RESUMO
The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 µM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.
Assuntos
Cafeína , Glutationa , Receptores de Detecção de Cálcio , Receptores de Detecção de Cálcio/metabolismo , Humanos , Regulação Alostérica/efeitos dos fármacos , Cafeína/química , Cafeína/farmacologia , Cafeína/metabolismo , Glutationa/metabolismo , Glutationa/química , Cálcio/metabolismo , Marcadores de Fotoafinidade/química , Sítios de Ligação , Células HEK293 , Ligantes , Cinacalcete/química , Cinacalcete/farmacologiaRESUMO
The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.
Assuntos
Benzofenonas , Peptídeo Sintases , Marcadores de Fotoafinidade , Benzofenonas/química , Benzofenonas/síntese química , Benzofenonas/farmacologia , Benzofenonas/metabolismo , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/síntese química , Peptídeo Sintases/metabolismo , Peptídeo Sintases/química , Estrutura MolecularRESUMO
Gangliosides, sialic acid bearing glycosphingolipids, are components of the outer leaflet of plasma membranes of all vertebrate cells. They contribute to cell regulation by interacting with proteins in their own membranes (cis) or their extracellular milieu (trans). As amphipathic membrane constituents, gangliosides present challenges for identifying their ganglioside protein interactome. To meet these challenges, we synthesized bifunctional clickable photoaffinity gangliosides, delivered them to plasma membranes of cultured cells, then captured and identified their interactomes using proteomic mass spectrometry. Installing probes on ganglioside lipid and glycan moieties, we captured cis and trans ganglioside-protein interactions. Ganglioside interactomes varied with the ganglioside structure, cell type, and site of the probe (lipid or glycan). Gene ontology revealed that gangliosides engage with transmembrane transporters and cell adhesion proteins including integrins, cadherins, and laminins. The approach developed is applicable to other gangliosides and cell types, promising to provide insights into molecular and cellular regulation by gangliosides.
Assuntos
Química Click , Gangliosídeos , Gangliosídeos/química , Gangliosídeos/metabolismo , Humanos , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/síntese química , Sondas Moleculares/química , Sondas Moleculares/síntese química , Membrana Celular/metabolismo , Membrana Celular/químicaRESUMO
Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan-protein networks and is poised to help elucidate the glycan-protein interactome, providing novel targets for therapeutic intervention.
Assuntos
Marcadores de Fotoafinidade , Polissacarídeos , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/metabolismo , Polissacarídeos/metabolismo , Polissacarídeos/química , Humanos , Ligação Proteica , Proteínas/metabolismo , Proteínas/química , Ligantes , Animais , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismoRESUMO
Understanding the mechanisms of drug action in malarial parasites is crucial for the development of new drugs to combat infection and to counteract drug resistance. Proteomics is a widely used approach to study host-pathogen systems and to identify drug protein targets. Plasmodione is an antiplasmodial early-lead drug exerting potent activities against young asexual and sexual blood stages inâ vitro with low toxicity to host cells. To elucidate its molecular mechanisms, an affinity-based protein profiling (AfBPP) approach was applied to yeast and P. falciparum proteomes. New (pro-) AfBPP probes based on the 3-benz(o)yl-6-fluoro-menadione scaffold were synthesized. With optimized conditions of both photoaffinity labeling and click reaction steps, the AfBPP protocol was then applied to a yeast proteome, yielding 11 putative drug-protein targets. Among these, we found four proteins associated with oxidoreductase activities, the hypothesized type of targets for plasmodione and its metabolites, and other proteins associated with the mitochondria. In Plasmodium parasites, the MS analysis revealed 44 potential plasmodione targets that need to be validated in further studies. Finally, the localization of a 3-benzyl-6-fluoromenadione AfBPP probe was studied in the subcellular structures of the parasite at the trophozoite stage.
