RESUMO
Enzyme-assisted posttranslational modifications (PTMs) constitute a major means of signaling across different cellular compartments. However, how nonenzymatic PTMs-despite their direct relevance to covalent drug development-impinge on cross-compartment signaling remains inaccessible as current target-identification (target-ID) technologies offer limited spatiotemporal resolution, and proximity mapping tools are also not guided by specific, biologically-relevant, ligand chemotypes. Here we establish a quantitative and direct profiling platform (Localis-rex) that ranks responsivity of compartmentalized subproteomes to nonenzymatic PTMs. In a setup that contrasts nucleus- vs. cytoplasm-specific responsivity to reactive-metabolite modification (hydroxynonenylation), â¼40% of the top-enriched protein sensors investigated respond in compartments of nonprimary origin or where the canonical activity of the protein sensor is inoperative. CDK9-a primarily nuclear-localized kinase-was hydroxynonenylated only in the cytoplasm. Site-specific CDK9 hydroxynonenylation-which we identified in untreated cells-drives its nuclear translocation, downregulating RNA-polymerase-II activity, through a mechanism distinct from that of commonly used CDK9 inhibitors. Taken together, this work documents an unmet approach to quantitatively profile and decode localized and context-specific signaling/signal-propagation programs orchestrated by reactive covalent ligands.
Assuntos
Proteínas/genética , Proteínas/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Células RAW 264.7 , Transdução de Sinais/fisiologia , Transcrição Gênica/genéticaRESUMO
Protein trafficking and protein-protein interactions (PPIs) are central to regulatory processes in cells. Induced dimerization systems have been developed to control PPIs and regulate protein trafficking (localization) or interactions. Chemically induced dimerization (CID) has proven to be a robust approach to control protein interactions and localization. The most recent embodiment of this technology relies on CID conjugates that react with a self-labeling protein on one side and a photocaged ligand on the other side to provide spatiotemporal control of the interaction with the protein of interest. Advancing this technology further is limited by the light delivery problem and the phototoxicity of intense irradiation necessary to achieve photouncaging. Herein, we designed a novel chemically induced dimerization system that was triggered by bioluminescence, instead of external light. Protein dimerization showed fast kinetics and was validated by an induced change of localization of a target protein (to and from the nucleus or plasma membrane) upon trigger. The technology was used transiently to activate the phosphatidylinositol 3-kinase (PI3K)/mTOR pathway and measure the impact on lipid synthesis/metabolism, assessed by lipidomics.
Assuntos
Luciferases/metabolismo , Proteínas/metabolismo , Dimerização , Ligação ProteicaRESUMO
Detection of specific oligonucleotide sequences is central to numerous applications, and technologies amenable to point-of-care diagnostics or end users are needed. Here, we report a technology making use of a bioluminescent readout and smartphone quantification. The sensor is a semisynthetic luciferase (H-Luc-PNA conjugate) that is turned on by a strand-displacement reaction. We demonstrated sensing of three different microRNAs (miRs), as representative cancer biomarkers, and demonstrate the possibility to integrate an AND gate to sense two sequences simultaneously.
Assuntos
Biomarcadores Tumorais/análise , Técnicas Biossensoriais , Lógica , MicroRNAs/análise , Smartphone , DNA , Luciferases , Medições LuminescentesRESUMO
Bioluminescence resonance energy transfer (BRET) has been widely used for studying dynamic processes in biological systems such as protein-protein interactions and other signaling events. Aside from acting as a reporter, BRET can also turn on functions in living systems. Herein, we report the application of BRET to performing a biorthogonal reaction in living cells; namely, releasing functional molecules through energy transfer to a coumarin molecule, a process termed bioluminolysis. An efficient BRET from Nanoluc-Halotag chimera protein (H-Luc) to a coumarin substrate yields the excited state of coumarin, which in turn triggers hydrolysis to uncage a target molecule. Compared to the conventional methods, this novel uncaging system requires no external light source and shows fast kinetics (t1/2 <2â min). We applied this BRET uncaging system to release a potent kinase inhibitor, ibrutinib, in living cells, highlighting its broad utility in controlling the supply of bioactive small molecules inâ vivo.
