RESUMO
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. OP-resistance in the olive fly was previously shown to be associated with two mutations in the acetylcholinesterase (AChE) enzyme that, apparently, hinder the entrance of the OP into the active site. The search for additional mutations in the ace gene that encodes AChE revealed a short deletion of three glutamines (Δ3Q) from a stretch of five glutamines, in the C-terminal peptide that is normally cleaved and substituted by a GPI anchor. We verified that AChEs from B. oleae and other Dipterans are actually GPI-anchored, although this is not predicted by the "big-PI" algorithm. The Δ3Q mutation shortens the unusually long hydrophilic spacer that follows the predicted GPI attachment site and may thus improve the efficiency of GPI anchor addition. We expressed the wild type B. oleae AChE, the natural mutant Δ3Q and a constructed mutant lacking all 5 consecutive glutamines (Δ5Q) in COS cells and compared their kinetic properties. All constructs presented identical K(m) and k(cat) values, in agreement with the fact that the mutations did not affect the catalytic domain of the enzyme. In contrast, the mutants produced higher AChE activity, suggesting that a higher proportion of the precursor protein becomes GPI-anchored. An increase in the number of GPI-anchored molecules in the synaptic cleft may reduce the sensitivity to insecticides.
Assuntos
Acetilcolinesterase/genética , Resistência a Inseticidas , Tephritidae/genética , Algoritmos , Substituição de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Glucose-6-Fosfato Isomerase/metabolismo , Inseticidas/metabolismo , Dados de Sequência Molecular , Mutação , Compostos Organofosforados/metabolismo , Deleção de Sequência , Tephritidae/enzimologiaRESUMO
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)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.
Assuntos
Acetilcolinesterase/biossíntese , Encéfalo/embriologia , Butirilcolinesterase/biossíntese , Galinhas , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas de Membrana/biossíntese , Complexos Multienzimáticos/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Multimerização Proteica/fisiologia , Regulação para Cima/fisiologia , Acetilcolinesterase/genética , Animais , Encéfalo/citologia , Encéfalo/enzimologia , Butirilcolinesterase/genética , Células Cultivadas , Embrião de Galinha , Proteínas de Membrana/genética , Complexos Multienzimáticos/genética , Mutação , Proteínas do Tecido Nervoso/genética , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transmissão Sináptica/fisiologiaRESUMO
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. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChE(T) and PRiMA, PRiMA-linked G(4) AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G(2) AChE or G(4) Q(N)-H(C)-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G(4) AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.
Assuntos
Acetilcolinesterase/metabolismo , Encéfalo/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Prolina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Humanos , Masculino , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de AminoácidosRESUMO
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. To explore these differences, we studied human AChE (Aa) and BChE (Bb), and chimeras in which the t peptides (a and b) were exchanged (Ab and Ba). We found that secretion was increased by deletion of the t peptides, and that it was more efficient with a than with b. The patterns of oligomers were similar for Aa and Ab, as well as for Ba and Bb, indicating a predominant influence of the catalytic domains. However, addition of a cysteine within the aromatic-rich segment of the t peptides modified the oligomeric patterns: with a cysteine at position 19, the proportion of tetramers was markedly increased for Aa(S19C) and Ba(S19C), and to a lesser extent for Bb(N19C); the Ab(N19C) mutant produced all oligomeric forms, from monomers to hexamers. These results indicate that both the catalytic domains and the C-terminal t peptides contribute to the capacity of cholinesterases to form and secrete various oligomers. Sequence comparisons show that the differences between the t peptides of AChE and BChE are remarkably conserved among all vertebrates, suggesting that they reflect distinct functional adaptations.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Estrutura Secundária de Proteína , Acetilcolinesterase/genética , Sequência de Aminoácidos , Animais , Butirilcolinesterase/genética , Domínio Catalítico , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Fragmentos de Peptídeos/química , Conformação Proteica , Vertebrados/metabolismoRESUMO
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). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.