Assuntos
Antimaláricos , Plasmodium falciparum , Proteômica , Vitamina K 3 , Antimaláricos/farmacologia , Antimaláricos/química , Plasmodium falciparum/efeitos dos fármacos , Vitamina K 3/farmacologia , Vitamina K 3/química , Vitamina K 3/metabolismo , Proteínas de Protozoários/metabolismo , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/farmacologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteoma/análise , Proteoma/metabolismo , Estrutura MolecularRESUMO
Photoaffinity probes are routinely utilized to identify proteins that interact with small molecules. However, despite this common usage, resolving the specific sites of these interactions remains a challenge. Here we developed a chemoproteomic workflow to determine precise protein binding sites of photoaffinity probes in cells. Deconvolution of features unique to probe-modified peptides, such as their tendency to produce chimeric spectra, facilitated the development of predictive models to confidently determine labeled sites. This yielded an expansive map of small-molecule binding sites on endogenous proteins and enabled the integration with multiplexed quantitation, increasing the throughput and dimensionality of experiments. Finally, using structural information, we characterized diverse binding sites across the proteome, providing direct evidence of their tractability to small molecules. Together, our findings reveal new knowledge for the analysis of photoaffinity probes and provide a robust method for high-resolution mapping of reversible small-molecule interactions en masse in native systems.
Assuntos
Marcadores de Fotoafinidade , Bibliotecas de Moléculas Pequenas , Sítios de Ligação , Humanos , Marcadores de Fotoafinidade/química , Bibliotecas de Moléculas Pequenas/química , Ligação Proteica , Proteômica/métodos , Proteoma/metabolismo , Proteínas/química , Proteínas/metabolismo , Peptídeos/química , Peptídeos/metabolismoRESUMO
As a vital project of forward chemical genetic research, target deconvolution aims to identify the molecular targets of an active hit compound. Chemoproteomics, either with chemical probe-facilitated target enrichment or probe-free, provides a straightforward and effective approach to profile the target landscape and unravel the mechanisms of action. Canonical methods rely on chemical probes to enable target engagement, enrichment, and identification, whereas click chemistry and photoaffinity labeling techniques improve the efficiency, sensitivity, and spatial accuracy of target recognition. In comparison, recently developed probe-free methods detect protein-ligand interactions without the need to modify the ligand molecule. This review provides a comprehensive overview of different approaches and recent advancements for target identification and highlights the significance of chemoproteomics in investigating biological processes and advancing drug discovery processes.
Assuntos
Descoberta de Drogas , Marcadores de Fotoafinidade , Ligantes , Descoberta de Drogas/métodos , Marcadores de Fotoafinidade/química , Química ClickRESUMO
Target identification studies are a major hurdle in probe and drug discovery pipelines due to the need to chemically modify small molecules of interest, which can be time intensive and have low throughput. Here, we describe a versatile and scalable method for attaching chemical moieties to a small molecule, isocyanate-mediated chemical tagging (IMCT). By preparation of a template resin with an isocyanate capture group and a cleavable linker, nucleophilic groups on small molecules can be modified with an enforced one-to-one stoichiometry. We demonstrate a small molecule substrate scope that includes primary and secondary amines, thiols, phenols, benzyl alcohols, and primary alcohols. Cheminformatic analyses predict that IMCT is reactive with more than 25% of lead-like compounds in publicly available databases. To demonstrate that the method can produce biologically active molecules, we generated FKBP12 photoaffinity labeling (PAL) compounds with a wide range of affinities and showed that purified and crude cleavage products can bind to and label FKBP12. This method could be used to rapidly modify small molecules for many applications, including the synthesis of PAL probes, fluorescence polarization probes, pull-down probes, and degraders.
