RESUMEN
Protein mutations alter protein-protein interactions that can lead to a number of illnesses. Mutations in lamin A (LMNA) have been reported to cause laminopathies. However, the proteins associated with the LMNA mutation have mostly remained unexplored. Herein, a new chemical tool for proximal proteomics is reported, developed by a combination of proximity chemical tagging and a bio-orthogonal supramolecular latching based on cucurbit[7]uril (CB[7])-based host-guest interactions. As this host-guest interaction acts as a noncovalent clickable motif that can be unclicked on-demand, this new chemical tool is exploited for reliable detection of the proximal proteins of LMNA and its mutant that causes laminopathic dilated cardiomyopathy (DCM). Most importantly, a comparison study reveals, for the first time, mutant-dependent alteration in LMNA proteomic environments, which allows to identify putative laminopathic DCM-linked proteins including FOXJ3 and CELF2. This study demonstrates the feasibility of this chemical tool for reliable proximal proteomics, and its immense potential as a new research platform for discovering biomarkers associated with protein mutation-linked diseases.
Asunto(s)
Cardiomiopatía Dilatada , Neoplasias Cutáneas , Humanos , Proteómica , Cardiomiopatía Dilatada/complicaciones , Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/genética , Mutación , Biomarcadores , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas CELF/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
Here we report synthetic monosaccharide channels built with shape-persistent organic cages, porphyrin boxes (PBs), that allow facile transmembrane transport of glucose and fructose through their windows. PBs show a much higher transport rate for glucose and fructose over disaccharides such as sucrose, as evidenced by intravesicular enzyme assays and molecular dynamics simulations. The transport rate can be modulated by changing the length of the alkyl chains decorating the cage windows. Insertion of a linear pillar ligand into the cavity of PBs blocks the monosaccharide transport. In vitro cell experiment shows that PBs transport glucose across the living-cell membrane and enhance cell viability when the natural glucose transporter GLUT1 is blocked. Time-dependent live-cell imaging and MTT assays confirm the cyto-compatibility of PBs. The monosaccharide-selective transport ability of PBs is reminiscent of natural glucose transporters (GLUTs), which are crucial for numerous biological functions.
Asunto(s)
Fructosa , Glucosa , Glucosa/metabolismo , Monosacáridos , Proteínas de Transporte de Monosacáridos/metabolismo , Transporte Biológico , Proteínas Facilitadoras del Transporte de la GlucosaRESUMEN
Aggregation of amyloidogenic proteins causing neurodegenerative diseases is an uncontrollable and contagious process that is often associated with lipid membranes in a highly complex physiological environment. Although several approaches using natural cells and membrane models have been reported, systematic investigations focusing on the association with the membranes are highly challenging, mostly because of the lack of proper molecular tools. Here, we report a new supramolecular approach using a synthetic cell system capable of controlling the initiation of protein aggregation and mimicking various conditions of lipid membranes, thereby enabling systematic investigations of membrane-dependent effects on protein aggregation by visualization. Extending this strategy through concurrent use of synthetic cells and natural cells, we demonstrate the potential of this approach for systematic and in-depth studies on interrogating inter- and intracellularly transmittable protein aggregation. Thus, this new approach offers opportunities for gaining insights into the pathological implications of contagious protein aggregation associated with membranes for neurotoxicity.
Asunto(s)
Células Artificiales , Proteínas Amiloidogénicas/metabolismo , Membrana Celular/metabolismo , Humanos , Lípidos , Agregado de Proteínas , Agregación Patológica de ProteínasRESUMEN
The identification of each cell type is essential for understanding multicellular communities. Antibodies set as biomarkers have been the main toolbox for cell-type recognition, and chemical probes are emerging surrogates. Herein we report the first small-molecule probe, CDgB, to discriminate B lymphocytes from T lymphocytes, which was previously impossible without the help of antibodies. Through the study of the origin of cell specificity, we discovered an unexpected novel mechanism of membrane-oriented live-cell distinction. B cells maintain higher flexibility in their cell membrane than T cells and accumulate the lipid-like probe CDgB more preferably. Because B and T cells share common ancestors, we tracked the cell membrane changes of the progenitor cells and disclosed the dynamic reorganization of the membrane properties over the lymphocyte differentiation progress. This study casts an orthogonal strategy for the small-molecule cell identifier and enriches the toolbox for live-cell distinction from complex cell communities.
