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Understanding how protein-protein interaction networks in the brain give rise to cognitive functions necessitates their characterization in live animals. However, tools available for this purpose require potentially disruptive genetic modifications and lack the temporal resolution necessary to track rapid changes in vivo. Here we leverage affinity-based targeting and photocatalyzed singlet oxygen generation to identify neurotransmitter receptor-proximal proteins in the live mouse brain using only small-molecule reagents and minutes of photoirradiation. Our photooxidation-driven proximity labeling for proteome identification (named PhoxID) method not only recapitulated the known interactomes of three endogenous neurotransmitter receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), inhibitory γ-aminobutyric acid type A receptor and ionotropic glutamate receptor delta-2) but also uncovered age-dependent shifts, identifying NECTIN3 and IGSF3 as developmentally regulated AMPAR-proximal proteins in the cerebellum. Overall, this work establishes a flexible and generalizable platform to study receptor microenvironments in genetically intact specimens with an unprecedented temporal resolution.
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Copper is an essential trace element that participates in many biological processes through its unique redox cycling between cuprous (Cu+) and cupric (Cu2+) oxidation states. To elucidate the biological functions of copper, chemical biology tools that enable selective visualization and detection of copper ions and proteins in copper-rich environments are required. Herein, we describe the design of Cu+-responsive reagents based on a conditional protein labeling strategy. Upon binding Cu+, the probes generated quinone methide via oxidative bond cleavage, which allowed covalent labeling of surrounding proteins with high Cu+ selectivity. Using gel- and imaging-based analyses, the best-performing probe successfully detected changes in the concentration of labile Cu+ in living cells. Moreover, conditional proteomics analysis suggested intramitochondrial Cu+ accumulation in cells undergoing cuproptosis. Our results highlight the power of Cu+-responsive protein labeling in providing insights into the molecular mechanisms of Cu+ metabolism and homeostasis.
Assuntos
Cobre , Cobre/química , Cobre/metabolismo , Humanos , Proteínas/metabolismo , Proteínas/química , Coloração e Rotulagem/métodos , Oxirredução , Proteômica/métodos , Células HeLaRESUMO
Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.
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Neurônios , Proteínas , Camundongos , Animais , Indicadores e Reagentes , Ligantes , EncéfaloRESUMO
Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed "fixation-driven chemical cross-linking of exogenous ligands (FixEL)," which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules.
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The covalent inhibition of a target protein has gained widespread attention in the field of drug discovery. Most of the current covalent drugs utilize the high reactivity of cysteines toward modest electrophiles. However, there is a growing need for warheads that can target lysine residues to expand the range of covalently druggable proteins and to deal with emerging proteins with mutations resistant to cysteine-targeted covalent drugs. We have recently developed an N-acyl-N-alkyl sulfonamide (NASA) as a lysine-targeted electrophile. Despite its successful application, this NASA warhead suffered from instability in physiological environments, such as serum-containing medium, because of its high intrinsic reactivity. In this study, we sought to modify the structure of the NASA warhead and found that N-acyl-N-aryl sulfonamides (ArNASAs) are promising electrophiles for use in a lysine-targeted covalent inhibition strategy. We prepared a focused library of ArNASA derivatives with diverse structures and reactivity and identified several warhead candidates with suppressed hydrolysis-mediated inactivation and reduced nonspecific reactions with off-target proteins, without sacrificing the reactivity toward the target. These reaction properties enabled the improved covalent inhibition of intracellular heat shock protein 90 (HSP90) in the presence of serum and the development of the first irreversible inhibitor for ibrutinib-resistant Bruton's tyrosine kinase (BTK) bearing the C481S mutation. This study clearly demonstrated the use of a set of ArNASA warheads to create highly potent covalent drugs and highlighted the importance of enriching the current arsenal of lysine-reactive warheads.
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Lisina , Piperidinas , Lisina/química , Tirosina Quinase da Agamaglobulinemia/metabolismo , Piperidinas/farmacologia , Cisteína/química , Sulfanilamida , Sulfonamidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/químicaRESUMO
Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Metabolismo dos Lipídeos , Citometria de Fluxo , Fosfatidilcolinas/metabolismo , Organelas/metabolismo , Sistemas CRISPR-Cas/genéticaRESUMO
Organocatalyst-mediated acyl transfer reactions hold promise for selective protein labeling in biological milieu. However, they often suffer from off-target reactions and high background signals because of the requirement of high concentrations of substrates. Here, we report a new catalytic protein acylation strategy promoted by the His-tag/NiNTA interaction. The recognition-assisted activation mechanism allows efficient protein labeling even with >10-fold lower substrate concentrations than conventional reactions, thereby enabling highly selective and efficient cell-surface receptor modification in live cells.
