Altered GPI modification of insect AChE improves tolerance to organophosphate insecticides.

The olive fruit fly Bactrocera oleae is the most destructive and intractable pest of olives. The management of B. oleae has been based on the use of organophosphate (OP) insecticides, a practice that induced resistance.

The PRiMA-linked cholinesterase tetramers are assembled from homodimers: hybrid molecules composed of acetylcholinesterase and butyrylcholinesterase dimers are up-regulated during development of chicken brain.

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)).

Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons.

In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100.

Unusual transfer of CutA into the secretory pathway, evidenced by fusion proteins with acetylcholinesterase.

The mouse CutA protein exists as long and short components of 20 and 15 kDa, produced by the use of different in-frame ATGs initiation codons, and by proteolytic cleavage. We recently showed that, surprisingly, the longer, uncleaved component resides mostly in the secretory pathway and is secreted, whereas the shorter component resides mostly in the cytoplasm. To confirm these subcellular localizations, we constructed fusion proteins in which the catalytic domain of rat acetylcholinesterase was placed downstream of the CutA variants.

Cholinesterases and the basal lamina at vertebrate neuromuscular junctions.

Macromolecules of the cholinergic basal lamina are essential elements of the complex signaling processes governing development, function, and repair of the vertebrate neuromuscular junction. One special form of acetylcholinesterase (AChE) is anchored within BL through a collagen tail (ColQ) that binds heparan sulfate proteoglycans, such as perlecan, and the post-synaptic muscle specific kinase MuSK. New experimental approaches are probing the spatio-temporal dynamics of ColQ-AChE over days or weeks in vivo, thereby unraveling its interactions with other BL components, as well as pre-and post-synaptic elements.

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").

Respective roles of the catalytic domains and C-terminal tail peptides in the oligomerization and secretory trafficking of human acetylcholinesterase and butyrylcholinesterase.

Butyrylcholinesterase (BChE) and the T splice variant of acetylcholinesterase that is predominant in mammalian brain and muscles (AChE(T)) possess a characteristic C-terminal tail (t) peptide. This t peptide allows their assembly into tetramers associated with the anchoring proteins ColQ and PRiMA. Although the t peptides of all vertebrate cholinesterases are remarkably similar and, in particular, contain seven strictly conserved aromatic residues, these enzymes differ in some of their oligomerization properties.

Old and new questions about cholinesterases.

Cholinesterases have been intensively studied for a long time, but still offer many fascinating and fundamental questions regarding their evolution, activity, biosynthesis, folding, post-translational modifications, association with structural proteins (ColQ, PRiMA and maybe others), export or degradation. They constitute an excellent model to study these processes, particularly because of the sensitivity and specificity of enzymic assays. In addition, a number of provocative ideas concerning their proposed non-conventional, or non-catalytic functions deserve to be further documented.

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA.

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer).

The C-terminal T peptide of cholinesterases: structure, interactions, and influence on protein folding and secretion.

Mammalian cholinergic tissues mostly express the T splice variant of acetylcholinesterase, in which the catalytic domain is associated with a C-terminal peptide of 40 residues, called the t peptide (Massoulié, 2002). Homologous t peptides exist in all vertebrate cholinesterases, acetylcholinesterases (AChEs), and butyrylcholinesterases (BChEs): they contain a series of seven conserved aromatic residues, including three tryptophans, and a cysteine at position-4 of their C-terminus. The major AChE isozyme of the nematode Caenorhabditis elegans also contains a similar peptide.

Readthrough acetylcholinesterase expression remains minor after stress or exposure to inhibitors.

The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002).

Assembly of acetylcholinesterase tetramers by peptidic motifs from the proline-rich membrane anchor, PRiMA: competition between degradation and secretion pathways of heteromeric complexes.

The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChE(T)), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain.

The C-terminal peptides of acetylcholinesterase: cellular trafficking, oligomerization and functional anchoring.

In vertebrates, the catalytic domain of acetylcholinesterase (AChE) may be associated with several C-terminal peptides generated by alternative splicing in the 3' region of transcripts. The "readthrough" (R) variant results from a lack of splicing after the last exon encoding the catalytic domain. Such a variant has been observed in Torpedo and in mammals; its C-terminal r peptide, also called "AChE Related Peptide" (ARP), is poorly conserved between rodents and humans.

The readthrough variant of acetylcholinesterase remains very minor after heat shock, organophosphate inhibition and stress, in cell culture and in vivo.

