Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.756
Filtrar
1.
ACS Chem Biol ; 19(9): 2070-2080, 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-39186040

RESUMO

Interest in affinity-based probes (AfBPs) as novel tools to interrogate G protein-coupled receptors (GPCRs) has gained traction in recent years. AfBPs represent an interesting and more versatile alternative to antibodies. In the present study, we report the development and validation of AfBPs that target the intracellular allosteric pocket of CCR2, a GPCR of interest for the development of therapies targeting autoimmune and inflammatory diseases and also cancer. Owing to the two-step labeling process of these CCR2 AfBPs through the incorporation of a click handle, we were successful in applying our most efficient probe in a variety of in vitro experiments and making use of multiple different detection techniques, such as SDS-PAGE and LC/MS-based proteomics. Collectively, this novel probe shows high selectivity, versatility, and applicability. Hence, this is a valuable alternative for CCR2-targeting antibodies and other traditional tool compounds and could aid in target validation and engagement in drug discovery.


Assuntos
Receptores CCR2 , Humanos , Marcadores de Afinidade/química , Regulação Alostérica , Sítio Alostérico , Células HEK293 , Sondas Moleculares/química , Receptores CCR2/metabolismo , Receptores CCR2/química , Mercaptoetanol/química
2.
Org Biomol Chem ; 22(37): 7659-7663, 2024 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-39193651

RESUMO

A new series of multivalent gold nanoparticle probes bearing different electrophilic groups were synthesized and their affinity labeling reactivities were evaluated. The dichlorotriazine group was identified as a useful protein-reactive label, allowing selective capture of a target protein at nanomolar probe concentrations.


Assuntos
Ouro , Triazinas , Triazinas/química , Ouro/química , Marcadores de Afinidade/química , Nanopartículas Metálicas/química , Estrutura Molecular , Carboidratos/química
3.
Brief Bioinform ; 25(1)2023 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-38084920

RESUMO

Protein-ligand binding affinity (PLBA) prediction is the fundamental task in drug discovery. Recently, various deep learning-based models predict binding affinity by incorporating the three-dimensional (3D) structure of protein-ligand complexes as input and achieving astounding progress. However, due to the scarcity of high-quality training data, the generalization ability of current models is still limited. Although there is a vast amount of affinity data available in large-scale databases such as ChEMBL, issues such as inconsistent affinity measurement labels (i.e. IC50, Ki, Kd), different experimental conditions, and the lack of available 3D binding structures complicate the development of high-precision affinity prediction models using these data. To address these issues, we (i) propose Multi-task Bioassay Pre-training (MBP), a pre-training framework for structure-based PLBA prediction; (ii) construct a pre-training dataset called ChEMBL-Dock with more than 300k experimentally measured affinity labels and about 2.8M docked 3D structures. By introducing multi-task pre-training to treat the prediction of different affinity labels as different tasks and classifying relative rankings between samples from the same bioassay, MBP learns robust and transferrable structural knowledge from our new ChEMBL-Dock dataset with varied and noisy labels. Experiments substantiate the capability of MBP on the structure-based PLBA prediction task. To the best of our knowledge, MBP is the first affinity pre-training model and shows great potential for future development. MBP web-server is now available for free at: https://huggingface.co/spaces/jiaxianustc/mbp.


Assuntos
Descoberta de Drogas , Proteínas , Ligantes , Proteínas/química , Ligação Proteica , Marcadores de Afinidade
4.
Chemistry ; 28(70): e202202397, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36082977

RESUMO

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 µM phomoxanthone A.


Assuntos
ATPases Mitocondriais Próton-Translocadoras , Espectrometria de Massas em Tandem , Cromatografia Líquida , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Marcadores de Afinidade , Trifosfato de Adenosina/metabolismo
5.
Methods Mol Biol ; 2446: 345-356, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35157282

RESUMO

Antigen-binding proteins such as nanobodies are extensively used in biomedicine, diagnostics, and as tools for molecular biology. Often such applications require modification of the nanobodies with fluorophores or drugs. Here, we describe a robust method for introduction of aldehyde handles into His-tagged nanobodies and further derivatization of these proteins with hydroxylamine functionalized compounds of interest. The method allows for isolation of nanobodies containing one or more labels.


Assuntos
Anticorpos de Domínio Único , Marcadores de Afinidade , Aldeídos/química , Corantes Fluorescentes , Proteínas/química , Anticorpos de Domínio Único/química
6.
Chemistry ; 28(11): e202103925, 2022 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-35023607

RESUMO

Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.


