RESUMEN
We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.
Asunto(s)
Ciclooctanos/análisis , Dendrímeros/metabolismo , Ácidos Grasos/metabolismo , Glioblastoma/metabolismo , Análisis de la Célula Individual , Azidas/química , Azidas/metabolismo , Proliferación Celular , Ciclooctanos/metabolismo , Dendrímeros/química , Ácidos Grasos/química , Fluorescencia , Glioblastoma/patología , Humanos , Estructura Molecular , Propiedades de SuperficieRESUMEN
We present here a cyclic peptide ligand, cy(WQETR), that binds to the terbium ion (Tb3+) and enhances Tb3+ luminescence intensity through the antenna effect. This peptide was identified through screening a cyclic peptide library against Tb3+ with an apparent EC50 of 540 µM. The tryptophan residue from the peptide directly interacts with the Tb3+ ion, which provides access to a low-lying triplet excited state of the tryptophan. Direct excitation of this triplet state enables energy transfer to the Tb3+ ion and enhances Tb3+ luminescence intensity by 150 fold. We further showcase the application of this cy(WQETR)-Tb3+ system by demonstrating the detection of tromethamine with a detection limit of 0.5 mM.
Asunto(s)
Luminiscencia , Terbio , Transferencia de Energía , Ligandos , Péptidos CíclicosRESUMEN
Analyzing intracellular signalling protein activities in living cells promises a better understanding of the signalling cascade and related biological processes. We have previously developed cyclic peptide-based probes for analyzing intracellular AKT signalling activities, but these peptide probes were not cell-permeable. Implementing fusogenic liposomes as delivery vehicles could circumvent the problem when analyzing adherent cells, but it remained challenging to study suspension cells using similar approaches. Here, we present a method for delivering these imaging probes into suspension cells using digitonin, which could transiently perforate the cell membrane. Using U87, THP-1, and Jurkat cells as model systems representing suspended adherent cells, myeloid cells, and lymphoid cells, we demonstrated that low concentrations of digitonin enabled a sufficient amount of probes to enter the cytosol without affecting cell viability. We further combined this delivery method with a microwell single-cell chip and interrogated the AKT signalling dynamics in THP-1 and Jurkat cells, followed by immunofluorescence-based quantitation of AKT expression levels. We resolved the cellular heterogeneity in AKT signalling activities and showed that the kinetic patterns of AKT signalling and the AKT expression levels were related in THP-1 cells, but decoupled in Jurkat cells. We expect that our approach can be adapted to study other suspension cells.
Asunto(s)
Fenómenos Biológicos , Proteínas Proto-Oncogénicas c-akt , Digitonina , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Análisis de la Célula IndividualRESUMEN
We present here a novel chemical method to continuously analyze intracellular AKT signaling activities at single-cell resolution, without genetic manipulations. A pair of cyclic peptide-based fluorescent probes were developed to recognize the phosphorylated Ser474 site and a distal epitope on AKT. A Förster resonance energy transfer signal is generated upon concurrent binding of the two probes onto the same AKT protein, which is contingent upon the Ser474 phosphorylation. Intracellular delivery of the probes enabled dynamic measurements of the AKT signaling activities. We further implemented this detection strategy on a microwell single-cell platform, and interrogated the AKT signaling dynamics in a human glioblastoma cell line. We resolved unique features of the single-cell signaling dynamics following different perturbations. Our study provided the first example of monitoring the temporal evolution of cellular signaling heterogeneities and unveiled biological information that was inaccessible to other methods.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Línea Celular Tumoral , Humanos , Modelos Moleculares , FosforilaciónRESUMEN
We report on a cyclic peptide that inhibits matrix metalloproteinase-2 (MMP2) activation with a low-nM-level potency. This inhibitor specifically binds to the D570-A583 epitope on proMMP2 and interferes with the protein-protein interaction (PPI) between proMMP2 and tissue inhibitor of metalloproteinases-2 (TIMP2), thereby preventing the TIMP2-assisted proMMP2 activation process. We developed this cyclic peptide inhibitor through an epitope-targeted library screening process and validated its binding to proMMP2. Using a human melanoma cell line, we demonstrated the cyclic peptide's ability to modulate cellular MMP2 activities and inhibit cell migration. These results provide the first successful example of targeting the PPI between proMMP2 and TIMP2, confirming the feasibility of an MMP2 inhibition strategy that has been sought after for 2 decades.