RESUMO
Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking.
Assuntos
Autofagossomos/metabolismo , Azidas/química , Colina/análogos & derivados , Retículo Endoplasmático/metabolismo , Fosfatidilcolinas/metabolismo , Coloração e Rotulagem/métodos , Autofagossomos/ultraestrutura , Transporte Biológico , Carbocianinas/metabolismo , Química Click/métodos , Retículo Endoplasmático/ultraestrutura , Corantes Fluorescentes/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Imagem Molecular/métodos , Fosfatidilcolinas/química , Rodamina 123/metabolismo , Proteína Vermelha FluorescenteRESUMO
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. H2O2-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with H2O2 in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.
Assuntos
Corantes Fluorescentes/química , Peróxido de Hidrogênio/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Imagem Óptica , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Células RAW 264.7RESUMO
This study reports the X-ray crystallographic structure of the glycyl-tRNA synthetase (GlyRS) of Nanoarchaeum equitans - a hyperthermophilic archaeal species. This is the first archaeal GlyRS crystal structure elucidated. The GlyRS comprises an N-terminal catalytic domain and a C-terminal anticodon-binding domain with a long ß-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNAGly was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNAGly with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNAGly elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNAGly G71 substitution mutants and a GlyRS mutant where Arg223 is mutated to alanine strengthen the possibility that the carbonyl oxygen at position 6 of G71 would hydrogen-bond with the guanidine nitrogen of Arg223 in N. equitans GlyRS.
Assuntos
Proteínas Arqueais/química , Glicina-tRNA Ligase/química , Nanoarchaeota/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Glicina-tRNA Ligase/metabolismo , Modelos Moleculares , Nanoarchaeota/química , Nanoarchaeota/metabolismo , Conformação Proteica , RNA de Transferência/química , RNA de Transferência/metabolismo , Alinhamento de SequênciaRESUMO
The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa cells and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA, as ER-associated proteins. The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.
Assuntos
Retículo Endoplasmático/química , Sondas Moleculares/química , Proteoma/análise , Xantenos/química , Carboxiliases/análise , Carboxiliases/química , Cromatografia Líquida , Ciclofilina A/análise , Ciclofilina A/química , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Sondas Moleculares/síntese química , Enzimas Multifuncionais/análise , Enzimas Multifuncionais/química , Peptídeo Sintases/análise , Peptídeo Sintases/química , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem , Tiorredoxinas/análise , Tiorredoxinas/química , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Xantenos/síntese químicaRESUMO
Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography-mass spectrometry (LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases.
Assuntos
Encéfalo/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteoma/metabolismo , Proteômica/métodos , Sítios de Ligação , Técnicas de Cultura de Células , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Marcação por Isótopo , Microscopia Confocal , Modelos Moleculares , Rodaminas/química , Espectrometria de Massas em TandemRESUMO
Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~104 M-1 s-1, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.