Assuntos
Azidas , Marcadores de Fotoafinidade , Marcadores de Afinidade , Alcinos , Marcadores de Fotoafinidade/metabolismo , Proteínas
7.
Angew Chem Int Ed Engl ; 61(3): e202113515, 2022 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-34758183

RESUMO

Small-molecule binding assays to target proteins are a core component of drug discovery and development. While a number of assay formats are available, significant drawbacks still remain in cost, sensitivity, and throughput. To improve assays by capitalizing on the power of DNA sequence analysis, we have developed an assay method that combines DNA encoding with split-and-pool sample handling. The approach involves affinity labeling of DNA-linked ligands to a protein target. Critically, the labeling event assesses ligand binding and enables subsequent pooling of several samples. Application of a purifying selection on the pool for protein-labeled DNAs allows detection of ligand binding by quantification of DNA barcodes. We demonstrate the approach in both ligand displacement and direct binding formats and demonstrate its utility in determination of relative ligand affinity, profiling ligand specificity, and high-throughput small-molecule screening.


Assuntos
Marcadores de Afinidade/química , Análise de Sequência de DNA/métodos , Bibliotecas de Moléculas Pequenas/química , DNA/química , DNA/metabolismo , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Ligantes , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo
8.
Acc Chem Res ; 54(17): 3491-3503, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34427078

RESUMO

In the past two decades, a DNA-encoded chemical library (DEL or DECL) has emerged and has become a major technology platform for ligand discovery in drug discovery as well as in chemical biology research. Although based on a simple concept, i.e., encoding each compound with a unique DNA tag in a combinatorial chemical library, DEL has been proven to be a powerful tool for interrogating biological targets by accessing vast chemical space at a fraction of the cost of traditional high-throughput screening (HTS). Moreover, the recent technological advances and rapid developments of DEL-compatible reactions have greatly enhanced the chemical diversity of DELs. Today, DELs have been adopted by nearly all major pharmaceutical companies and are also gaining momentum in academia. However, this field is heavily biased toward library encoding and synthesis, and an underexplored aspect of DEL research is the selection methods. Generally, DEL selection is considered to be a massive binding assay conducted over an immobilized protein to identify the physical binders using the typical bind-wash-elute procedure. In recent years, we and other research groups have developed new approaches that can perform DEL selections in the solution phase, which has enabled the selection against complex biological targets beyond purified proteins. On the one hand, these methods have significantly widened the target scope of DELs; on the other hand, they have enabled the functional and potentially phenotypic assays of DELs beyond simple binding. An overview of these methods is provided in this Account.Our laboratory has been using DNA-programmed affinity labeling (DPAL) as the main strategy to develop new DEL selection methods. DPAL is based on DNA-templated synthesis; by using a known ligand to guide the target binding, DPAL is able to specifically establish a stable linkage between the target protein and the ligand. The DNA tag of the target-ligand conjugates serves as a programmable handle for protein characterization or hit compound decoding in the case of DEL selections. DPAL also takes advantage of the fast reaction kinetics of photo-cross-linking to achieve high labeling specificity and fidelity, especially in the selection of DNA-encoded dynamic libraries (DEDLs). DPAL has enabled DEL selections not only in buffer and cell lysates but also with complex biological systems, such as large protein complexes and live cells. Moreover, this strategy has also been employed in other biological applications, such as site-specific protein labeling, protein detection, protein profiling, and target identification. In the Account, we describe these methods, highlight their underlying principles, and conclude with perspectives of the development of the DEL technology.


Assuntos
DNA/química , Bibliotecas de Moléculas Pequenas , Marcadores de Afinidade , Descoberta de Drogas , Ligantes , Processos Fotoquímicos
9.
Acc Chem Res ; 54(16): 3215-3227, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34319695

