Real-time Analysis of Cellular Trafficking of APE1 in Cancer Cells
Disciplines
Cell Biology
Abstract (300 words maximum)
The apurinic/apyrimidinic endonuclease (APE1) is a multifunctional protein widely studied for its role in DNA repair, inhibition of reactive oxidative species and transcriptional co-activation. APE1 primarily localizes to the nucleus though redistribution of APE1 to the cytosol is a common phenomenon in many diseases. In cancer biopsies, an increase in cytosolic versus nuclear APE1 correlates to increased metastasis and poor patient outcome. The goal of this work was to study APE1’s subcellular localization in living cancer cells following direct delivery of APE1 protein via a novel cell penetrating peptide-adaptor (CPP). We created chimeric CPP-adaptor constructs that could readily bind APE1 in presence of calcium and dissociate in the absence of calcium. These constructs were expressed in E. coli and purified via fast protein liquid chromatography. Binding kinetics of APE1 with our CPP adaptor were determined by optical biosensing. Live cellular uptake was assayed by confocal microscopy following introduction of fluorescently labeled APE1 into living cancer cells. Free APE1 was readily delivered into living cells via our CPP adaptor and quantified by mean fluorescent intensity, 72.6 MFI with our CPP and 2.7 without. In cancer cells, over 90% of APE1 localized to the cytoplasm. Our model system allows us to analyze APE1’s cellular distribution in real time. Currently, are investigating localization of APE1 to different cellular compartments under conditions, such as oxidative stress, associated with carcinogenesis.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Jonathan McMurry
Additional Faculty
Julia LeCher, Department of Molecular and Cellular Biology, jwand@kennesaw.edu
Real-time Analysis of Cellular Trafficking of APE1 in Cancer Cells
The apurinic/apyrimidinic endonuclease (APE1) is a multifunctional protein widely studied for its role in DNA repair, inhibition of reactive oxidative species and transcriptional co-activation. APE1 primarily localizes to the nucleus though redistribution of APE1 to the cytosol is a common phenomenon in many diseases. In cancer biopsies, an increase in cytosolic versus nuclear APE1 correlates to increased metastasis and poor patient outcome. The goal of this work was to study APE1’s subcellular localization in living cancer cells following direct delivery of APE1 protein via a novel cell penetrating peptide-adaptor (CPP). We created chimeric CPP-adaptor constructs that could readily bind APE1 in presence of calcium and dissociate in the absence of calcium. These constructs were expressed in E. coli and purified via fast protein liquid chromatography. Binding kinetics of APE1 with our CPP adaptor were determined by optical biosensing. Live cellular uptake was assayed by confocal microscopy following introduction of fluorescently labeled APE1 into living cancer cells. Free APE1 was readily delivered into living cells via our CPP adaptor and quantified by mean fluorescent intensity, 72.6 MFI with our CPP and 2.7 without. In cancer cells, over 90% of APE1 localized to the cytoplasm. Our model system allows us to analyze APE1’s cellular distribution in real time. Currently, are investigating localization of APE1 to different cellular compartments under conditions, such as oxidative stress, associated with carcinogenesis.