Molecular Forms of Acetylcholinesterase

Several molecular forms of acetylcholinesterase are obtained from Electrophorus or Torpedo electric organs. They have been characterized by physico-chemical methods and observed by electron microscopy. The most complex D form is made up of a globular »head« containing probably twelve subunits, or three tetrameric groups of subunits, attached to a rod like tail. Two other asymmetric forms, C and A, may be derived from it by removal of one or two tetramers from the »head«. These forms can ultimately be degraded by proteolytic digestion or sonication into tetrameric and dimeric active enzymes, G and G\u27. No striking difference in the catalytic properties of these forms could be demonstrated. An analysis of their thermal denaturation suggests · that internal breaks may exist in the polypeptide chains without being revealed ,in catalytic or sedimentation properties of the molecules. /\u27:,, H=I= values demonstrate stabilizing interactions in the more complex molecules. Analysis of subunits by SDS polyacrylamide gel electrophoresis shows that one main 90 000 subunit is progressively split into a 60 000 DFP-labelled chain together with smaller peptides jn the 30 000 range. No difference could be found between D and G subunit patterns which could ibe identified to the tail component. Comparing the molecular weight of A (one tetramer plus ta il) and G (tetramer), one finds that the mass of the tail should be in the 60 000-80 000 range. Recent micrographs suggest that it consists of three strands linked to the three tetramers in the head of D. We therefore propose a three stranded collagen like structure for the tail. We discuss the possible physiological role of the asymmetric structure o.f acetylcholinesterase and its implication with the membrane association of the enzyme. Multiple forms of acetylcholinesterase are not genetically determined i,sozymes but rather represent different states of association of the active monomern. The significance of multiple forms of acetylcholinesterase, especially in mammals, is considered

Heterologous Amyloid Seeding: Revisiting the Role of Acetylcholinesterase in Alzheimer's Disease

Neurodegenerative diseases associated with abnormal protein folding and ordered aggregation require an initial trigger which may be infectious, inherited, post-inflammatory or idiopathic. Proteolytic cleavage to generate vulnerable precursors, such as amyloid-β peptide (Aβ) production via β and γ secretases in Alzheimer's Disease (AD), is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Aβ in the central nervous system are regulated by several catabolic proteases, including insulysin (IDE) and neprilysin (NEP). The known association of human acetylcholinesterase (hAChE) with pathological aggregates in AD together with its ability to increase Aβ fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE575-614) is an exposed amphiphilic α-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR) with high propensity for conversion to non-native (hidden) β-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE586-599) encompassing the CSR region shares homology with Aβ and forms β-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE β-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE586-599, or versus Aβ, differed. Moreover, IDE digestion of T40 triggered the conformational switch from α- to β-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers). Crucially, these CSR species significantly increased Aβ fibril formation both by seeding the energetically unfavorable formation of amyloid nuclei and by enhancing the rate of amyloid elongation. Hence, these results may offer an explanation for observations that implicate hAChE in the extent of Aβ deposition in the brain. Furthermore, this process of heterologous amyloid seeding by a proteolytic fragment from another protein may represent a previously underestimated pathological trigger, implying that the abundance of the major amyloidogenic species (Aβ in AD, for example) may not be the only important factor in neurodegeneration

Rôle du peptide C-terminal de l'acétylcholinestérase dans l'oligomérisation et la dégradation

L'acétylcholinestérase joue un rôle crucial dans la transmission cholinergique, en hydrolysant le neurotransmetteur, l'acétylcholine. Pour une transmission cholinergique efficace, il est nécessaire que l'AChE soit correctement localisée à la synapse ; ceci est assuré par son association avec les deux protéines d'ancrage ColQ et PRiMA. Ces protéines contiennent un domaine riche en prolines nommé PRAD qui organise les sous-unités T en tétramères, par interaction avec leur peptide C-terminal t nommé WAT. Le peptide t organisé en hélice amphiphile contient 40 résidus dont une série de résidus aromatiques conservés chez tous les vertébrés. L'objectif de cette thèse a été d'identifier les éléments du peptide t qui déterminent l'organisation, l'association avec les protéines d'ancrage et le devenir des sous-unités de type T. Les résidus aromatiques W et F sont impliqués dans l'interaction avec le PRAD et la dégradation. Les sous-unités d'AChE non assemblées au PRAD sont dégradées par la voie ERAD, contrairement aux hétéro-oligomères fonctionnels efficacement sécrétés.Acetylcholinesterase plays a crucial role in cholinergic transmission, by hydrolyzing acetylcholine ; It depends on the precise localisation of the enzyme at the synapses, ensured by its association with two anchoring proteins, CdQ and PRiMA. These anchors contain a proline rich motif, named PRAD, which organise acetylcholinesterase tetramers by interacting with their C-terminal peptide, named WAT. The t peptide is organised in amphiphilic helix which contains 40 residues, and a serie of conserved aromatic residues. Aromatic residues are involved in the interaction with a PRAD and the degradation of the enzyme. Unassembled subunits are degraded by the ERAD mechanism, contrary to the functionnal hetero-oligomers which are officiently secreted.PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

Etude de l'association entre l'acétylcholinestérase et sa protéine d'ancrage membranaire "PRiMA"