RESUMO

The fundamental repeating unit of chromatin, the nucleosome, is composed of DNA wrapped around two copies each of four canonical histone proteins. Nucleosomes possess 2-fold pseudo-symmetry that is subject to disruption in cellular contexts. For example, the post-translational modification (PTM) of histones plays an essential role in epigenetic regulation, and the introduction of a PTM on only one of the two "sister" histone copies in a given nucleosome eliminates the inherent symmetry of the complex. Similarly, the removal or swapping of histones for variants or the introduction of a histone mutant may render the two faces of the nucleosome asymmetric, creating, if you will, a type of "Janus" bioparticle. Over the past decade, many groups have detailed the discovery of asymmetric species in chromatin isolated from numerous cell types. However, in vitro biochemical and biophysical investigation of asymmetric nucleosomes has proven synthetically challenging. Whereas symmetric nucleosomes are readily formed via a stochastic combination of their histone and DNA components, asymmetric nucleosome assembly demands the selective incorporation of a single modified/mutant histone copy alongside its wild-type counterpart.Herein we describe the chemical biology tools that we and others have developed in recent years for investigating nucleosome asymmetry. Such approaches, each with its own benefits and shortcomings, fall into five broad categories. First, we discuss affinity tag-based purification methods. These enable the assembly of theoretically any asymmetric nucleosome of interest but are frequently labor-intensive and suffer from low yields. Second, we detail transient cross-linking strategies that are amenable to the preparation of histone H3- or H4-modified/mutant asymmetric species. These yield asymmetric nucleosomes in a traceless fashion, albeit through the use of more complicated synthesis techniques. Third, we describe a synthetic biology technique based on the generation of bump-hole mutant H3 histones that selectively heterodimerize. Although currently developed only for H3 and a related isoform, this method uniquely allows for the interrogation of nucleosome asymmetry in yeast. Fourth, we outline a method for generating H2A- or H2B-modified/mutant asymmetric nucleosomes that relies on the differential DNA-histone contact strength inherent in the Widom 601 DNA sequence. This technique involves the initial formation of hexasomes which are then complemented with distinct H2A/H2B dimers. Finally, we review an approach that utilizes split intein technology to isolate asymmetric H2A- or H2B-modified/mutant nucleosomes. This method shares steps in common with the former but exploits tagged, intein-fused dimers for the facile purification of asymmetric products.Throughout the Account, we highlight various biological questions that drove the development of these methods and ultimately were answered by them. Though each technique has its own shortcomings, collectively these chemical biology tools provide a means to biochemically interrogate a plethora of asymmetric nucleosome species. We conclude with a discussion of remaining challenges, particularly that of endogenous asymmetric nucleosome detection.


Assuntos
Nucleossomos/metabolismo , Marcadores de Afinidade , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/química
10.
Int J Mol Sci ; 22(14)2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34298982

RESUMO

Engineering of biomimetic motives have emerged as promising approaches to improving cells' binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface's receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5ß1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.


Assuntos
Marcadores de Afinidade , Materiais Biomiméticos , Fibronectinas , Oligopeptídeos , Fragmentos de Peptídeos , Proteínas Recombinantes de Fusão/química , Adsorção , Adesão Celular , Materiais Revestidos Biocompatíveis , Escherichia coli , Matriz Extracelular/química , Vetores Genéticos , Integrina alfa5beta1/metabolismo , Ligantes , Espectrometria de Massas , Poliestirenos , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
11.
Bioprocess Biosyst Eng ; 44(10): 2129-2139, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34036441

RESUMO

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.


Assuntos
Endopeptidases/metabolismo , Corpos de Inclusão/enzimologia , Mutação , Marcadores de Afinidade , Sequência de Aminoácidos , Cromatografia de Afinidade , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/isolamento & purificação , Enzimas Imobilizadas/metabolismo
12.
Angew Chem Int Ed Engl ; 60(24): 13542-13547, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-33768725

RESUMO

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.


Assuntos
Acetiltransferases/antagonistas & inibidores , Marcadores de Afinidade/química , Bibliotecas de Moléculas Pequenas/química , Acetiltransferases/metabolismo , Sítios de Ligação , Humanos , Cinética , Luz , Palmitoil Coenzima A/antagonistas & inibidores , Palmitoil Coenzima A/metabolismo , Piridinas/química , Piridinas/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade
13.
Sci Signal ; 14(675)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758061

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Sinalização do Cálcio/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADP/análogos & derivados , SARS-CoV-2/fisiologia , Marcadores de Afinidade , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Química Click/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transcriptoma , Internalização do Vírus
14.
Chem Commun (Camb) ; 57(31): 3769-3772, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33730131

RESUMO

Membrane proteins are important drug targets; however, ligand discovery for membrane proteins is highly challenging due to their hydrophobic nature. We show that membrane proteins may be specifically labelled with a DNA tag by DNA-programmed affinity labelling (DPAL), thereby enabling the screening of chemical compounds against membrane proteins directly on live cells.


Assuntos
Marcadores de Afinidade/química , DNA/análise , Proteínas de Membrana/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química
15.
ACS Chem Biol ; 16(2): 404-413, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33543920

RESUMO

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.


Assuntos
Marcadores de Afinidade/química , Azidas/química , Reagentes de Ligações Cruzadas/química , Diazometano/análogos & derivados , Hidrocarbonetos Clorados/química , Proteômica/métodos , Marcadores de Afinidade/efeitos da radiação , Azidas/efeitos da radiação , Cromatografia Líquida , Reagentes de Ligações Cruzadas/efeitos da radiação , Dasatinibe/análogos & derivados , Dasatinibe/farmacologia , Dasatinibe/efeitos da radiação , Diazometano/efeitos da radiação , Histona Desacetilases/análise , Histona Desacetilases/química , Humanos , Hidrocarbonetos Clorados/efeitos da radiação , Hidrolases/química , Células K562 , Espectrometria de Massas , Propranolol/análogos & derivados , Propranolol/farmacologia , Propranolol/efeitos da radiação , Proteínas Quinases/análise , Proteínas Quinases/química , Receptores Adrenérgicos alfa 2/análise , Receptores Adrenérgicos alfa 2/química , Raios Ultravioleta , Vorinostat/análogos & derivados , Vorinostat/farmacologia , Vorinostat/efeitos da radiação
16.
Neurosci Lett ; 750: 135766, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33639221

