Development of High Sensitive and Quantitative FRET Based Biosensor to Detect Atg4A Kinetics in Autophagy Cell Death Pathway

Abstract

Cell death is a major process in a biological cell that occurs during development, homeostasis and immune regulation in multicellular organisms. Dysregulation of cell death pathway has been implicated in many diseases. Principal cell death pathways include apoptosis, autophagy, necrosis, mitotic catastrophe, etc. Knowledge of cell death pathways and the reason the cell chooses to die are key factors to understand the disease, the way it affects the cellular system and subsequent drug discovery. This study is focused on developing genetically encoded Förster Resonance Energy Transfer (FRET) based biosensors to identify autophagy pathways in vitro. FRET is an energy transfer phenomenon that occurs between two spectrum-overlapping fluorophores that are within 10nm of each other. The design of the sensor is based on enzyme-substrate dynamics and consists of a reporter gene fused between fluorescent proteins. Additionally, FRET-based protease assay has been used to determine the kinetics of Atg4A, an enzyme involved in autophagy. The kinetic parameters Km, kcat, kcat /Km were derived using real-time detection methods. A further aim of this research is to transfect the sensor in H460 lung cancer cell line to identify the type of death that the cell chooses on treatment with drugs.