Asunto(s)
Linfocitos B/citología , Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Linfocitos T/citología , Animales , Linfocitos B/química , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Membrana Celular/química , Citometría de Flujo , Lipidómica , Ratones , Linfocitos T/química , Linfocitos T/inmunologíaRESUMEN
Hierarchical self-assembly of building blocks over multiple length scales is ubiquitous in living organisms. Microtubules are one of the principal cellular components formed by hierarchical self-assembly of nanometer-sized tubulin heterodimers into protofilaments, which then associate to form micron-length-scale, multi-stranded tubes. This peculiar biological process is now mimicked with a fully synthetic molecule, which forms a 1:1 host-guest complex with cucurbit[7]uril as a globular building block, and then polymerizes into linear poly-pseudorotaxanes that associate laterally with each other in a self-shape-complementary manner to form a tubular structure with a length over tens of micrometers. Molecular dynamic simulations suggest that the tubular assembly consists of eight poly-pseudorotaxanes that wind together to form a 4.5â nm wide multi-stranded tubule.
Asunto(s)
Microtúbulos/química , Polímeros/química , Hidrocarburos Aromáticos con Puentes/química , Imidazoles/química , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Rotaxanos/químicaRESUMEN
Herein we report a facile transformation of hydroxylated cucurbit[n]uril (CB[n], n = 6 and 7) to other functionality-conjugated CB[n]s by nucleophilic substitution of the hydroxyl group with a wide range of nitriles and alcohols. The reaction proceeds efficiently via generation of a superelectrophilic carbocation on the CB framework from hydroxylated CB[n]s under superacidic conditions. One of the resulting CB[n] derivatives with reactive functionality, monocarboxylated CB[7], is efficiently conjugated to an enzyme (horseradish peroxidase, HRP) by amide coupling. This provides a CB[7]-conjugated functional biomaterial (CB[7]-HRP) that selectively detects proteins labeled with a guest, adamantylammonium (AdA), based on bioorthogonal high-affinity host-guest interactions between CB[7] and AdA. We demonstrated the potential of overcoming the limitations in preparing reactive functional CB[n] derivatives, enabling the exploration of novel bioapplications of CB[n]-based host-guest chemistry with new CB[n]-conjugated functional materials.
RESUMEN
Here we report the endocytosis and excretion pathways of two different dye-conjugated cucurbit[7]urils, (cyanine 3-conjugated CB[7] and rhodamine X-conjugated CB[7]), which have great potential as molecular probes for live cell imaging. The dye-CB[7]s are translocated into live cells (human breast carcinoma cells, MCF-7) via multiple pathways, predominantly by clathrin-mediated endocytosis, and excreted from cells via lysosome-associated exocytosis. Interestingly, the CB[7] moiety has a substantial influence on the uptake and excretion pathways. These findings may widen the applications of the dyes conjugated to CB[7] and assist in the design of new molecular probes for live cell imaging.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/metabolismo , Carbocianinas/metabolismo , Endocitosis/fisiología , Exocitosis/fisiología , Colorantes Fluorescentes/metabolismo , Imidazoles/metabolismo , Rodaminas/metabolismo , Hidrocarburos Aromáticos con Puentes/química , Carbocianinas/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Imidazoles/química , Lisosomas/fisiología , Células MCF-7 , Rodaminas/químicaRESUMEN
In this Commentary, we discuss cucurbit[7]uril-based ultrastable artificial binding pairs as a supramolecular latching system and how we envision this becoming important tools in proteomics. The limitations of current proteomic techniques are described with an emphasis on the lack of tools to answer questions about the complex and dynamic nature of the proteome. Our thoughts as to how artificial ultrastable binding pairs may be able to address these questions are given especially when they are combined with existing methods.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/química , Imidazoles/química , Proteómica , Sitios de UniónRESUMEN
