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

Chitra Hariharan,Fan Zhang,Jiayu Liao,Lei Xu,Fatma Zehra Kadayifci,Jun Li,Shasha Shasha

doi:10.11159/jbeb.2021.001

Abstract

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

Highlights

The major cell death pathways, classified as autophagic, apoptotic, and necrotic, work to maintain homeostasis in the organism
Förster Resonance Energy Transfer (FRET)-based protease assay has been used to determine the kinetics of Atg4A, an enzyme involved in autophagy
Digestion by Atg4A releases the products CyPet-Gate16 and C-terminus of Gate16-YPet, and the FRET signal decreases corresponding to the amount of digested substrate (Figure 1)

Summary

Introduction

The major cell death pathways, classified as autophagic, apoptotic, and necrotic, work to maintain homeostasis in the organism. Dysregulation of these pathways leads to several pathologies. There is a need for a specific and sensitive technology to identify and modulate the pathway [1]-[4]. Based on enzyme-substrate dynamics and consists of a reporter gene fused between fluorescent proteins. 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

Methods

Results

Discussion

Conclusion