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Oximas , ProteínasRESUMO
The endoplasmic reticulum (ER) is an essential organelle responsible for many cellular functions, including protein synthesis and folding, lipid synthesis, membrane trafficking, and storage of Ca2+. Therefore, global profiling of ER-associated proteins should be invaluable for understanding these biological processes. However, the difficulty of isolating the intact ER hampered proteome-wide analysis of ER proteins. This chapter describes a chemoproteomic approach for ER proteome analysis using ER-localizable reactive molecules (ERMs), which need neither ER fractionation nor genetic transformation. ERMs spontaneously accumulate in the ER of live cells, and the resultant high concentration of ERMs facilitates spatially limited chemical modification of ER-localized proteins with a detection/purification tag via simple intermolecular reactions. This enables the tag-mediated enrichment and quantitative analysis of the ER-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with SILAC technology.
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Proteoma , Espectrometria de Massas em Tandem , Proteoma/metabolismo , Cromatografia Líquida , Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , Fatores de Transcrição/metabolismo , Estresse do Retículo Endoplasmático , Resposta a Proteínas não DobradasRESUMO
For light-induced stabilization of microtubules (MTs) to manipulate cells, a photo-reactive diazirine group was conjugated to a Tau-derived peptide, a motif binding on the inside of MTs. Ultraviolet (UV) light irradiation induced significant stabilization of MTs via the formation of a covalent bond of the peptide and showed toxicity.
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Microtúbulos , Proteínas tau , Microtúbulos/metabolismo , Peptídeos/metabolismo , Raios Ultravioleta , Proteínas tau/metabolismoRESUMO
Membrane biology studies have revealed that in addition to providing structural support for compartment formation and membrane protein function, subcellular biomembranes are also critically involved in many biological events. To facilitate our understanding of the functions, biophysical properties and structural dynamics of organelle membranes, various exciting chemical biology tools have recently emerged. This short review aims to describe the latest molecular probes for organelle membrane studies. In particular, we will feature chemical strategies to visualize and quantitatively analyze the dynamic propeties of organelle membranes and lipids and discuss current limitations and potential future directions of this challenging research area.
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Proteínas de Membrana , Organelas , Biologia , Lipídeos/química , Proteínas de Membrana/metabolismo , Sondas Moleculares/metabolismo , Organelas/metabolismoRESUMO
Choline-containing phospholipids (CPLs), including phosphatidylcholine (PC) and sphingomyelin (SM), are the major components of mammalian cell membranes and play critical roles during a variety of cellular processes. However, intracellular dynamics of CPLs is poorly understood due to a lack of methods to trace CPL trafficking at organelle resolution. Here, we describe protocols that make it possible to fluorescently label CPLs at the targeted organelles and to monitor their movement within living cells using confocal microscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: ER-Golgi-selective labeling of azide-tagged CPLs for confocal imaging Basic Protocol 2: Mitochondria-selective labeling of azide-tagged CPLs for confocal imaging.
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Colina , Fosfolipídeos , Animais , Complexo de Golgi , Organelas , FosfatidilcolinasRESUMO
Protein-protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.