A supramolecular FRET pair based on the ultrahigh binding affinity between cyanineâ 3 conjugated cucurbit[7]uril (CB[7]-Cy3) and cyanineâ 5 conjugated adamantylamine (AdA-Cy5) was exploited as a new synthetic tool for imaging cellular processes in live cells. Confocal laser scanning microscopy revealed that CB[7]-Cy3 and AdA-Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. CB[7]-Cy3 and AdA-Cy5 then formed a host-guest complex, reported by a FRET signal, as a result of the fusion of lysosomes and mitochondria. This observation not only indicated that CB[7] forms a stable complex with AdA in a live cell, but also suggested that this FRET pair can visualize dynamic organelle fusion processes, such as those involved in the degradation of mitochondria through autophagy (mitophagy), by virtue of its small size, chemical stability, and ease of use.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/fisiología , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Lisosomas/metabolismo , Amantadina/química , Autofagosomas/química , Hidrocarburos Aromáticos con Puentes/química , Carbocianinas/química , Humanos , Imidazoles/química , Lisosomas/química , Células MCF-7 , Fusión de Membrana , Microscopía ConfocalRESUMEN
Serendipitously, mono-allyloxylated cucurbit[7]uril (AO1 CB[7]) was discovered to act as an unconventional amphiphile which self-assembles into light-responsive vesicles (AO1 CB[7]VC) in water. Although the mono-allyloxy group, directly tethered on the periphery of CB[7], is much shorter (C4) than the hydrophobic tails of conventional amphiphiles, it played an important role in vesicle formation. Light-activated transformation of the allyloxy group by conjugation with glutathione was exploited as a remote tool to disrupt the vesicle. The vesicle showed on-demand release of cargo upon irradiation by a laser, after they were internalized into cancer cells. This result demonstrated the potential of AO1 CB[7]VC as a new type of light-responsive intracellular delivery vehicle for the release of therapeutic cargo, within cells, on demand.
RESUMEN
The design of synthetic, monovalent host-guest molecular recognition pairs is still challenging and of particular interest to inquire into the limits of the affinity that can be achieved with designed systems. In this regard, cucurbit[7]uril (CB[7]), an important member of the host family cucurbit[n]uril (CB[n], n = 5-8, 10, 14), has attracted much attention because of its ability to form ultra-stable complexes with multiple guests. The strong hydrophobic effect between the host cavity and guests, ion-dipole and dipole-dipole interactions of guests with CB portals helps in cooperative and multiple noncovalent interactions that are essential for realizing such strong complexations. These highly selective, strong yet dynamic interactions can be exploited in many applications including affinity chromatography, biomolecule immobilization, protein isolation, biological catalysis, and sensor technologies. In this review, we summarize the progress in the development of high affinity guests for CB[7], factors affecting the stability of complexes, theoretical insights, and the utility of these high affinity pairs in different challenging applications.
Asunto(s)
Biotecnología/métodos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/metabolismo , Adamantano , Avidina , Bioensayo , Biotina , Compuestos Bicíclicos con Puentes , Cromatografía de Afinidad , Compuestos Ferrosos , Metalocenos , OctanosRESUMEN
A facile method has been developed for synthesizing polymer nanocapsules and thin films using multiple in-plane stitching of monomers by the formation of reversible disulfide linkages. Owing to the reversibility of the disulfide linkages, the nanostructured materials readily transform their structures in response to environmental changes at room temperature. For example, in reducing environments, the polymer nanocapsules release loaded cargo molecules. Moreover, reversible morphological transformations between these structures can be achieved by simple solvent exchanges. This work is a novel approach for the formation of robust nano/microstructured materials that dynamically respond to environmental stimuli.