Le système nerveux et les muscles des mammifères expriment le variant T de l'acétylcholinestérase (AChET), associé à des protéines d'ancrage ColQ et PRiMA produisant respectivement des formes avec des queues collagéniques et des tétramères membranaires. Les formes collagéniques sont ancrés dans la lame basale de la jonction neuro-musculaire, tandis que les tétramères membranaires sont ancrés à la surface cellulaire par le domaine transmembranaire de PRiMA. Les tétramères membranaires constituent la forme majoritaire de l'enzyme dans le cerveau. L'association de l'AChET avec ColQ a été bien étudiée : elle est basée sur l'interaction de 4 peptides t avec un motif riche en pralines, appelé PRAD ("Proline-Rich Attachment Domain"), localisé dans la région N-terminale de ColQ. L'association de l'AChET avec PRiMA paraît similaire puisque cette protéine transmembranaire contient aussi un PRAD, mais qui est significativement différent par le nombre de pralines (8 dans ColQ, 14 dans PRiMA) et par le nombre et positions des cystéines qui peuvent former des ponts disulfures avec les cystéines C-terminales des peptides t. Donc, nous avons analysé l'association de l'AChET avec PRiMA. En effectuant des délétions et des mutations dans PRiMA, nous avons défini un motif peptidique, suffisant pour l'interaction avec les sous-unités AChET.The nervous tissue and muscles of mammals express the T splice variant of acetylcholinesterase (ACHET), associatied with anchoring proteins, ColQ and PRiMA, producing respectively collagen-tailed forms and membrane-bound tetramers. These interactions are important since they condition the functional anchoring of AChE in cholinergic tissues. The collagen-tailed forms are inserted in the basal lamina at neuromuscular junction, while the membrane-bound tetramers are anchored at the cell surface through the transmembrane domain of PRiMA. The membrane-bound tetramers represent the major enzyme species in the brain. The association of AChEr subunits with ColQ has been extensively studied : it is based on an tight interaction between four t peptides and a praline-rich motif, called PRAD ("Proline-Rich Attachment Domain"), located in the N-terminal region of ColQ. The association of AChEj subunits with PRiMA appears similar because this transmembrane protein also contains a proline-rich motif, but there are significant differences in the number of pralines (8 in ColQ, 14 in PRiMA) and in the number and positions of cysteines that might form intercatenary disulfide bonds with the cysteine located near the C-terminus of the t peptides. Therefore, we have undertaken an analysis of the association of AChET with PRiMA. Using deletions and point mutations in PRiMA, we defined a minimal motif in PRiMA, which could associate with AChEr subunits.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Expression et accumulation fonctionnelle de l'acétylcholinestérase dans le système nerveux central

PARIS5-BU Saints-Pères (751062109) / SudocSudocFranceF

Etude de protéines impliquées dans l'oligomérisation, la sécrétion, l'ancrage membranaire et la localisation fonctionnelle de l'acétylcholinestérase dans le cerveau des mammifères

Both the catalytic domains and the C-terminal t peptides contribute to the capacity of cholinesterases to form and secrete various oligomers. 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 potential phosphorylation sites and palmitoylation sites are not required for recruitment of the complex in the rafts. We undertook a search for such potential partners by two-hybrid strategies, both in yeast and in E. coli. 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. The CutA protein affects the processing of proteins in the secretory pathway, facilitating tetramerization of AChEt.Nous avons construit des protéines chimères dans lesquelles les peptides t ont été échangés entre les deux cholinestérases, et nous avons introduit diverses mutations dans ces peptides. Nous avons montré que le domaine catalytique et le peptide t contribuent à la capacité d'oligomérisation et à la sécrétion des cholinestérases. Nous avons entrepris de rechercher de tels partenaires potentiels, en utilisant la stratégie de double-hybride, chez la levure et chez E. coli, avec la région C-terminale de PRiMA I comme appât et des banques d'ADNc issues de cerveau humain. Cinq partenaires potentiels ont été obtenus plusieurs fois chez la levure Le complexe AChE-PRiMA est au moins partiellement intégré dans des sous-domaines membranaires appelés rafts , aussi bien dans le cerveau de rat que dans des cellules de neuroblastomes ou des cellules COS transfectées. En mutant ces résidus, nous avons montré que les modifications post tranductionelle ne sont pas nécessaires pour le recrutement dans les rafts . Nous avons trouvé que l'expression de la forme longue de mCutA réduit la production d'AChEt et que ceci dépend du peptide C-terminal t. Nous avons également observé que la co-expression avec mCutA augmente la formation de tétramères et facilite la tétramérisation de l'AChEy de manière directe ou indirectePARIS5-BU Méd.Cochin (751142101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Formes moleculaires de l'acetylcholinesterase

SIGLECNRS-CDST / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Protein CutA Undergoes an Unusual Transfer into the Secretory Pathway and Affects the Folding, Oligomerization, and Secretion of Acetylcholinesterase

The mammalian protein CutA was first discovered in a search for the membrane anchor of mammalian brain acetylcholinesterase (AChE). It was co-purified with AChE, but it is distinct from the real transmembrane anchor protein, PRiMA. CutA is a ubiquitous trimeric protein, homologous to the bacterial CutA1 protein that belongs to an operon involved in resistance to divalent ions (''copper tolerance A''). The function of this protein in plants and animals is unknown, and several hypotheses concerning its subcellular localization have been proposed. We analyzed the expression and the subcellular localization of mouse CutA variants, starting at three in-frame ATG codons, in transfected COS cells. We show that CutA produces 20-kDa (H) and 15-kDa (L) components. The H component is transferred into the secretory pathway and secreted, without cleavage of a signal peptide, whereas the L component is mostly cytosolic. We show that expression of the longer CutA variant reduces the level of AChE, that this effect depends on the AChE C-terminal peptides, and probably results from misfolding. Surprisingly, CutA increased the secretion of a mutant possessing a KDEL motif at its C terminus; it also increased the formation of AChE homotetramers. We found no evidence for a direct interaction between CutA and AChE. The longer CutA variant seems to affect the processing and trafficking of secretory proteins, whereas the shorter one may have a distinct function in the cytoplasm