Assuntos
Isocianatos , Proteína 1A de Ligação a Tacrolimo , Descoberta de Drogas , Compostos de Sulfidrila , Marcadores de Fotoafinidade/químicaRESUMO
Understanding how small molecules bind to specific protein complexes in living cells is critical to understanding their mechanism-of-action. Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules would provide advantages over target-focused approaches, including the ability to detect previously unknown ligand targets and complexes. However, there are few current methods for unbiased profiling of small molecule interactomes. To address this, we envisioned a technology that would combine the sensitivity and live-cell compatibility of proximity labelling coupled to mass spectrometry, with the specificity and unbiased nature of chemoproteomics. In this manuscript, we describe the BioTAC system, a small-molecule guided proximity labelling platform that can rapidly identify both direct and complexed small molecule binding proteins. We benchmark the system against µMap, photoaffinity labelling, affinity purification coupled to mass spectrometry and proximity labelling coupled to mass spectrometry datasets. We also apply the BioTAC system to provide interactome maps of Trametinib and analogues. The BioTAC system overcomes a limitation of current approaches and supports identification of both inhibitor bound and molecular glue bound complexes.
Assuntos
Biotina , Proteínas , Proteínas/metabolismo , Cromatografia de Afinidade , Espectrometria de Massas/métodos , Marcadores de Fotoafinidade/químicaRESUMO
Glycan recognition by glycan-binding proteins is central to the biology of all living organisms. The efficient capture and characterization of relatively weak non-covalent interactions remains an important challenge in various fields of research. Photoaffinity labeling strategies can create covalent bonds between interacting partners, and photoactive scaffolds such as benzophenone, diazirines and aryl azides have proved widely useful. Since their first introduction, relatively few improvements have been advanced and products of photoaffinity labeling remain difficult to detect. We report a fluorinated azido-coumarin scaffold which enables photolabeling under fast and mild activation, and which can leave a fluorescent tag on crosslinked species. Coupling this scaffold to an α-fucoside, we demonstrate fluorogenic photolabeling of glycan-protein interactions over a wide range of affinities. We expect this strategy to be broadly applicable to other chromophores and we envision that such "fluoro-crosslinkers" could become important tools for the traceable capture of non-covalent binding events.
Assuntos
Proteínas de Transporte , Proteínas , Proteínas/química , Marcadores de Fotoafinidade/química , Cumarínicos , Azidas/metabolismo , PolissacarídeosRESUMO
Target identification involves deconvoluting the protein target of a pharmacologically active, small-molecule ligand, a process that is critical for early drug discovery yet technically challenging. Photoaffinity labelling strategies have become the benchmark for small-molecule target deconvolution, but covalent protein capture requires the use of high-energy ultraviolet light, which can complicate downstream target identification. Thus, there is a strong demand for alternative technologies that allow for controlled activation of chemical probes to covalently label their protein target. Here we introduce an electroaffinity labelling platform that leverages the use of a small, redox-active diazetidinone functional group to enable chemoproteomic-based target identification of pharmacophores within live cell environments. The underlying discovery to enable this platform is that the diazetidinone can be electrochemically oxidized to reveal a reactive intermediate useful for covalent modification of proteins. This work demonstrates the electrochemical platform to be a functional tool for drug-target identification.
Assuntos
Descoberta de Drogas , Proteínas , Proteínas/metabolismo , Marcadores de Fotoafinidade/química , Ligantes , FarmacóforoRESUMO
Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.
Assuntos
Ácido Aspártico Endopeptidases , Catepsina D , Pepstatinas , Marcadores de Fotoafinidade , Humanos , Ácido Aspártico Endopeptidases/química , Catepsina D/química , Diazometano , Pepstatinas/química , Pepstatinas/farmacologia , Marcadores de Fotoafinidade/química , Proteína Sequestossoma-1/químicaRESUMO
In materials (polymer) science and medicinal chemistry, heteroaromatic derivatives play the role of the central skeleton in development of novel devices and discovery of new drugs. On the other hand, (3-trifluoromethyl)phenyldiazirine (TPD) is a crucial chemical method for understanding biological processes such as ligand-receptor, nucleic acid-protein, lipid-protein, and protein-protein interactions. In particular, use of TPD has increased in recent materials science to create novel electric and polymer devices with comparative ease and reduced costs. Therefore, a combination of heteroaromatics and (3-trifluoromethyl)diazirine is a promising option for creating better materials and elucidating the unknown mechanisms of action of bioactive heteroaromatic compounds. In this review, a comprehensive synthesis of (3-trifluoromethyl)diazirine-substituted heteroaromatics is described.