Asunto(s)
Disulfuros/química , Nanocápsulas/química , Polímeros/síntesis química , Termodinámica , Conformación Molecular , Tamaño de la Partícula , Polímeros/química , Propiedades de SuperficieRESUMEN
Despite a wide investigation of hydrogels as an artificial extracellular matrix, there are few scaffold systems for the facile spatiotemporal control of mesenchymal stem cells (MSCs). Here, we report 3D tissue engineered supramolecular hydrogels prepared with highly water-soluble monofunctionalized cucurbit[6]uril-hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug conjugated CB[6] (drug-CB[6]) for the controlled chondrogenesis of human mesenchymal stem cells (hMSCs). The mechanical property of supramolecular HA hydrogels was modulated by changing the cross-linking density for the spatial control of hMSCs. In addition, the differentiation of hMSCs was temporally controlled by changing the release profiles of transforming growth factor-ß3 (TGF-ß3) and/or dexamethasone (Dexa) from the hydrolyzable Dexa-CB[6]. The effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogel with TGF-ß3 and Dexa-CB[6] was confirmed by biochemical glycosaminoglycan content analysis, real-time quantitative PCR, histological, and immunohistochemical analyses. Taken together, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels as a platform scaffold with controlled drug delivery for cartilage regeneration and other various tissue engineering applications.
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Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células Madre Mesenquimatosas/efectos de los fármacos , Cartílago/citología , Matriz Extracelular/química , Humanos , Ácido Hialurónico/química , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Células Madre Mesenquimatosas/citología , Ingeniería de TejidosRESUMEN
Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report "OrthoID", a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were-to the best of our knowledge-previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.
Asunto(s)
Proteoma , Proteómica , Proteoma/metabolismo , Proteómica/métodos , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
In this study, the distinctive behavior of cucurbit[n]uril (CB[n]), which captures a variety of alkali halide clusters inside the cavity during the droplet evaporation, has been investigated by using ion mobility spectrometry-mass spectrometry. Complexes of CB[7] with various alkali chloride cluster cations or anions generated during the electrospray ionization were studied, and their collision cross-section (CCS) values were obtained to determine whether these clusters were trapped inside the cavity or not. It was found that the clusters smaller than a specific critical size were trapped inside the CB[7] cavity in the gas phase, although trapping of alkali halide clusters at the given concentration is supposed to be unfavorable in solution. We suggest that the rapid solvent evaporation rapidly increases ion concentrations and subsequently forms alkali-chloride contact ion pairs; therefore, it may provide a specific environment to enable the formation of the inclusion complexes.
RESUMEN
Spatiotemporal control of chemical cascade reactions within compartmentalized domains is one of the difficult challenges to achieve. To implement such control, scientists have been working on the development of various artificial compartmentalized systems such as liposomes, vesicles, polymersomes, etc. Although a considerable amount of progress has been made in this direction, one still needs to develop alternative strategies for controlling cascade reaction networks within spatiotemporally controlled domains in a solution, which remains a non-trivial issue. Herein, we present the utilization of audible sound induced liquid vibrations for the generation of transient domains in an aqueous medium, which can be used for the control of cascade chemical reactions in a spatiotemporal fashion. This approach gives us access to highly reproducible spatiotemporal chemical gradients and patterns, in situ growth and aggregation of gold nanoparticles at predetermined locations or domains formed in a solution. Our strategy also gives us access to nanoparticle patterned hydrogels and their applications for region specific cell growth.
Asunto(s)
Oro , Nanopartículas del Metal , Liposomas , Sonido , VibraciónRESUMEN
Exploiting the orthogonal molecular interactions of natural (phospholipids) and synthetic (mono-allyloxylated cucurbit[7]uril) amphiphiles to form their own vesicles, the formation of two different types of compartments in a self-sorted manner mimicking cellular compartments is demonstrated. Even after simultaneous extrusion of both vesicles through small pore membranes, which transformed them into smaller vesicles, both vesicles were not fused but still appeared as independent compartments in sucrose solution. The simultaneous use of natural and synthetic amphiphiles, forming independent compartments, holds great potential for in-depth investigation of self-sorted multi-compartments and their structures as prototype cells.
