RESUMEN
α-Ketoglutarate (α-KG) also known as 2-oxoglutarate (2-OG) is an intermediate metabolite in the tricarboxylic acid (TCA) cycle and is also produced by the deamination of glutamate. It is an indispensable cofactor for a series of 2-oxoglutarate-dependent oxygenases including epigenetic modifiers such as ten-eleven translocation DNA demethylases (TETs) and JmjC domain-containing histone demethylases (JMJDs). Since these epigenetic enzymes target genomic DNA and histone in the nucleus, the nuclear concentration of α-KG would affect the levels of transcription by modulating the activity of the epigenetic enzymes. Thus, it is of great interest to measure the nuclear concentration of α-KG to elucidate the regulatory mechanism of these enzymes. Here, we report a novel fluorescence resonance energy transfer (FRET)-based biosensor with multiple nuclear localization signals (NLSs) to measure the nuclear concentration of α-KG. The probe contains the α-KG-binding GAF domain of NifA protein from Azotobacter vinelandii fused with EYFP and ECFP. Treatment of 3T3-L1 preadipocytes expressing this probe with either dimethyl-2-oxoglutarate (dimethyl-2-OG), a cell-permeable 2-OG derivative, or citrate elicited time- and dose-dependent changes in the FRET ratio, proving that this probe functions as an α-KG sensor. Measurement of the nuclear α-KG levels in the 3T3-L1 cells stably expressing the probe during adipocyte differentiation revealed that the nuclear concentration of α-KG increased in the early stage of differentiation and remained high thereafter. Thus, this nuclear-localized α-KG probe is a powerful tool for real-time monitoring of α-KG concentrations with subcellular resolution in living cells and is useful for elucidating the regulatory mechanisms of epigenetic enzymes.
Asunto(s)
Técnicas Biosensibles , Ácidos Cetoglutáricos , Adipocitos/metabolismo , Diferenciación Celular , Transferencia Resonante de Energía de Fluorescencia , Ácidos Cetoglutáricos/metabolismo , Señales de Localización NuclearRESUMEN
Dead cells always accompany with live cells in vivo and in cell culture. It is important to distinguish dead cells from live cells in various biological studies. Currently, the probes for dead cells are mainly nucleic acid-intercalators, most of which have low affinity and potential toxicity to live cells. In this work, we report a novel aptameric probe (Ch4-1) for the first time, which binds cell nuclei with high affinity (apparent Kd = 6.65 ± 3.40 nM). Ch4-1 was generated by Cell-SELEX process, it was identified to target nucleoproteins in cell nuclei. As an oligonucleotide, Ch4-1 cannot penetrate the integrated cell membrane; therefore, it only binds to dead cells rather than live cells. Compared with traditional DNA-targeting nuclear dyes, Ch4-1 possesses a high affinity to the nucleus, no toxicity to live cells, and can be easily labeled with different fluorescent dyes. It was demonstrated to serve as a probe for distinguishing dead cells from live cells in apoptosis assay, as well as for the nuclear staining of tissue sections.
Asunto(s)
Apoptosis , Aptámeros de Nucleótidos/química , Núcleo Celular/química , ADN/química , Neoplasias/clasificación , Nucleoproteínas/análisis , Animales , Muerte Celular , Línea Celular Tumoral , Humanos , Ratones , Neoplasias/patología , Nucleoproteínas/química , Oligodesoxirribonucleótidos/química , Técnica SELEX de Producción de AptámerosRESUMEN
Recalcitrant relationships are characterized by very short internodes that can be found among shallow and deep phylogenetic scales all over the tree of life. Adding large amounts of presumably informative sequences, while decreasing systematic error, has been suggested as a possible approach to increase phylogenetic resolution. The development of enrichment strategies, coupled with next generation sequencing, resulted in a cost-effective way to facilitate the reconstruction of recalcitrant relationships. By applying the anchored hybrid enrichment (AHE) genome partitioning strategy to Aristolochia using an universal angiosperm probe set, we obtained 231-233 out of 517 single or low copy nuclear loci originally contained in the enrichment kit, resulting in a total alignment length of 154,756bp to 160,150bp. Since Aristolochia (Piperales; magnoliids) is distantly related to any angiosperm species whose genome has been used for the plant AHE probe design (Amborella trichopoda being the closest), it serves as a proof of universality for this probe set. Aristolochia comprises approximately 500 species grouped in several clades (OTUs), whose relationships to each other are partially unknown. Previous phylogenetic studies have shown that these lineages branched deep in time and in quick succession, seen as short-deep internodes. Short-shallow internodes are also characteristic of some Aristolochia lineages such as Aristolochia subsection Pentandrae, a clade of presumably recent diversification. This subsection is here included to test the performance of AHE at species level. Filtering and subsampling loci using the phylogenetic informativeness method resolves several recalcitrant phylogenetic relationships within Aristolochia. By assuming different ploidy levels during bioinformatics processing of raw data, first hints are obtained that polyploidization contributed to the evolution of Aristolochia. Phylogenetic results are discussed in the light of current systematics and morphology.