RESUMO
Three new bisindole alkaloids, 3'-(2-oxopropyl)-19,20-dihydrotabernamine (1: ), 3'-(2-oxopropyl)-ervahanine B (2: ), 19,20-dihydrovobparicine (3: ), and 20 known compounds were isolated from the aerial parts of Tabernaemontana bufalina. The structures of these alkaloids were elucidated using spectroscopic methods. The absolute configurations of 1: -3: were determined by the circular dichroic exciton chirality method. Compounds 1: -23: were screened for their cytotoxicity against two human cancer cell lines, A-549 and MCF-7. Ten compounds (1: -3, 10, 14, 16, 17, 19, 22: , and 23: ) exhibited inhibitory effects against the two human cancer cells with IC50 values of 1.19 ~ 6.13 µM.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Alcaloides Indólicos/química , Monoterpenos/química , Tabernaemontana/química , Hidrocarbonetos Aromáticos com Pontes/isolamento & purificação , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/farmacologia , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Modelos Estruturais , Monoterpenos/isolamento & purificação , Monoterpenos/farmacologia , Componentes Aéreos da Planta/químicaRESUMO
Proteomics at single-cell resolution can help to identify the heterogeneity among cell populations, shows more and more significance in current chemistry and biology. In this work, we demonstrated a new single cell chemical proteomic (SCCP) strategy with a membrane-permeable activity-based probe (ABP) to characterize the functional proteins in lysosome located in the cytosol. The ABP targeted to the cysteine cathepsin family protein, CpFABP-G, was designed for cysteine cathepsins labeling. The labeled HeLa cell of a cancer cell line was injected into a capillary and was lysed by SDS solution with heating. The lysate was then online readout by capillary electrophoresis-laser-induced fluorescence method. Due to the employment of highly specified ABP kicking out the uncorrelated proteins, the expression of cysteine cathepsins in individual HeLa cells was easily detected, and heterogeneity among those HeLa cells was readily discriminated. Further work was concentrated on SCCP analysis of the mouse leukemia cell of monocyte macrophage (RAW264.7). It was for the first time identifying two expression modes of cysteine cathepsins in RAW264.7, which could be undermined by the analysis of cell populations. We believed that SCCP would be one of the powerful alternatives for proteomics at single-cell resolution.
Assuntos
Membranas Intracelulares/metabolismo , Lisossomos/química , Sondas Moleculares/análise , Proteínas de Neoplasias/análise , Neoplasias/química , Neoplasias/patologia , Proteômica , Análise de Célula Única , Animais , Catepsinas/análise , Catepsinas/metabolismo , Linhagem Celular Tumoral , Eletroforese Capilar , Fluorescência , Células HeLa , Humanos , Lasers , Camundongos , Sondas Moleculares/metabolismo , Proteínas de Neoplasias/metabolismo , Permeabilidade , Espectrometria de FluorescênciaRESUMO
Cell-permeable activity-based probes (ABPs) are capable of labeling target proteins in living cells, thereby providing a powerful tool for profiling active enzymes in their native environment. In this study, we describe the synthesis and use of a novel trifunctional cell-permeable activity-based probe (TCpABP) for proteomic profiling of active cysteine cathepsins in living cells. We demonstrate that although TCpABP contains cell-impermeable tags, it was able to enter living cells efficiently via the delivery of a cell-penetrating peptide. TCpABP also allowed simultaneous detection and affinity isolation of labeled proteins with a fluorophore and a biotin motif, respectively. We optimized the enrichment protocol to minimize contaminants and identified 7 cathepsins, 2 of which have never been identified using existing ABPs. We also used a label-free quantification approach to quantify the relative abundances of active cathepsins and compared them with their previously published mRNA expression levels. A high degree of correlation between the mRNA expression levels and protein relative activities was observed for most of the identified cathepsins except cathepsin H. The results herein indicate that TCpABP is valuable for the detection of active cathepsins in living cells and provides useful guidelines for designing novel cell-permeable ABPs for in vivo labeling and their applications in in vivo proteomics studies.