Assuntos
Ácidos Nucleicos , Marcadores de Fotoafinidade , Marcadores de Fotoafinidade/química , Diazometano/química , Química Farmacêutica , Proteínas/químicaRESUMO
Photoaffinity ligands are best known as tools used to identify the specific binding sites of drugs to their molecular targets. However, photoaffinity ligands have the potential to further define critical neuroanatomic targets of drug action. In the brains of WT male mice, we demonstrate the feasibility of using photoaffinity ligands in vivo to prolong anesthesia via targeted yet spatially restricted photoadduction of azi-m-propofol (aziPm), a photoreactive analog of the general anesthetic propofol. Systemic administration of aziPm with bilateral near-ultraviolet photoadduction in the rostral pons, at the border of the parabrachial nucleus and locus coeruleus, produced a 20-fold increase in the duration of sedative and hypnotic effects compared with control mice without UV illumination. Photoadduction that missed the parabrachial-coerulean complex also failed to extend the sedative or hypnotic actions of aziPm and was indistinguishable from nonadducted controls. Paralleling the prolonged behavioral and EEG consequences of on target in vivo photoadduction, we conducted electrophysiologic recordings in rostral pontine brain slices. Using neurons within the locus coeruleus to further highlight the cellular consequences of irreversible aziPm binding, we demonstrate transient slowing of spontaneous action potentials with a brief bath application of aziPm that becomes irreversible on photoadduction. Together, these findings suggest that photochemistry-based strategies are a viable new approach for probing CNS physiology and pathophysiology.SIGNIFICANCE STATEMENT Photoaffinity ligands are drugs capable of light-induced irreversible binding, which have unexploited potential to identify the neuroanatomic sites of drug action. We systemically administer a centrally acting anesthetic photoaffinity ligand in mice, conduct localized photoillumination within the brain to covalently adduct the drug at its in vivo sites of action, and successfully enrich irreversible drug binding within a restricted 250 µm radius. When photoadduction encompassed the pontine parabrachial-coerulean complex, anesthetic sedation and hypnosis was prolonged 20-fold, thus illustrating the power of in vivo photochemistry to help unravel neuronal mechanisms of drug action.
Assuntos
Anestésicos Intravenosos , Encéfalo , Hipnose , Hipnóticos e Sedativos , Ligantes , Marcadores de Fotoafinidade , Propofol , Animais , Masculino , Camundongos , Neurônios Adrenérgicos/efeitos dos fármacos , Anestesia Intravenosa , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/efeitos da radiação , Eletrocorticografia , Eletroencefalografia , Hipnose/métodos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/química , Hipnóticos e Sedativos/farmacologia , Hipnóticos e Sedativos/efeitos da radiação , Locus Cerúleo/citologia , Locus Cerúleo/efeitos dos fármacos , Locus Cerúleo/metabolismo , Locus Cerúleo/efeitos da radiação , Camundongos Endogâmicos C57BL , Núcleos Parabraquiais/efeitos dos fármacos , Núcleos Parabraquiais/metabolismo , Núcleos Parabraquiais/efeitos da radiação , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/efeitos da radiação , Propofol/administração & dosagem , Propofol/análogos & derivados , Propofol/farmacologia , Propofol/efeitos da radiação , Fatores de Tempo , Raios Ultravioleta , Anestésicos Intravenosos/administração & dosagem , Anestésicos Intravenosos/química , Anestésicos Intravenosos/farmacologia , Anestésicos Intravenosos/efeitos da radiaçãoRESUMO
Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.