RESUMEN
Recently, chemical interface damping (CID) has been proposed as a new plasmon damping pathway based on interfacial hot-electron transfer from metal to adsorbate molecules. It has been considered essential, owing to its potential implications in efficient photochemical processes and sensing experiments. However, thus far, studies focusing on controlling CID in single gold nanoparticles have been very limited, and in situ reversible tuning has remained a considerable challenge. In these scanning electron microscopy-correlated dark-field spectroscopic measurements and density functional theory calculations, cucurbit[7]uril (CB[7])-based host-guest supramolecular interactions were employed to examine and control the CID process using monoamine-functionalized CB[7] (CB[7]-NH2) attached to single gold nanorods (AuNRs). In situ tuning of CID through the CB[7]-oxaliplatin complexation, which can result in the variation of the chemical nature and electronic properties of adsorbates, was presented. In addition, in situ tuning of CID was demonstrated through the competitive release of the oxaliplatin guest from the oxaliplatin@CB[7] complex, which was then replaced by a competitor guest of spermine in sufficient amounts. Furthermore, nuclear magnetic resonance experiments confirmed that the release of the guest is the consequence of adding salt (NaCl). Thus, in situ reversible tuning of CID in single AuNRs was achieved through successive steps of encapsulation and release of the guest on the same AuNR in a flow cell. Finally, single CB[7]-NH2@AuNRs were presented as a recyclable platform for CID investigations after the complete release of guest molecules from their host-guest inclusion complexes. Therefore, this study has paved a new route to achieve in situ reversible tuning of CID in the same AuNR and to investigate the CID process using CB-based host-guest chemistry with various guest molecules in single AuNRs for efficient hot-electron photochemistry and biosensing applications.
RESUMEN
A rationally designed supramolecular FRET pair consisting of cyanine3-cucurbit[7]uril (Cy3-CB[7]) and boron-dipyrromethene 630/650-adamantylammonium (BDP-AdA) can be used to visualize organelle-specific autophagy events. The intracellular accumulations of Cy3-CB[7] in lysosomes and BDP-AdA in lipid droplets (LDs) and the formation of an intracellular host-guest complex between Cy3-CB[7] and BDP-AdA resulting in FRET signals allow us to visualize the fusion of LDs with lysosomes, namely, lipophagy. This study demonstrates the potential of supramolecular imaging based on bio-orthogonal host-guest interactions in the investigation of selective autophagy events.
Asunto(s)
Autofagia , Hidrocarburos Aromáticos con Puentes/química , Transferencia Resonante de Energía de Fluorescencia , Imidazoles/química , Células HeLa , Humanos , Sustancias Macromoleculares/química , Estructura MolecularRESUMEN
The design and synthesis of a novel reduction-sensitive, robust, and biocompatible vesicle (SSCB[6]VC) are reported, which is self-assembled from an amphiphilic cucurbit[6]uril (CB[6]) derivative that contains disulfide bonds between hexaethylene glycol units and a CB[6] core. The remarkable features of SSCB[6]VC include: 1) facile, non-destructive, non-covalent, and modular surface modification using exceptionally strong host-guest chemistry; 2) high structural stability; 3) facile internalization into targeted cells by receptor-mediated endocytosis, and 4) efficient triggered release of entrapped drugs in a reducing environment such as cytoplasm. Furthermore, a significantly increased cytotoxicity of the anticancer drug doxorubicin to cancer cells is demonstrated using doxorubicin-loaded SSCB[6]VC, the surface of which is decorated with functional moieties such as a folate-spermidine conjugate and fluorescein isothiocyanate-spermidine conjugate as targeting ligand and fluorescence imaging probe, respectively. SSCB[6]VC with such unique features can be used as a highly versatile multifunctional platform for targeted drug delivery, which may find useful applications in cancer therapy. This novel strategy based on supramolecular chemistry and the unique properties of CB[6] can be extended to design smart multifunctional materials for biomedical applications including gene delivery.