Assuntos
Acetilcolinesterase/química , Colágeno/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Acetilcolinesterase/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Colágeno/química , Cisteína/química , Dimerização , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/químicaRESUMO
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. Expression of AChE(T) subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (P(stp54) mutant), produced secreted heteromeric complexes (T4-P(stp54)), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChE(T) subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChE(T) subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChE(T) subunits induces their intracellular degradation.
Assuntos
Acetilcolinesterase/química , Proteínas de Membrana/fisiologia , Complexos Multiproteicos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Peptídeos/química , Transdução de Sinais , Acetilcolinesterase/metabolismo , Acetilcolinesterase/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Células COS , Domínio Catalítico/genética , Chlorocebus aethiops , Cricetinae , Espaço Extracelular/química , Espaço Extracelular/genética , Espaço Extracelular/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/fisiologia , Transporte Proteico/genética , Ratos , Deleção de Sequência/genética , Transdução de Sinais/genéticaRESUMO
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). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).
Assuntos
Acetilcolinesterase/genética , Encéfalo/enzimologia , Inibidores da Colinesterase/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Estresse Psicológico/enzimologia , Animais , Linhagem Celular Tumoral , Masculino , Camundongos , Neuroblastoma , RNA Mensageiro/genética , Restrição Física , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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. Although the C-terminal t peptides do not seem to affect the catalytic activity of cholinesterases, they determine their physiological function, because they allow cholinesterase subunits of type T to form oligomers and to associate with structural anchoring proteins. When reduced to their catalytic domain, AChE subunits without a t peptide are active but remain monomeric and soluble.
Assuntos
Colinesterases/metabolismo , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Colinesterases/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Dobramento de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
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. In rodents, it is significantly expressed in embryonic tissues and at a very low level in the brain of adult mice; it may be increased under various stress conditions, but remains very low. The "hydrophobic" (H) variant generates glycolipid (GPI)-anchored dimers, which are expressed in muscles of Torpedo, and in blood cells of mammals; H variants exist in Torpedo and in mammals, but apparently not in other vertebrate classes, suggesting that they were lost during evolution of early vertebrates and re-appeared independently in mammals. The "tailed" (T) variant exists in all vertebrate cholinesterases and their C-terminal t peptides are strongly conserved; in mammals, AChE(T) subunits represent the major type of acetylcholinesterase in cholinergic tissues. They produce a wide variety of oligomeric forms, ranging from monomers to heteromeric assemblies containing the anchoring proteins ColQ (collagen-tailed forms) and PRiMA (membrane-bound tetramers), which constitute the major functional enzyme species in mammalian muscles and brain, respectively. The oligomerization of AChE(T) subunits depends largely on the properties of their C-terminal t peptide. These peptides contain seven conserved aromatic residues, including three tryptophans, and are organized in an amphiphilic alpha helix in which these residues form a hydrophobic cluster. The presence of a cysteine is required for dimerization, while aromatic residues are necessary for tetramerization. In the collagen-tailed molecules, four t peptides form a coiled coil around a proline-rich motif (PRAD) located in the N-terminal region of ColQ. The t peptide also strongly influences the folding and cellular trafficking of AChE(T) subunits: the presence of hydrophobic residues induces partial misfolding leading to inactive protein, while aromatic residues, organized or not in an amphiphilic helix, induce intracellular degradation through the "Endoplasmic Reticulum Associated Degradation" (ERAD) pathway, rather than secretion. It has been proposed that the r and t C-terminal peptides, or fragments of these peptides, may exert independent, non cholinergic biological functions: this interesting possibility still needs to be documented, especially in view of their various degrees of evolutionary conservation.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Acetilcolinesterase/genética , Animais , Humanos , Fragmentos de Peptídeos/genética , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Transporte ProteicoRESUMO
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. To generalize this observation, we attempted to quantify AChE(R) and AChE(T) after organophosphate intoxication in the mouse brain and compared the observed effects with those of stress induced by swimming or immobilization; we also analyzed the effects of heat shock and AChE inhibition on neuroblastoma cells. Active AChE molecular forms were characterized by sedimentation and non-denaturing electrophoresis, and AChE transcripts were quantified by real-time PCR. We observed a moderate increase of the AChE(R) transcript in some cases, both in the mouse brain and in neuroblastoma cultures, but we did not detect any increase of the corresponding active enzyme.