Assuntos
Ciclobutanos , Diazometano , Diazometano/química , Alcinos , Marcadores de Fotoafinidade/química , Ligantes , Proteínas de MembranaRESUMO
Photoaffinity labeling is a powerful technique to interrogate drug-protein interactions in native cellular environments. Photo-cross-linkers are instrumental for this technique. However, the introduction of unnatural photo-cross-linkers may significantly reduce the bioactivity of the drug, thus impairing the chemoproteomic outcomes. Herein, we developed a common pharmacophore, isoxazole, into a natively embedded photo-cross-linker for chemoproteomics, which minimally perturbs the drug structure. The photo-cross-linking reactions of the isoxazole were thoroughly investigated for the first time. Functionalized isoxazoles were then designed and applied to protein labeling, demonstrating the superior photo-cross-linking efficiency. Subsequently, two isoxazole-based drugs, Danazol and Luminespib, were employed in chemoproteomic studies, revealing their potential cellular targets. These results provide valuable strategies for future chemoproteomic study and drug development.
Assuntos
Marcadores de Fotoafinidade , Proteínas , Marcadores de Fotoafinidade/química , Proteínas/química , Isoxazóis , Reagentes de Ligações Cruzadas/químicaRESUMO
Small molecules remain an important category of therapeutic agents. Their binding to different proteins can lead to both desired and undesired biological effects. Identification of the proteins that a drug binds to has become an important step in drug development because it can lead to safer and more effective drugs. Parent bioactive molecules can be converted to appropriate probes that allow for visualization and identification of their target proteins. Typically, these probes are designed and synthesized utilizing some or all of five major tools; a photoactivatable group, a reporter tag, a linker, an affinity tag, and a bioorthogonal handle. This review covers two of the most challenging tools, photoactivation and bioorthogonal ligation. We provide a historical and theoretical background along with synthetic routes to prepare them. In addition, the review provides comparative analyses of the available tools that can assist decision making when designing such probes. A survey of most recent literature reports is included as well to identify recent trends in the field.
Assuntos
Marcadores de Fotoafinidade , Proteínas , Animais , Marcadores de Fotoafinidade/química , Proteínas/químicaRESUMO
2'3'-cyclic GMP-AMP (2'3'-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2'3'-cGAMP, it is important to find out whether 2'3'-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2'3'-cGAMP-based photoaffinity probes that capture and isolate 2'3'-cGAMP-binding proteins. These probes enable the identification of some potential 2'3'-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2'3'-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2'3'-cGAMP interactome, discover unknown functions of 2'3'-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.
Assuntos
Nucleotídeos Cíclicos/química , Fator 1 de Elongação de Peptídeos/análise , Marcadores de Fotoafinidade/química , Linhagem Celular , Humanos , Estrutura Molecular , Nucleotídeos Cíclicos/imunologia , Fator 1 de Elongação de Peptídeos/imunologiaRESUMO
The nucleotides diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) are formed in prokaryotic and eukaryotic cells. Since their concentrations increase significantly upon cellular stress, they are considered to be alarmones triggering stress adaptive processes. However, their cellular roles remain elusive. To elucidate the proteome-wide interactome of Ap3A and Ap4A and thereby gain insights into their cellular roles, we herein report the development of photoaffinity-labeling probes and their employment in chemical proteomics. We demonstrate that the identified ApnA interactors are involved in many fundamental cellular processes including carboxylic acid and nucleotide metabolism, gene expression, various regulatory processes and cellular response mechanisms and only around half of them are known nucleotide interactors. Our results highlight common functions of these ApnAs across the domains of life, but also identify those that are different for Ap3A or Ap4A. This study provides a rich source for further functional studies of these nucleotides and depicts useful tools for characterization of their regulatory mechanisms in cells.
Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Proteômica , Trifosfato de Adenosina/metabolismo , Fosfatos de Dinucleosídeos/química , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Humanos , L-Lactato Desidrogenase/metabolismo , Fosfoglicerato Quinase/metabolismo , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.