Assuntos
Cumarínicos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Luciferases/metabolismo , Medições Luminescentes/métodos , Proteínas Luminescentes/metabolismo , Pirazóis/metabolismo , Pirimidinas/metabolismo , Adenina/análogos & derivados , Células HeLa , Humanos , Piperidinas , Ligação ProteicaRESUMO
DNA-based circuitry empowers logic gated operations and amplifications but is restricted to nucleic acid output. Templated reactions enable the translation of nucleic acid cues into diverse small-molecule outputs but are more limited in their amplification. Herein, we demonstrate the coupling of a DNA circuit to templated reactions in order to achieve high levels of amplification in the output of small molecules, in response to nucleic acid input. We demonstrate that the coupling of the DNA circuit to templated reactions allows for the detection of the fM concentration of analyte and can respond with the release of a cytotoxic drug.
Assuntos
DNA/química , Moldes Genéticos , DNA/metabolismo , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Peptídeos/síntese químicaRESUMO
Lipids are essential components of eukaryotic cell membranes and play crucial roles in cellular signaling and metabolism. While increasing evidence shows that the activities of lipids are dependent upon subcellular localization, tools to study local lipid metabolism and signaling are limited. Herein, we report an approach that enabled us to selectively deliver photo-caged lipids into lysosomes and thereafter to quickly release the lipid molecules by illumination. On combining this method with genetic techniques and lipidomics, we were able to investigate the localization-dependent metabolism of an important intermediate of sphingolipid metabolism, sphingosine. Our data reveal a distinct metabolic pattern of lysosomal sphingosine. In general, this method has the potential to serve as a platform to study lysosomal metabolism and signaling of various lipids and metabolites in living cells.
RESUMO
Nucleic acid templated reactions have attracted attention as an important technology to sense oligonucleotides and to translate nucleic acid-based instructions into diverse outputs. Great progress has been made in accelerating the reaction in order to improve signal amplification, reaching rates where substrate turnover rather than chemical reaction is rate limiting. Herein we explore the utility of architectures inspired by three-way junction that yield a cleavage of a strand thus accelerating substrate turnover. We demonstrate that such design can overcome product inhibition in templated reactions and operate close to the rate of hybridization.
Assuntos
Hibridização de Ácido Nucleico/métodos , Ácidos Nucleicos/química , Ácidos Nucleicos Peptídicos/química , Cumarínicos/química , Fluorescência , Corantes Fluorescentes/química , Oligonucleotídeos/química , Vancomicina/químicaRESUMO
Nucleic acid-templated reactions have attracted significant attention for nucleic acid sensing and imaging. The level of signal amplification obtained from templated reactions is a function of the template turnover, wherein the template acts as the catalyst. Herein, we report the application of a pyridinium linker that immolates upon photocatalytic reduction with a ruthenium complex to yield the fastest nucleic acid templated reaction reported to date. We show that the templated reaction turnover is limited by the duplex dissociation kinetics beyond probes longer than a 6-mer and proceeded fastest for a 5-mer PNA probe. Using a beacon architecture that masks the catalytic template, we show that this methodology can be used for nucleic acid sensing extending the analyte recognition beyond a 5-mer. The system proceeds with a catalytic efficiency of 105 M-1 s-1 and achieves turnover frequency of >100 h-1.
Assuntos
Ácidos Nucleicos/análise , Compostos Organometálicos/química , Compostos de Piridínio/química , Rutênio/química , Estrutura MolecularRESUMO
Pd-catalyzed difluoromethylation of di-, tri- or tetra-substituted vinyl bromides, triflates, tosylates and nonaflates under mild conditions is described. The reaction tolerates a wide range of functional groups, such as bromide, chloride, fluoride, ester, amine, nitrile, and protected carbonyl, thus providing a general route for the preparation of difluoromethylated alkenes.