Identifying potential drug candidates by way of coupling luciferase gene expressions and the interaction of TREM2, TYROBP proteins
Alzheimer's Disease (AD) is the leading cause of dementia and is currently the sixth leading cause of death in the United States. For the past 113 years AD has seen minimal progress in delaying progression and preventing disease onset. Our strategy is to target the TREM2 (Triggering Receptor Expressed on Myeloid cells 2) protein found in microglial cells to induce activation and potentially increase uptake of toxic aggregates. For this response to happen we need to identify potential drug candidates that trigger TREM2 receptors to activate the TYROBP (TYRO Kinase Binding Protein) signaling pathway. This project was to design and construct a TREM2/TYROBP split luciferase assay. Through the use of polymerase chain reaction (PCR), restriction digestion, ligation, transformation, and transfection cloning methods our construct can be created. When the TREM2 receptor becomes agonised by an introduced drug, the receptor will move through the phospholipid bi layer until reaching close proximity to the TYROBP structure. The oxidative enzyme known as luciferase will activate its bioluminescent capabilities when the C-terminal luciferase enzyme (attached to TREM2) couples with its counterpart, N-terminal luciferase (attached to TYROBP). The luminescence produced will signify a successful triggering of the desired pathway, and the amount of luminescence produced can be used to judge a potential drug agonists effectiveness. An effective agonist of the TREM2 signaling receptor through the TYROBP signaling pathway will make for a contender in future clinical trials.