Assuntos
Acetilcolinesterase/metabolismo , Processamento Alternativo/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Temperatura Alta , Soman/farmacologia , Estresse Fisiológico/enzimologia , Acetilcolinesterase/química , Acetilcolinesterase/genética , Acetilcolinesterase/farmacologia , Processamento Alternativo/fisiologia , Animais , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Ácido Desoxicólico/farmacologia , Detergentes/farmacologia , Interações Medicamentosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neuroblastoma , Octoxinol/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/biossíntese , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
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. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.
Assuntos
Acetilcolinesterase/química , Peptídeos/química , Conformação Proteica , Sinapses/química , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Colágeno/química , Cristalografia por Raios X , Dissulfetos/química , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/química , Peptídeos/metabolismo , Mutação Puntual , Estrutura Terciária de Proteína , Análise Espectral Raman , Sinapses/metabolismo , Triptofano/químicaRESUMO
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 formation of homo-tetramers and Q(N)-linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32-40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T(4) and T(4)-Q(N) tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with Q(N), indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the alpha-helical structure of the t peptide were found to enhance degradation. Co-expression with Q(N) generally increased secretion, mostly as T(4)-Q(N) complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChE(T) subunits and the choice between secretion and degradation.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Acetilcolinesterase/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Animais , Células COS , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Torpedo , TransfecçãoRESUMO
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. After in vivo DNA transfection, pColQ-1 showed strong activity in slow twitch muscle (e.g. soleus), whereas pColQ-1a was preferably expressed in fast twitch muscle (e.g. tibialis). Mutation analysis of the ColQ promoters suggested that the muscle fiber type-specific expression pattern of ColQ transcripts were regulated by a slow upsteam regulatory element (SURE) and a fast intronic regulatory element (FIRE). These regulatory elements were responsive to a calcium ionophore and to calcineurin inhibition by cyclosporine A. The slow fiber type-specific expression of ColQ-1 was abolished by the mutation of an NFAT element in pColQ-1. Moreover, both the ColQ promoters contained N-box element that was responsible for the synapse-specific expression of ColQ transcripts. These results explain the specific expression patterns of collagen-tailed acetylcholinesterase in slow and fast muscle fibers.
Assuntos
Acetilcolinesterase/genética , Difosfato de Adenosina/análogos & derivados , Colágeno/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Proteínas Musculares/genética , Proteínas Nucleares , Acetilcolinesterase/biossíntese , Acetilcolinesterase/metabolismo , Difosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Linhagem Celular , Galinhas , Colágeno/biossíntese , Colágeno/metabolismo , Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Genes Reporter/genética , Humanos , Camundongos , Dados de Sequência Molecular , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares de Contração Lenta/enzimologia , Proteínas Musculares/biossíntese , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Fatores de Transcrição NFATC , Neurregulinas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ratos , Sequências Reguladoras de Ácido Nucleico , Transmissão Sináptica/efeitos dos fármacos , Tionucleotídeos/farmacologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , TransfecçãoRESUMO
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. We also analyzed the formation of disulfide bonds between two different AChET subunits, and between AChET subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (QN)] possessing two cysteines upstream or downstream of the PRAD. This shows that, in the complex formed by four T subunits with QN (T4-QN), the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD. The formation of disulfide bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/isolamento & purificação , Acetilcolinesterase/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Electrophorus , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Estrutura Secundária de ProteínaRESUMO
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. Replacement of two, four or five aromatic residues gradually suppressed degradation and increased secretion. Co-expression with a PRAD- containing protein induced the assembly of PRAD-linked tetramers in the endoplasmic reticulum (ER) and allowed partial secretion of a dimerization- defective mutant; by masking the aromatic side chains, hetero-oligomerization rescued this enzyme from degradation. Degradation was due to ERAD, since it was not blocked by brefeldin A but was sensitive to proteasome inhibitors. Kifunensine reduced degradation, suggesting a cooperativity between the glycosylated catalytic domain and the non-glycosylated T peptide. This system appears particularly well suited to analyze the mechanisms which determine the degradation of correctly folded multidomain proteins in the ER.
