RESUMEN
DNA nanotechnology has revolutionized the ability to position matter at the nanoscale, but the preparation of DNA-based architectures remains laborious. To facilitate the formation of custom structures, a fully automated method is reported to produce sequence- and size-defined DNA nanotubes. By programming the sequential addition of desired building blocks, rigid DX-tile-based DNA nanotubes and flexible wireframe DNA structures are attained, where the total number of possible constructs increases as a power function of the number of different units available. Using single-molecule fluorescence imaging, the kinetics and yield of each synthetic step can be quantitatively determined, revealing differences in self-assembly dynamics as the nanotube is built up from the solid support and providing new insights into DNA self-assembly. The exploitation of automation for both assembly and analysis (through an ad-hoc developed K-means clustering algorithm) facilitates a workflow wherein the synthesis parameters may be iteratively improved upon, demonstrating how a single-molecule "assembly-analysis-optimization" sequence can be used to generate complex, noncovalent materials in good yield. The presented synthetic strategy is generalizable, making use of equipment already available in most standard laboratories and represents the first fully automated supramolecular assembly on a solid support.
Asunto(s)
Automatización , ADN , ADN/química , Nanotubos/química , Nanotecnología/métodos , Nanoestructuras/químicaRESUMEN
Intercellular communication in the nervous system occurs through the release of neurotransmitters into the synaptic cleft between neurons. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single-particle electron cryomicroscopy allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzyme's rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. ATP hydrolysis during vesicle loading results in a loss of the V1 region of V-ATPase from the SV membrane, suggesting that loading is sufficient to induce dissociation of the enzyme.
Asunto(s)
Vesículas Sinápticas , ATPasas de Translocación de Protón Vacuolares , Animales , Ratas , Proteínas Bacterianas/química , Encéfalo/ultraestructura , Encéfalo/enzimología , Colesterol/química , Microscopía por Crioelectrón , Hidrólisis , Vesículas Sinápticas/enzimología , Vesículas Sinápticas/ultraestructura , Sinaptofisina/metabolismo , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/aislamiento & purificación , ATPasas de Translocación de Protón Vacuolares/ultraestructura , Conformación ProteicaRESUMEN
Suitably configured allyl ethers of unsaturated cyclitols act as substrates of ß-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of ß-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus ß-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.
Asunto(s)
Ciclitoles , Ciclitoles/química , Glicósido Hidrolasas/metabolismo , Glicósidos , Dominio Catalítico , Inhibidores Enzimáticos/farmacologíaRESUMEN
Periodontitis is a chronic inflammatory disease characterized by the release of matrix metalloproteinases (MMPs) from resident connective tissue cells in tooth-supporting tissues (periodontium). Platelet activation, and the attendant release of pro-inflammatory chemokines such as platelet factor 4 (CXCL4/PF4), are associated with periodontitis although the associated biochemical pathways remain undefined. Here we report that recombinant PF4 is internalized by cultured human gingival fibroblasts (hGFs), resulting in significant (p < 0.05) upregulation in both the production and release of MMP-2 (gelatinase A). This finding was corroborated by elevated circulating levels of MMP-2 (p < 0.05) in PF4-overexpressing transgenic mice, relative to controls. We also determined that PF4 induces the phosphorylation of NF-κB; notably, the suppression of NF-κB signaling by the inhibitor BAY 11-7082 abrogated PF4-induced MMP-2 upregulation. Moreover, the inhibition of surface glycosaminoglycans (GAGs) blocked both PF4 binding and NF-κB phosphorylation. Partial blockade of PF4 binding to the cells was achieved by treatment with either chondroitinase ABC or heparinase III, suggesting that both chondroitin sulfate and heparan sulfate mediate PF4 signaling. These results identify a novel pathway in which PF4 upregulates MMP-2 release from fibroblasts in an NF-κB- and GAG-dependent manner, and further our comprehension of the role of platelet signaling in periodontal tissue homeostasis.
Asunto(s)
Metaloproteinasa 2 de la Matriz , Periodontitis , Ratones , Animales , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Factor Plaquetario 4/metabolismo , FN-kappa B/metabolismo , Encía , Fibroblastos/metabolismo , Periodontitis/metabolismo , Inhibidores de la Angiogénesis/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismoRESUMEN
The central mechanism in ferroptosis linking lipid hydroperoxide accumulation with cell death remains poorly understood. Although lipid hydroperoxides are known to break down to reactive lipid-derived electrophiles (LDEs), the ability of cells to detoxify increasing LDE levels during ferroptosis has not been studied. Here, we developed an assay (ElectrophileQ) correlating the cellular retention vs. excretion of a fluorogenic lipophilic electrophile (AcroB) that enables live-cell assessment of the glutathione-mediated LDE conjugation and adduct export steps of the LDE detoxification pathway. This method revealed that during ferroptosis, LDE detoxification failure occurs through decreased conjugation or export impairment, amplifying cellular electrophile accumulation. Notably, ferroptosis susceptibility was increased following exacerbation of LDE-adduct export impairment through export channel inhibition. Our results expand understanding of the ferroptosis molecular cell death mechanism to position the LDE detoxification pathway as a ferroptosis-relevant therapeutic target. We envision the ElectrophileQ assay becoming an invaluable tool for studying ferroptosis and cellular health.
RESUMEN
α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining ß-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available. We designed such a reagent by incorporating a highly reactive fluorogenic leaving group onto unsaturated cyclitol ethers, which have been recently shown to act as slow substrates for retaining glycosidases that operate via a covalent 'glycosyl'-enzyme intermediate. By appending this warhead onto the appropriate oligosaccharide, we developed efficient active site titration reagents for α-amylases that effect quantitation down to low nanomolar levels.