RESUMO
N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Mitocôndrias/genética , Biossíntese de Proteínas , RNA de Transferência de Treonina/genética , Acetilação , Adenosina/análogos & derivados , Adenosina/genética , Anticódon/genética , Escherichia coli/genética , Proteínas de Ligação ao GTP/genética , Humanos , Mutação , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Emulsion droplets can serve as ideal compartments for reactions. In fact, in many cases, the chemical reactions are supposed to be triggered at a desired position and time without change of the system environment. Here, we present a type of light and magnetic dual-responsive Pickering emulsion microreactor by coadsorption of light-sensitive titania (TiO2) and super paramagnetic iron oxide (Fe3O4) nanoparticles at the oil-water interface of emulsion droplets. The droplets encapsulating different reactants in advance can be driven close to each other by an external magnetic field, and then the chemical reaction is triggered by UV illumination due to the contact of the isolated reactants as a result of droplet coalescence. An insight into the incorporation of hydrophobic TiO2 and hydrophilic Fe3O4 nanoparticles simultaneously at the emulsion interface is achieved. On the basis of that, an account is given of the coalescence mechanism of the Pickering emulsion microreactors. Our work not only provides a novel Pickering emulsion microreactor platform for triggering chemical reactions in a nonintrusive and well-controlled way but also opens a promising avenue to construct multifunctional Pickering emulsions by assembly of versatile building block nanoparticles at the interface of emulsion droplets.
RESUMO
In this work, a new strategy for developing light-triggered Pickering emulsions as smart soft vehicles for on-demand release is proposed. Initially, UV-induced tailored wettability allows anchoring of TiO2 nanoparticles at the interface to prepare stable water in oil emulsions. Such emulsions show the efficacy of microencapsulation and controlled release by demulsification due to the hydrophilic conversion of the TiO2 nanoparticles using a noninvasive light irradiation trigger. A molecule of interest is selected as a model cargo to quantitatively evaluate the as-prepared Pickering emulsions for their encapsulation and release behaviors. Moreover, light-responsive emulsion destabilization mechanism is studied as a function of particle concentration, light wavelength, and light intensity, respectively, determined by drop diameter evolution and droplet coalescence kinetics plots. For consideration of application in life sciences, Pickering emulsions sensitive to visible light are also established based on nitrogen doping of TiO2 nanoparticle emulsifiers.
RESUMO
Water-in-water (w/w) emulsions are attractive microcompartmentalized platforms due to their outstanding biocompatibility. To address the main disadvantage of poor stability that hampers their practical application, here we report a novel type of polymer-protein conjugate emulsifier obtained by Schiff base synthesis to stabilize w/w emulsions. In particular, the proposed mild approach benefits the modification of proteins of suitable size and wettability as particulate emulsifiers retaining their bioactivity. As demonstrated in a model system, the methoxy polyethylene glycol (mPEG)-urease conjugate particles anchor at the w/w interfaces, where they serve as an effective emulsifier-combined-catalyst and catalyze the hydrolysis of urea in water to ammonium carbonate. Our study is unique in that it employs bioactive particles to stabilize w/w emulsions. Considering the characteristics of all-aqueous, compartmental and interfacial biocatalysis of the system, it will open up new possibilities in the life sciences.