Assuntos
Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Retículo Endoplasmático/metabolismo , Peptídeos/metabolismo , Subunidades Proteicas/metabolismo , Acetilcolinesterase/genética , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/genética , Animais , Células COS , Chlorocebus aethiops , Sequência Conservada , Cisteína/metabolismo , Dimerização , Humanos , Lactonas/farmacologia , Leupeptinas/farmacologia , Dados de Sequência Molecular , Nippostrongylus/enzimologia , Ratos , Serina/metabolismoRESUMO
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. We show that an 80-residue segment located downstream of the collagenous regions contains the trimerization domain, that it can form trimers without the collagenous regions, and that a pair of cysteines located at the N-boundary of this domain facilitates oligomerization, although it is not absolutely required. We further show that AChE subunits can associate with nonhelical collagen ColQ monomers, forming ColQ-associated tetramers (G4-Q), which are secreted or are anchored at the cell surface when the C-terminal domain of ColQ is replaced by a GPI-addition signal.
Assuntos
Acetilcolinesterase/química , Colágeno/química , Proteínas Musculares , Acetilcolinesterase/genética , Sequência de Aminoácidos/genética , Animais , Células COS , Colágeno/genética , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína/fisiologia , Torpedo/genética , TransfecçãoRESUMO
Oligonucleotides induce various cellular responses which are not due to the blockade of protein synthesis by an antisense mechanism. Oligonucleotides presenting double-stranded or G-quartet structures (ribo- or deoxyribonucleotides, phosphodiester or phosphorothioated) induce retraction of neurites and aggregation of chicken retinal cells within 10-20 h. This effect is reversible, non-toxic; it appears to require internalization and can be mimicked by treatment of the cells with an RGDS peptide. The oligonucleotides appear to trigger a cascade of intracellular events, affecting the adhesive properties of integrins. In addition, a subset of oligonucleotides induced platelet aggregation, probably through their interaction with membrane receptors. Recognition of these effects is important for the design and interpretation of antisense experiments.
Assuntos
Oligonucleotídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Retina/efeitos dos fármacos , Animais , Plaquetas/efeitos dos fármacos , Cálcio/metabolismo , Agregação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Humanos , Integrinas/metabolismo , Líquido Intracelular/metabolismo , Neuritos/efeitos dos fármacos , Conformação de Ácido Nucleico , Oligonucleotídeos/farmacocinética , Oligopeptídeos/farmacologia , Peptídeos , Inibidores da Agregação Plaquetária/farmacologia , Retina/citologia , Retina/embriologia , TransfecçãoRESUMO
As a tetramer, acetylcholinesterase (AChE) is anchored to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses. We have previously shown that collagen Q (ColQ) anchors AChE at the neuromuscular junction. We have now cloned the gene PRiMA (proline-rich membrane anchor) encoding the AChE anchor in mammalian brain. We show that PRiMA is able to organize AChE into tetramers and to anchor them at the surface of transfected cells. Furthermore, we demonstrate that AChE is actually anchored in neural cell membranes through its interaction with PRiMA. Finally, we propose that only PRiMA anchors AChE in mammalian brain and muscle cell membranes.