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Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Sulfonamidas/síntese química , Sulfonamidas/química , Proteína Supressora de Tumor p53/químicaRESUMO
For many years of Japan's long history, Japanese surnames have been handed down patrilineally. This study investigated relations between major surnames and Y chromosomal polymorphism among the Japanese male population. To analyze genetic phylogeny in namesakes, the Y-single nucleotide polymorphism (SNP) plus Y-short tandem repeat (STR) approach was employed. A haplogroup based on SNPs and haplotypes at 17 STR loci were typed in 567 unrelated volunteers recruited in Kanagawa, Japan. Samples covered 27 common surnames such as Satoh and Suzuki, each name having 10-55 bearers. Significant difference was found for SNP haplogroup compositions and a multidimensional scaling plot using STR haplotypes in several surname groups. By contrast, these common surnames displayed wide diversity with phylogenetic networks, suggesting that no genetic drift event has occurred in their history. In all, 22 descent clusters were found, as judgcriteria ed by ad hoc of groups within five mutational steps in the 15 STR loci with the same haplogroup. The times of the most recent common ancestor ranged from 279 to over 2577 years. According to the approximate millennium span of Japanese surname history, descent criteria are expected to be reasonable for grouping within four step-neighbors. High heterogeneity of common surnames resembles that observed for England and Spain, but not for Ireland. Our results highlight that common Japanese surnames consist of descent clusters and many singletons, reflecting a mixture of long-term bearers and short-term bearers among the population. The genetic study of this population revealed characteristic features of Japanese surnames.
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Cromossomos Humanos Y/genética , Genética Populacional , Haplótipos/genética , Filogenia , Deriva Genética , Variação Genética/genética , Genótipo , Humanos , Japão , Masculino , Repetições de Microssatélites/genética , Nomes , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+-NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.
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Histidina/química , Indicadores e Reagentes/química , Sondas Moleculares/química , Proteínas/química , Coloração e Rotulagem , Sulfonamidas/química , Células HEK293 , Histidina/metabolismo , Humanos , Indicadores e Reagentes/metabolismo , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Sondas Moleculares/metabolismo , Estrutura Molecular , Níquel/química , Níquel/metabolismo , Ácido Nitrilotriacético/química , Ácido Nitrilotriacético/metabolismo , Proteínas/metabolismo , Sulfonamidas/metabolismoRESUMO
Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.
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Autofagossomos/metabolismo , Azidas/química , Colina/análogos & derivados , Retículo Endoplasmático/metabolismo , Fosfatidilcolinas/metabolismo , Coloração e Rotulagem/métodos , Autofagossomos/ultraestrutura , Transporte Biológico , Carbocianinas/metabolismo , Química Click/métodos , Retículo Endoplasmático/ultraestrutura , Corantes Fluorescentes/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Imagem Molecular/métodos , Fosfatidilcolinas/química , Rodamina 123/metabolismo , Proteína Vermelha FluorescenteRESUMO
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. H2O2-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with H2O2 in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.
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Corantes Fluorescentes/química , Peróxido de Hidrogênio/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Imagem Óptica , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Células RAW 264.7RESUMO
Protein analysis under biological conditions is now regarded as indispensable for understanding the structure and function of proteins, in addition to in vitro studies using purified target proteins. Because there are many molecules other than the protein-of-interest (POI) under live cell conditions, selective labeling of a POI is critical to distinguish the POI from other proteins for precise analysis. Protein labeling strategies utilizing genetically encoded tags have been used in POI modification in the complex environment of live cells. However, genetic manipulation may often induce overexpression of the POI and/or perturb the cellular context, resulting in unexpected artifacts in the protein analysis. Alternatively, recent progress in chemical biology has produced two major chemical approaches for analyzing endogenous proteins under native conditions. In this review, we summarize these techniques that utilize either protein-selective chemical labeling or proteome-directed chemical modification.
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Proteínas/química , Benzenossulfonatos/química , Humanos , Ligantes , Proteínas/metabolismo , Proteoma/metabolismo , Proteômica , Piridinas/química , Coloração e RotulagemRESUMO
Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.
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Proteínas de Choque Térmico HSP90/metabolismo , Sulfonamidas/metabolismo , Descoberta de Drogas , Flavonoides/metabolismo , Células HeLa , Humanos , Ligantes , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Nitric oxide (NO) is a pleiotropic signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in cardiovascular, nervous, and immunological systems. To understand the biological functions of NO in detail, comprehensive characterization of proteins found in high-NO concentration environments is crucial. Herein, we describe the design of NO-responsive protein labeling reagents based on N-alkoxyacyl- o-phenylenediamine as an optimal reactive scaffold. The designed molecules can label proteins in murine macrophage cells in response to endogenously produced NO. The combination of NO-responsive protein labeling and liquid chromatography-tandem mass spectrometry technology allowed the characterization of the proteome under NO-generated conditions. Moreover, we demonstrated that our reagent was able to selectively mark and be used to fluorescently visualize NO-producing cells in a mixed cell culture system.