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Development of Highly Sensitive FRET Based Biosensor to Detect Cell Death Pathways

  • Author(s): Hariharan, Chitra
  • Advisor(s): Liao, Jiayu
  • et al.
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. Principle cell death pathways include

apoptosis, autophagy, necrosis, mitotic catastrophe etc. Knowledge of cell death pathways and the reason the cell chooses to die one way or the other, 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 apoptosis and autophagy pathways in vitro.

FRET is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores that are in close proximity. 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 method. To take this forward, the sensor will be transfected in H460 lung cancer cell line to identify the type of death the cell chooses on treatment with sumoylation inhibitors that were previously developed in the lab. MTS assay was conducted to establish the supremacy of sumoylation inhibitors over other commercially available drugs.

In conclusion, the biosensor developed in this study is highly sensitive in detecting apoptosis and autophagy and can be used to derive quantitative data using FRET technology. It can be used both in vitro and in mammalian cells and can differentiate between apoptosis and autophagy. The results of this study can help to expand biomedical knowledge by illuminating the mechanisms of different cell death pathways. This will pave way for simple and non-invasive ways to modulate cell death pathways for therapeutic intervention in the future.

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