Acetylcholinesterase (AChE) exists in various molecular forms, depending on alternative splicing of its transcripts and association with structural proteins. Tetramers of the 'tailed' variant (AChE(T)), which are anchored in the cell membrane of neurons by the PRiMA (Proline Rich Membrane Anchor) protein, constitute the main form of AChE in the mammalian brain. In the mouse brain, stress and anticholinesterase inhibitors have been reported to induce expression of the unspliced 'readthrough' variant (AChE(R)) mRNA which produces a monomeric form.

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix.

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD.

Determinants of the t peptide involved in folding, degradation, and secretion of acetylcholinesterase.

The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic alpha helix with a cluster of seven aromatic residues. It allows oligomerization and induces a partial degradation of AChE subunits through the endoplasmic reticulum-associated degradation pathway. We show that the t peptide induces the misfolding of a fraction of AChE subunits, even when mutations disorganized the cluster of aromatic residues or when these residues were replaced by leucines, indicating that this effect is due to hydrophobic residues.

Transcriptional regulation of acetylcholinesterase-associated collagen ColQ: differential expression in fast and slow twitch muscle fibers is driven by distinct promoters.

The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase and butyrylcholinesterase at vertebrate neuromuscular junctions which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow and fast twitch muscles in mammals. Two distinct promoters, pColQ-1 and pColQ-1a, were isolated from the upstream sequences of human COLQ gene; they showed muscle-specific expression and were activated by myogenic transcriptional elements in cultured myotubes.

Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation.

The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic alpha-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChE(T) on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with Q(N), a PRAD-containing N-terminal fragment of ColQ.

The C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports homomeric and heteromeric interactions.

Acetylcholinesterase subunits of type T (AChET) possess an alternatively spliced C-terminal peptide (t peptide) which endows them with amphiphilic properties, the capacity to form various homo-oligomers and to associate, as a tetramer, with anchoring proteins containing a proline rich attachment domain (PRAD). The t peptide contains seven conserved aromatic residues. By spectroscopic analyses of the synthetic peptides covering part or all of the t peptide of Torpedo AChET, we show that the region containing the aromatic residues adopts an alpha helical structure, which is favored in the presence of lipids and detergent micelles: these residues therefore form a hydrophobic cluster in a sector of the helix.

Expression of PRiMA in the mouse brain: membrane anchoring and accumulation of 'tailed' acetylcholinesterase.

We analysed the expression of PRiMA (proline-rich membrane anchor), the membrane anchor of acetylcholinesterase (AChE), by in situ hybridization in the mouse brain. We compared the pattern of PRiMA transcripts with that of AChE transcripts, as well as those of choline acetyltransferase and M1 muscarinic receptors which are considered pre- and postsynaptic cholinergic markers. We also analysed cholinesterase activity and its molecular forms in several brain structures.

The C-terminal T peptide of acetylcholinesterase enhances degradation of unassembled active subunits through the ERAD pathway.

The catalytic domain of acetylcholinesterase AChE(T) subunits is followed by a C-terminal T peptide which mediates their association with the proline-rich attachment domain (PRAD) of anchoring proteins. Addition of the T peptide induced intracellular degradation and concomitantly reduced to variable degrees the secretion of AChE species differing in their oligomerization capacity and of human alkaline phosphatase. The T peptide forms an amphiphilic alpha-helix, containing a series of conserved aromatic residues.

Trimerization domain of the collagen tail of acetylcholinesterase.

In the collagen-tailed forms of cholinesterases, each subunit of a specific triple helical collagen, ColQ, may be attached through a proline-rich domain (PRAD) situated in its N-terminal noncollagenous region, to tetramers of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). This heteromeric assembly ensures the functional anchoring of AChE in extracellulare matrices, for example, at the neuromuscular junction. In this study, we analyzed the influence of deletions in the noncollagenous C-terminal region of ColQ on its capacity to form a triple helix.

Transcriptional regulation of gene expression by the coding sequence: An attempt to enhance expression of human AChE.

In a previous report, Morel and Massoulié showed that Bungarus AChE (bBAChE) is produced more efficiently than rat AChE in various expression systems, mainly because the Bungarus coding sequence exerts a stimulatory effect on transcription (Morel and Massoulié, 2000). They reported that a 5' Bungarus fragment could partially transfer this property to a CAT expression vector. This appeared to offer the possibility of increasing the production of recombinant proteins.

The origin of the molecular diversity and functional anchoring of cholinesterases.

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain.