources for the fermentation process. To enhance the bacterial growth and ectoine production, biotin was added to the Halomonas salina fermentation media. In addition, the effects addition of surfactants such as Tween 80 and saponin on the ectoine production were also investigated. Results showed that both the CSL and SH can be used as the nitrogen source substitutes in the fermentation media. Higher amount of ectoine (1781.9 mg L-1) was produced in shake flask culture with SH-containing media as compared to CSL-containing media. A total of 2537.0 mg L-1 of ectoine was produced at pH 7 when SH-containing media was applied in the 2 L batch fermentation. Moreover, highest amount of ectoine (1802.0 mg L-1) was recorded in the SH-containing shake flask culture with addition of 0.2 µm mL-1 biotin. This study demonstrated the efficacy of industrial waste as the nutrient supplement for the fermentation of ectoine production.


Assuntos
Diamino Aminoácidos/metabolismo , Fermentação , Halomonas/metabolismo , Microbiologia Industrial/métodos , Técnicas de Cultura Celular por Lotes , Biomassa , Biotina/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Resíduos Industriais , Nitrogênio/metabolismo , Soja/química , Zea mays/química
8.
Nat Commun ; 11(1): 2399, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404879

RESUMO

The ability to monitor molecules volumetrically throughout the body could provide valuable biomarkers for studies of healthy function and disease, but noninvasive detection of molecular targets in living subjects often suffers from poor sensitivity or selectivity. Here we describe a family of potent imaging probes that can be activated by molecules of interest in deep tissue, providing a basis for mapping nanomolar-scale analytes without the radiation or heavy metal content associated with traditional molecular imaging agents. The probes are reversibly caged vasodilators that induce responses detectable by hemodynamic imaging; they are constructed by combining vasoactive peptides with synthetic chemical appendages and protein blocking domains. We use this architecture to create ultrasensitive biotin-responsive imaging agents, which we apply for wide-field mapping of targets in rat brains using functional magnetic resonance imaging. We also adapt the sensor design for detecting the neurotransmitter dopamine, illustrating versatility of this approach for addressing biologically important molecules.


Assuntos
Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Vasodilatadores/metabolismo , Animais , Biotina/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Dopamina/metabolismo , Células HEK293 , Humanos , Imagem por Ressonância Magnética/métodos , Sondas Moleculares/química , Neurotransmissores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Ratos , Reprodutibilidade dos Testes , Vasodilatadores/química
9.
Appl Environ Microbiol ; 86(12)2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32276977

RESUMO

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiae BIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiae IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.


Assuntos
Biotina/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Ascomicetos/enzimologia , Ascomicetos/genética , Engenharia Metabólica , Microrganismos Geneticamente Modificados/enzimologia , Microrganismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Leveduras/enzimologia , Leveduras/genética
10.
Anal Chim Acta ; 1101: 120-128, 2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32029102

RESUMO

Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a protein's thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 µM for both metal species. The system can be used in a variety of high-throughput assay formats including for the screening of metal-binding proteins and chelators. Bp BirA-GFP has also the additional benefit of being useful in Cu(II) ion field-testing applications through simple visual observation of a temperature-dependent loss of fluorescence. Bp BirA-GFP is the first example of a 2protein-based dual purpose Cu(II) and Zn(II) ion sensor compatible with two different yet complementary signal-transduction detection systems.


Assuntos
Carbono-Nitrogênio Ligases/química , Cobre/análise , Proteínas de Fluorescência Verde/química , Proteínas Recombinantes de Fusão/química , Zinco/análise , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Biotina/metabolismo , Burkholderia pseudomallei/enzimologia , Carbono-Nitrogênio Ligases/metabolismo , Cobre/metabolismo , Fluorometria/métodos , Proteínas de Fluorescência Verde/metabolismo , Limite de Detecção , Estudo de Prova de Conceito , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Zinco/metabolismo
12.
Chem Commun (Camb) ; 56(18): 2787-2790, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32025667

RESUMO

Expanding the catalytic repertoire of ribozymes to include vitamin synthesis requires efficient labelling of RNA with the substrate of interest, prior to in vitro selection. For this purpose, we rationally designed and synthesized six GMP-conjugates carrying a synthetic pre-thiamine or biotin precursor and investigated their transcription incorporation properties by T7 RNA polymerase.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Guanosina Monofosfato/biossíntese , Proteínas Virais/metabolismo , Vitaminas/biossíntese , Biocatálise , Biotina/química , Biotina/metabolismo , Guanosina Monofosfato/química , Estrutura Molecular , Tiamina/química , Tiamina/metabolismo , Vitaminas/química
13.
Nat Chem Biol ; 16(4): 415-422, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042199

RESUMO

In biotin biosynthesis, the conversion of pimeloyl intermediates to biotin is catalyzed by a universal set of four enzymes: BioF, BioA, BioD and BioB. We found that the gene homologous to bioA, the product of which is involved in the conversion of 8-amino-7-oxononanoate (AON) to 7,8-diaminononanoate (DAN), is missing in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We provide structural and biochemical evidence showing that a novel dehydrogenase, BioU, is involved in biotin biosynthesis and functionally replaces BioA. This enzyme catalyzes three reactions: formation of covalent linkage with AON to yield a BioU-DAN conjugate at the ε-amino group of Lys124 of BioU using NAD(P)H, carboxylation of the conjugate to form BioU-DAN-carbamic acid, and release of DAN-carbamic acid using NAD(P)+. In this biosynthetic pathway, BioU is a suicide enzyme that loses the Lys124 amino group after a single round of reaction.


Assuntos
Biotina/biossíntese , Oxirredutases/ultraestrutura , Synechocystis/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Diamino Aminoácidos/química , Diamino Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Biotina/metabolismo , Catálise , Clonagem Molecular , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos , Oxirredutases/metabolismo , Synechocystis/genética , Transaminases/metabolismo
14.
Chemistry ; 26(22): 5085-5092, 2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32096262

RESUMO

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.


Assuntos
Biotina/química , DNA/metabolismo , Proteínas/metabolismo , Trombina/química , Biotina/metabolismo , Primers do DNA , Corantes Fluorescentes , Proteínas/química , Trombina/metabolismo
15.
Mikrochim Acta ; 187(2): 116, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31925569

RESUMO

Biotinylated peptide-Cu2+ nanoparticles (Cu-P NPs) were synthesized via "one-pot" self-assembly. The peptide P conststs of a hydrophobic dipeptide (FF), a tripeptide (KGH), and a biotin moiety attached to the terminal amino group of the Lys residue. The Cu-P NPs contain abundant catalytically active Cu2+ ions which are liberated by acid-induced dissolution. The released Cu2+ ions catalyze the oxidization of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 because of their intrinsic peroxidase activity, and this results in the formation of a blue-green coloration. Based on the streptavidin-biotin interaction, the Cu-P NPs were employed to establish an enzyme-free colorimetric method for determination of prostate-specific antigen (PSA) as a model biomarker. Under optimal conditions, the linear response range is 0.001-1 ng mL-1, with a limit of detection as low as 1 pg mL-1. Graphical abstract Schematic illustration of a colorimetric immunoassay for the prostate specific antigen (PSA) with biotinylated peptide-Cu2+ nanoparticle (Cu-P NP) as the signal label based on the streptavidin (SA)-biotin interaction. The signal was produced by Cu2+-catalyzed oxidization of 3,3',5,5'-tetramethylbenzidine (TMB). P: KGHFF.


Assuntos
Colorimetria/métodos , Nanopartículas Metálicas/química , Antígeno Prostático Específico/análise , Benzidinas/química , Biotina/metabolismo , Biotinilação , Cobre/química , Cobre/farmacologia , Humanos , Imunoensaio/métodos , Limite de Detecção , Masculino , Peptídeos/química , Estreptavidina/metabolismo
17.
Methods Mol Biol ; 2043: 113-123, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463907

RESUMO

Biotinylation is a versatile technique that has been used to label proteins for a variety of applications. Under alkaline conditions, the N-hydroxylsuccinimide (NHS) ester present on the biotinylation reagent reacts with primary amines such as the side chain of lysine residues or the N-termini of proteins to yield stable amide bonds. However, the effect of biotinylation on enzyme structure and function has not been generally appreciated. In this chapter, I describe specific issues involving biotinylation of proteoglycanases (e.g., ADAMTS-1, -4, and -5). Taking ADAMTS-5 as an example, I show how high incorporation of biotin molecules causes a decrease in aggrecanase activity, most likely by disrupting exosites present in the cysteine-rich and spacer domains. Such an effect is not evident when enzymatic activity is measured with synthetic peptides, since exosites are not strictly required for peptidolytic activity. Therefore, extreme care must be taken when labeling proteoglycanases and the appropriate enzyme/biotin ratio must be determined experimentally for each enzyme.


