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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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Synaptic AMPA receptors (AMPARs) on neuronal plasma membranes are correlated with learning and memory. Using a unique labeling and super-resolution imaging, we have visualized the nanoscale synaptic and extra-synaptic organization of native surface AMPARs for the first time in mouse brain slices as a function of brain region and tauopathy. We find that the fraction of surface AMPARs organized in synaptic clusters is two-times smaller in the hippocampus compared to the motor and somatosensory cortex. In 6 months old PS19 model of tauopathy, synaptic and extrasynaptic distributions are disrupted in the hippocampus but not in the cortex. Thus, this optimized super-resolution imaging tool allows us to observe synaptic deterioration at the onset of tauopathy before apparent neurodegeneration.
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Analyzing the quantity and distribution of molecules throughout intact biological tissue is crucial for understanding various biological phenomena. Traditional methods involving destructive extraction result in the loss of spatial information. Conversely, tissue-clearing techniques combined with fluorescence imaging have recently emerged as a powerful tool for deep tissue imaging without sacrificing spatial coverage. Key to this approach is the anchoring and labeling of targets in intact tissue. In this review, methods for anchoring and labeling proteins, lipids, carbohydrates, and small molecules are presented. Future directions include the development of activity-based probes that work in vivo and mark transient events with spatial information to enable a deeper understanding of biological phenomena.
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Colorantes Fluorescentes , Imagenología Tridimensional , Animales , Colorantes Fluorescentes/química , Imagenología Tridimensional/métodos , Humanos , Coloración y Etiquetado/métodos , Imagen Óptica/métodos , Proteínas/metabolismo , Proteínas/químicaRESUMEN
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.
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Cobre , Cobre/química , Cobre/metabolismo , Humanos , Proteínas/metabolismo , Proteínas/química , Coloración y Etiquetado/métodos , Oxidación-Reducción , Proteómica/métodos , Células HeLaRESUMEN
Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have limitations in in vivo applications. Here, we develop a click chemistry-based post-labeling method for flow cytometric measurement of glucose uptake with low background adsorption. This strategy relies on GLUT-mediated uptake of azide-tagged sugars, and subsequent intracellular labeling with a cell-permeable fluorescent reagent via a copper-free click reaction. Screening a library of azide-substituted monosaccharides, we discover 6-azido-6-deoxy-D-galactose (6AzGal) as a suitable substrate of GLUTs. 6AzGal displays glucose-like physicochemical properties and reproduces in vivo dynamics similar to 18F-FDG. Combining this method with multi-parametric immunophenotyping, we demonstrate the ability to precisely resolve metabolically-activated cells with various GLUT activities in ex vivo and in vivo models. Overall, this method provides opportunities to dissect the heterogenous metabolic landscape in complex tissue environments.
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Azidas , Glucosa , Azidas/química , Glucosa/metabolismo , Análisis de la Célula IndividualRESUMEN
Characterizing the protein constituents of a specific organelle and protein neighbors of a protein of interest (POI) is essential for understanding the function and state of the organelle and protein networks associated with the POI. Proximity labeling (PL) has emerged as a promising technology for specific and efficient spatial proteomics. Nevertheless, most enzymes adopted for PL still have limitations: APEX requires cytotoxic H2O2 for activation and thus is poor in biocompatibility for in vivo application, BioID shows insufficient labeling kinetics, and TurboID suffers from high background biotinylation. Here, we introduce a bacterial tyrosinase (BmTyr) as a new PL enzyme suitable for H2O2-free, fast (≤10 min in living cells), and low-background protein tagging. BmTyr is genetically encodable and enables subcellular-resolved PL and proteomics in living cells. We further designed a strategy of ligand-tethered BmTyr for in vivo PL, which unveiled the surrounding proteome of a neurotransmitter receptor (Grm1 and Drd2) in its resident synapse in a live mouse brain. Overall, BmTyr is one promising enzyme that can improve and expand PL-based applications and discoveries.
