Multi-approached structural analysis of ChAT Enzyme-interactions with micelles and ligand : SPA, Micro-ED, and Mass photometry experiments

Sammanfattning

Current treatments for neurodegenerative diseases such as Alzheimer’s’ Disease (AD) and Amyotrophic lateral sclerosis (ALS) mainly target Amyloid-β plaques or inhibit the enzyme Acetylcholinesterase (AchE) which breaks down Acetylcholine (Ach). A novel possible alternative method of action for treatment of these diseases is to potentiate the enzyme Choline acetyltransferase (ChAT) which synthesizes acetylcholine from choline and acetate. This thesis will investigate two potentiating agents, ChAT-Potentiating Ligand 75 (CPL75), and the micelle forming surfactant Tween 20 (TW20). Although they have both been shown to increase recombinant human ChAT (rhChAt) activity, the mechanism of which they do so is still unknown. This thesis therefore aims to investigate complexes between rhChAt and the two potentiators. To achieve this, two Cryogenic Electron Microscopy (Cryo-EM) techniques are utilized, namely Single Particle Analysis (SPA) and Microcrystal Electron Diffraction (Micro-ED), as well as mass photometry. In terms of results, microcrystals were grown and imaged on the electron microscope using wildtype (WT) rhChAt, and larger short-lived microcrystals were grown and captured on the optical microscope using surface-entropy reduced rhChAt. Crystallization conditions presented in this report therefore provide a good starting point for further research on rhChAt ligand binding. SPA attempts yielded anisotropic and low-resolution results, despite application of several computational and experimental tools. Although whether TW20 micelle-ChAt complexes occur is still unclear, a new hypothesis, regarding an increase in dimer composition of rhChAt in the presence of TW20 was conceived. This hypothesis was then tested using mass photometry. Results presented in this report therefore lay some of the groundwork of the path from ligand to drug candidate within this novel targeting of AD and ALS pathology.