Study Of Proteins Involved In The Oligomerization, Secretion, Membrane Anchoring And Functional Localization Of Acetylcholinesterase

The major role of acetylcholinesterase(AChE) is to hydrolyze the neurotransmitter acetylcholine after its release by cholinergic neurons.This function depends on the correct localization of AChE,which in turn depends on its oligomerization,on its association with anchoring proteins such as the transmembrane protein PRiMA,and probably on further interactions of these complexes with membrane sub-domains and/or extra-or intracellular macromolecular scaffolds.In this thesis,we explored several of these aspects.We first studied the respective roles of the catalytic domain and of the C-terminal t peptide in the oligomerization of cholinesterases.For this,we compared the T splice variant of human AChE,which represents the major AChE variant expressed in cholinergic tissues(AChE_T),and human butyrylcholinesterase(BChE).We constructed chimeras in which the t peptides were exchanged,and we introduced various mutations in these peptides.We thus found that both the catalytic domain and the t peptide contribute to the oligomerization and secretability of cholinesterases.In the mammalian brain,the major AChE species consists of a tetramer of AChE_T subunits,anchored in cell membranes through its association with PRiMA.PRiMA possesses two splice variants,which differ by their C-terminal intracellular regions (40 residues for the PRiMAâ… variant and 7 residues for the minor PRiMAâ…¡variant).The existence of two variants differing in their C-terminal regions,and the possibility of post-translational modifications suggest that these regions might interact with intracellular proteins,possibly with the cytoskeleton.We therefore undertook a search for such potential partners by two-hybrid strategies,both in yeast and in E. coli,using the C-terminal 40 residues of PRiMAâ… as bait,and cDNA libraries derived from human brain.Five potential partners were obtained several times,and one of those obtained from the yeast screen appeared also positive in the bacterial screen. This corresponds to a zinc-finger rich protein,which may act as a transcriptional regulator but was also shown to bind a short C-terminal peptide from a membrane receptor.However,our attempts to validate its binding to PRiMAâ… by co-immuno-precipitation were unsuccessful,so that this cannot be published.The AChE-PRiMA complex is at least partially contained in lipid rafts,both in the rat brain and in transfected neuroblastoma and COS cells.The C-terminal region of PRiMAâ… contains serine residues that may be phosphorylated,and a cysteine that may be palmitoylated.By mutating these residues,we showed that such modifications are nor required for recruitment of the complex in the rafts.Another potential partner of the AChE-PRiMA complex is the mCutA protein. This protein was discovered in mammals as a component of purified AChE from brain,and was first mistaken as the membrane anchor of this enzyme,before PRiMA was identified.The mCutA protein is homologous to a bacterial protein involved in resistance to divalent cations;it is ubiquitously expressed in mammalian tissues,but its function is unknown.It was thought to participate in the formation of the AChE-PRiMA complex.In an attempt to clarify this question,we first studied the subcellular localization of the two splice variants of mCutA that exist in mouse.These variants differ in their N-terminal region,by the use of two translation initiation sites.Using mutants in which we introduced epitopes at different positions and mutated the N-terminal methionines,we find that the longer form of mCutA is at least partly transferred in the endoplasmic reticulum and secreted,but without cleavage of its N-terminal peptide,while the shorter form is mostly cytoplasmic.This was illustrated by the fact that a fusion protein in which AChE was added,without its secretory signal peptide,at the C-terminus of mCutA was catalytically active and secreted,mCutA may thus interact with AChE in the secretory pathway.We found that co-expression of the longer form of mCutA reduces the production of active AChE_T,and that this effect depends on the C-terminal t peptide.We also found that co-expression with mCutA increased the formation of tetramers of AChE_T subunits,an effect which is particularly clear in the case of mutants lacking a C-terminal cysteine.Taken together, these results suggest that mCutA may act as a kind of chaperone in the endoplasmic reticulum,facilitating tetramerization of AChE_T in a direct or indirect manner.