RESUMO

Ischemic stroke is one of the major diseases that cause mortality and morbidity of human beings, but there is still lack of effective treatment and prevention. We found that 2-(2-Benzofuranyl)-2-Imidazoline (2-BFI) is potently protective against stroke and acute inflammatory immune disease. Moreover, the mammalian target of rapamycin (mTOR) signaling contributes effectively to the modulation of post-stroke neuroinflammatory response. However, whether the protection of 2-BFI against ischemic injury is through mTOR-mediated neuroinflammatory response remains unestablished. Here, we used 2-BFI to treat ischemic rats induced by distal middle cerebral artery occlusion (dMCAO). We found that 2-BFI administration after dMCAO improved the neurological deficits and decreased the infarct volume. 2-BFI reduced phosphorylation of mTOR and p70S6, increased IL-10 and TGF-ß, and decreased IFN-γ levels in ischemic rats. Our results demonstrated that 2-BFI attenuates ischemic injury by inhibiting the activation of mTOR signaling and modulating neuroinflammation after stroke in rats.


Assuntos
Marcadores de Afinidade/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Benzofuranos/uso terapêutico , Imidazóis/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismo , Marcadores de Afinidade/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Benzofuranos/farmacologia , Imidazóis/farmacologia , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
17.
Methods Mol Biol ; 2178: 107-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33128747

RESUMO

The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific toward particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications in proteins and peptides widen the application of affinity ligand-tag receptors pairs toward universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely the affinity tags and receptors employed on the production of recombinant fusion proteins, and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.


Assuntos
Marcadores de Afinidade/química , Cromatografia de Afinidade , Proteínas Recombinantes de Fusão , Biotecnologia , Proteômica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação
18.
Mass Spectrom Rev ; 40(3): 215-235, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32519381

RESUMO

GTP-binding proteins are among the most important enzyme families that are involved in a plethora of biological processes. However, owing to the enormous diversity of the nucleotide-binding protein family, comprehensive analyses of the expression level, structure, activity, and regulatory mechanisms of GTP-binding proteins remain challenging with the use of conventional approaches. The many advances in mass spectrometry (MS) instrumentation and data acquisition methods, together with a variety of enrichment approaches in sample preparation, render MS a powerful tool for the comprehensive characterizations of the activities and expression levels of various GTP-binding proteins. We review herein the recent developments in the application of MS-based techniques, together with general and widely used affinity enrichment approaches, for the proteome-wide and targeted capture, identification, and quantification of GTP-binding proteins. The working principles, advantages, and limitations of various strategies for profiling the expression level, activity, posttranslational modifications, and interactome of GTP-binding proteins are discussed. It can be envisaged that future applications of MS-based proteomics will lead to a better understanding about the roles of GTP-binding proteins in different biological processes and human diseases. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.


Assuntos
Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/metabolismo , Espectrometria de Massas/métodos , Proteômica/métodos , Marcadores de Afinidade/química , Animais , Biotinilação , Eletroforese/métodos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Guanina/química , Humanos , Processamento de Proteína Pós-Traducional
19.
PLoS One ; 15(12): e0243286, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33270761

RESUMO

In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.


Assuntos
Apoptose/fisiologia , Corantes Fluorescentes/química , Coloração e Rotulagem/métodos , Marcadores de Afinidade/química , Anexina A5/química , Anexina A5/metabolismo , Células Sanguíneas/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Humanos , Neoplasias/metabolismo , Proteínas/química , Proteínas/metabolismo , Tecnologia
20.
Sci Rep ; 10(1): 20253, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219273

RESUMO

5-methylcytosine is the most studied DNA epigenetic modification, having been linked to diverse biological processes and disease states. The elucidation of cytosine demethylation has drawn added attention the three additional intermediate modifications involved in that pathway-5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine-each of which may have distinct biological roles. Here, we extend a modular method for labeling base modifications in DNA to recognize all four bases involved in demethylation. We demonstrate both differential insertion of a single affinity tag (biotin) at the precise position of target elements and subsequent repair of the nicked phosphate backbone that remains following the procedure. The approach enables affinity isolation and downstream analyses without inducing widespread damage to the DNA.


Assuntos
Marcadores de Afinidade , Citosina/metabolismo , Metilação de DNA
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA