Résumé :
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Acetylcholinesterase (AChE) rapidly hydrolyzes the neurotransmitter ACh at central and peripheral synapses to restore the excitability of the postsynaptic membrane of neurons and muscles. AChE inhibitors are used to attenuate the cognitive or functional deficiencies associated with, eg. Alzheimer's disease or myasthenia gravis. The AChE active site is buried at the center of the molecule at the bottom of a deep, narrow gorge. This gorge is the only identified way for the opposite traffics of ACh and its hydrolysis products. The apparent discrepancy of this geometry with the AChE high activity has suggested that a back door (BD), distinct from the active site gorge entrance, must exist to permit a fast exit of the products and/or alternative ACh entry. This BD may also be involved in a new mechanism for regulation of AChE catalysis.Our aim is to prove the existence of the BD and document its structure and functioning as a regulatory site. This will allow us to reconcile the particular geometry and high activity of AChE, and to propose a new target site for new therapeutic drugs with higher specificity.Our model is eel AChE (EeAChE), because 1) the EeAChE subunit associates in tetramers similar to those found in mammals and has a high activity suggesting a very efficient BD, 2) we have inhibitors which bind EeAChE either at the gorge entrance or in the BD region. Such ligands are not available for other AChEs, 3) preliminary structural results obtained in our team point to several residues as belonging to the BD region.We designed a soluble wild type EeAChE (EeAChE-wt), produced it in HEK cells and purified it. Analysis of its structural and functional features, using biochemistry, pharmacology and enzymology techniques, showed that EeAChE-wt is representative of natural EeAChE. Then we mutated residues suspected to belong to the BD region. The mutants were produced and purified, and analyzed for homogeneity, stability and sensitivity to inhibitors. Four mutants display the expected properties while three others are partially misfolded and weakly active, suggesting that functional and structural determinants, respectively, were altered. We also produced sufficient EeAChE-wt amounts to start a structural study. A preliminary X-ray dataset has been collected. We are trying to improve the resolution and to crystallize a mutant with a possibly enlarged BD. Current and future data will allow us to describe, at the structural and functional levels, the BD of AChE.
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