Assuntos
Biotina/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Biotinilação , Lisina/metabolismo , Peptídeos/metabolismo , Domínios Proteicos , Coloração e Rotulagem
18.
Nat Prod Rep ; 37(1): 100-135, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31074473

RESUMO

Covering: up to 2019Metabolic production of CO2 is natural product chemistry on a mammoth scale. Just counting humans, among all other respiring organisms, the seven billion people on the planet exhale about 3 billion tons of CO2 per year. Essentially all of the biogenic CO2 arises by action of discrete families of decarboxylases. The mechanistic routes to CO2 release from carboxylic acid metabolites vary with the electronic demands and structures of specific substrates and illustrate the breadth of chemistry employed for C-COO (C-C bond) disconnections. Most commonly decarboxylated are α-keto acid and ß-keto acid substrates, the former requiring thiamin-PP as cofactor, the latter typically cofactor-free. The extensive decarboxylation of amino acids, e.g. to neurotransmitter amines, is synonymous with the coenzyme form of vitamin B6, pyridoxal-phosphate, although covalent N-terminal pyruvamide residues serve in some amino acid decarboxylases. All told, five B vitamins (B1, B2, B3, B6, B7), ATP, S-adenosylmethionine, manganese and zinc ions are pressed into service for specific decarboxylase catalyses. There are additional cofactor-independent decarboxylases that operate by distinct chemical routes. Finally, while most decarboxylases use heterolytic ionic mechanisms, a small number of decarboxylases carry out radical pathways.


Assuntos
Dióxido de Carbono/metabolismo , Carbono/metabolismo , Carboxiliases/metabolismo , Animais , Biotina/metabolismo , Carboxiliases/química , Ácidos Carboxílicos/metabolismo , Catálise , Coenzimas/química , Coenzimas/metabolismo , Humanos , Redes e Vias Metabólicas , NAD/metabolismo , Neurotransmissores/metabolismo , Niacinamida/metabolismo , Piruvato Descarboxilase/metabolismo , Zinco/metabolismo
19.
J Liposome Res ; 30(1): 21-36, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30741049

RESUMO

Mammary gland tumour has the highest incidence rate and mortality in women, worldwide. The present study envisaged a molecularly targeted nanostructured lipid carrier (NLCs) for doxorubicin (Dox) delivery capable of inducing cellular apoptosis in mammary gland tumour. NLCs were prepared utilizing Perilla frutescens oil (54-69% ω3-fatty acid) as liquid lipid to enhance entrapment of Dox through molecular ion pairing. Biotin decorated NLCs (b-Dox-NLCs) were evaluated in vitro and in vivo. The b-Dox-NLCs showed particle size of 105.2 ± 3.5 nm, zeta potential -35 ± 2 mV, entrapment 99.15 ± 1.71%, drug content 19.67 ± 2.6 mg.g-1, biotin content 5.85 ± 0.64 µg.g-1 and drug release 98.67 ± 2.43% (facilitated by acidic microenvironment) respectively. MTT assay and Flow cytometric analysis revealed higher anti-proliferative capability of b-Dox-NLCs to force apoptosis in MCF-7 cell line vis-à-vis marketed Dox, evidenced by reactive oxygen species level and mitochondrial membrane potential mediated apoptosis. Enhanced antitumor targeting, therapeutic safety and efficacy was exhibited by b-Dox-NLCs, as investigated through tumour volume, animal survival, weight variation, cardiotoxicity and biodistribution studies in 7,12-Dimethylbenz[a]anthracene induced mammary gland tumour. Immunoblotting assay demonstrated b-Dox-NLCs downregulated anti-apoptotic proteins, i.e. bcl-2, MMP-9 while upregulated pro-apoptotic proteins, i.e. caspase-9, p16 and BAX. The experimental results suggest that biotinylated ω3-fatty acid augmented NLCs loaded with Dox are capable of inducing programmed cell death in mammary tumour and can be utilized as safe and effective delivery system with enhanced potential for mammary gland carcinoma therapy.


Assuntos
Antineoplásicos/química , Biotina/química , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/química , Ácidos Graxos/química , Lipossomos/química , Nanoestruturas/química , Animais , Antracenos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Biotina/metabolismo , Cardiotoxicidade/metabolismo , Preparações de Ação Retardada/química , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Composição de Medicamentos , Liberação Controlada de Fármacos , Feminino , Humanos , Células MCF-7 , Membranas Mitocondriais/metabolismo , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Propriedades de Superfície , Distribuição Tecidual , Microambiente Tumoral/efeitos dos fármacos
20.
Methods Mol Biol ; 2041: 137-146, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646485

RESUMO

Covalent labeling of protein with biotin (biotinylation) is a versatile technique which enables the capture and analysis of labeled protein with streptavidin-coated beads. It is particularly useful for detecting and quantifying the cell-surface expression of membrane proteins and widely used to analyze protein trafficking and the effect of mutations (particularly those which render the protein nonfunctional) on cell-surface expression. Here I describe the procedure for biotinylation and capture of cell-surface rat P2X2 receptors expressed in mammalian cells, and outline the steps in data analysis required to measure the proportion of cell-surface expressed protein of single point mutants relative to a wild-type control.


Assuntos
Biotina/metabolismo , Biotinilação/métodos , Membrana Celular/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Estreptavidina/metabolismo , Animais , Células HEK293 , Humanos , Ratos
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