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Peróxido de Hidrógeno , Monofenol Monooxigenasa , Animales , Ratones , Monofenol Monooxigenasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Orgánulos/metabolismo , Proteoma/metabolismo , BiotinilaciónRESUMEN
Supramolecular chemistry currently faces the challenge of controlling nonequilibrium dynamics such as the dynamic instability of microtubules. In this study, we explored the emergence of dynamic instability through the hybridization of peptide-type supramolecular nanofibers with surfactant micelles. Using real-time confocal imaging, we discovered that the addition of micelles to nanofibers induced the simultaneous but asynchronous growth and shrinkage of nanofibers during which the total number of fibers decreased monotonically. This dynamic phenomenon unexpectedly persisted for 6 days and was driven not by chemical reactions but by noncovalent supramolecular interactions between peptide-type nanofibers and surfactant micelles. This study demonstrates a strategy for inducing autonomous supramolecular dynamics, which will open up possibilities for developing soft materials applicable to biomedicine and soft robotics.
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Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.
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Flavonoides , Quercetina , Humanos , Quercetina/farmacología , Flavonoides/farmacología , Flavonoides/química , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismoRESUMEN
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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Neuronas , Proteínas , Ratones , Animales , Indicadores y Reactivos , Ligandos , EncéfaloRESUMEN
Living cells are complex, nonequilibrium supramolecular systems capable of independently and/or cooperatively integrating multiple bio-supramolecules to execute intricate physiological functions that cannot be accomplished by individual biomolecules. These biological design strategies offer valuable insights for the development of synthetic supramolecular systems with spatially controlled hierarchical structures, which, importantly, exhibit cell-like responses and functions. The next grand challenge in supramolecular chemistry is to control the organization of multiple types of supramolecules in a single system, thus integrating the functions of these supramolecules in an orthogonal and/or cooperative manner. In this perspective, the recent progress in constructing multicomponent supramolecular soft materials through the hybridization of supramolecules, such as self-assembled nanofibers/gels and coacervates, with other functional molecules, including polymer gels and enzymes is highlighted. Moreover, results show that these materials exhibit bioinspired responses to stimuli, such as bidirectional rheological responses of supramolecular double-network hydrogels, temporal stimulus pattern-dependent responses of synthetic coacervates, and 3D hydrogel patterning in response to reaction-diffusion processes are presented. Autonomous active soft materials with cell-like responses and spatially controlled structures hold promise for diverse applications, including soft robotics with directional motion, point-of-care disease diagnosis, and tissue regeneration.
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Hidrogeles , Polímeros , Polímeros/química , Hidrogeles/químicaRESUMEN
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 , Agammaglobulinemia Tirosina Quinasa/metabolismo , Piperidinas/farmacología , Cisteína/química , Sulfanilamida , Sulfonamidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Here, we present fixation-driven chemical crosslinking of exogenous ligands, a protocol to visualize the distribution of exogenously administered small molecules in the mouse brain. We first describe the probe design of the small molecules of interest and the probe microinjection into a live mouse brain in detail. We then detail procedures for paraformaldehyde-perfusion fixation. This approach is especially useful for imaging-based evaluation of the small-molecule ligands distribution in mouse brain tissue relying on their interaction with endogenous proteins. For complete details on the use and execution of this protocol, please refer to Nonaka et al.1.
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Encéfalo , Técnicas Histológicas , Animales , Ratones , Microinyecciones , Perfusión , Encéfalo/diagnóstico por imagenRESUMEN
Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).1.
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Azidas , Aparato de Golgi , Animales , Citometría de Flujo/métodos , Mitocondrias , Fosfatidilcolinas , MamíferosRESUMEN
Coacervates, which are formed by liquid-liquid phase separation, have been extensively explored as models for synthetic cells and membraneless organelles, so their in-depth structural analysis is crucial. However, both the inner structure dynamics and formation mechanism of coacervates remain elusive. Herein, we demonstrate real-time confocal observation of a three-dimensional sponge-like network in a dipeptide-based coacervate. In situ generation of the dipeptide allowed us to capture the emergence of the sponge-like network via unprecedented membrane folding of vesicle-shaped intermediates. We also visualized dynamic fluctuation of the network, including reversible engagement/disengagement of cross-links and a stochastic network kissing event. Photoinduced transient formation of a multiphase coacervate was achieved with a thermally responsive phase transition. Our findings expand the fundamental understanding of synthetic coacervates and provide opportunities to manipulate their physicochemical properties by engineering the inner network for potential applications in development of artificial cells and life-like material fabrication.
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The sedative and anxiolytic-like activity of two coronaridine congeners, (+)-catharanthine and (-)-18-methoxycoronaridine (18-MC), was studied in male and female mice. The underlying molecular mechanism was subsequently determined by fluorescence imaging and radioligand binding experiments. The loss of righting reflex and locomotor activity results showed that both (+)-catharanthine and (-)-18-MC induce sedative effects at doses of 63 and 72 mg/kg in a sex-independent manner. At a lower dose (40 mg/kg), only (-)-18-MC induced anxiolytic-like activity in naïve mice (elevated O-maze test), whereas both congeners were effective in mice under stressful/anxiogenic conditions (light/dark transition test) and in stressed/anxious mice (novelty-suppressed feeding test), where the latter effect lasted for 24 h. Coronaridine congeners did not block pentylenetetrazole-induced anxiogenic-like activity in mice. Considering that pentylenetetrazole inhibits GABAA receptors, this result supports a role for this receptor in the activity mediated by coronaridine congeners. Functional and radioligand binding results showed that coronaridine congeners interact with a site different from that for benzodiazepines, increasing GABAA receptor affinity for GABA. Our study showed that coronaridine congeners induce sedative and anxiolytic-like activity in naïve and stressed/anxious mice in a sex-independent fashion, likely by a benzodiazepine-independent allosteric mechanism that increases GABAA receptor affinity for GABA.
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Ansiolíticos , Ratones , Masculino , Femenino , Animales , Ansiolíticos/farmacología , Hipnóticos y Sedantes/farmacología , Receptores de GABA-A/metabolismo , Pentilenotetrazol , Benzodiazepinas/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABAA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABAA and the nicotinic α7 receptor modulator (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary αßγ GABAA receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-N-phenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (ΔG â¼ -1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the α1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: We have synthesized and characterized the actions of novel acrylamide-derived compounds on the mammalian GABAA receptor. We show that the compounds have concurrent potentiating effects mediated by the classic anesthetic binding sites, and inhibitory actions that bear mechanistic resemblance to but do not share binding sites with, the inhibitory steroid pregnenolone sulfate.
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Anestésicos , Neuroesteroides , Animales , Receptores de GABA-A/metabolismo , Acrilamida/farmacología , Simulación del Acoplamiento Molecular , Sitios de Unión , Esteroides , Furanos/farmacología , Mamíferos/metabolismoRESUMEN
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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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Metabolismo de los Lípidos , Citometría de Flujo , Fosfatidilcolinas/metabolismo , Orgánulos/metabolismo , Sistemas CRISPR-Cas/genéticaRESUMEN
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 , ProteínasRESUMEN
Synthetic composite hydrogels comprising supramolecular fibers and covalent polymers have attracted considerable attention because their properties are similar to biological connective tissues. However, an in-depth analysis of the network structures has not been performed. In this study, we discovered the composite network can be categorized into four distinct patterns regarding morphology and colocalization of the components using in situ, real-time confocal imaging. Time-lapse imaging of the network formation process reveals that the patterns are governed by two factors, the order of the network formation and the interactions between the two different fibers. Additionally, the imaging studies revealed a unique composite hydrogel undergoing dynamic network remodeling on the scale of a hundred micrometers to more than one millimeter. Such dynamic properties allow for fracture-induced artificial patterning of a network three dimensionally. This study introduces a valuable guideline to the design of hierarchical composite soft materials.