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 α 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. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic α helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several “signals” in the catalytic domain and in the t peptide act cooperatively for AChE quality control. The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic α 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. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic α helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several “signals” in the catalytic domain and in the t peptide act cooperatively for AChE quality control. The major function of acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase (EC 3.1.1.7); AChET or T, AChE subunit of type T; DEPQ, 7-(O,O-diethylphosphinyloxy)-1-methylquinolinium methyl sulfate; ER, endoplasmic reticulum; PRAD, proline-rich attachment domain; t peptide, C-terminal peptide of the AChET splice variant; WT, wild type; DOC, Na+ deoxycholate; Scr, scrambled. is to regulate the cholinergic transmission by cleaving the neurotransmitter acetylcholine. In mammals, alternative splicing of a single gene gives rise to different variants (types R, H, and T), which possess the same catalytic activity but different tissue-specific distributions (1Massoulié J. Bon S. Ann. Rev. Neurosci. 1982; 5: 57-106Crossref PubMed Scopus (687) Google Scholar, 2Taylor P. Radic Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (616) Google Scholar, 3Massoulié J. NeuroSignals. 2002; 11: 130-143Crossref PubMed Scopus (245) Google Scholar). The catalytic subunits of type T (AChET), which are predominant in the nervous system and muscles, are characterized by a C-terminal peptide of 40 amino acids called the t peptide (4Massoulié J. Anselmet A. Bon S. Krejci E. Legay C. Morel N. Simon S. J. Physiol. (Paris). 1998; 92: 183-190Crossref PubMed Scopus (67) Google Scholar). The t peptide enables AChET subunits to assemble into dimers and tetramers and to associate with structural proteins possessing a proline-rich attachment domain (PRAD), the collagen ColQ, and the transmembrane protein PRiMA (proline-rich membrane anchor) (5Krejci E. Thomine S. Boschetti N. Legay C. Sketelj J. Massoulié J. J. Biol. Chem. 1997; 272: 22840-22847Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 6Bon S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Perrier A.L. Massoulié J. Krejci E. Neuron. 2002; 33: 275-285Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). These proteins anchor PRAD-linked tetramers of cholinesterase in the basal lamina of neuromuscular junctions and in neuronal membranes, respectively. The t peptide behaves as an autonomous interaction domain in its association with a PRAD and therefore has been called the tryptophan amphiphilic tetramerization domain (WAT) (8Simon S. Krejci E. Massoulié J. EMBO J. 1998; 17: 6178-6187Crossref PubMed Scopus (76) Google Scholar). Its homo- and hetero-oligomerization properties depend on two major features, which are conserved in all vertebrates: 1) a series of seven aromatic residues (Trp10, Phe14, Trp17, Tyr20, Trp24, Phe28, and Tyr31) and 2) a cysteine near its C terminus (Cys37) (9Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar). Spectroscopic and crystallographic studies showed that the t peptide is organized as an amphiphilic α-helix in which all of the aromatic residues are grouped on one side (10Bon S. Dufourcq J. Leroy J. Cornut I. Massoulié J. Eur. J. Biochem. 2004; 271: 33-47Crossref PubMed Scopus (22) Google Scholar). 2Dvir, H., Harel, M., Bon, S., Liu, W. Q., Vidal, M., Garbay, C., Sussman, J. L., Massoulié, J., and Silman, I. (2004) EMBO J.23, 4394–4405. Cys37 may form disulfide bridges between two t peptides in homodimers and homotetramers or between two t peptides and the two PRAD cysteines in the heteromeric PRAD-linked tetramers (10Bon S. Dufourcq J. Leroy J. Cornut I. Massoulié J. Eur. J. Biochem. 2004; 271: 33-47Crossref PubMed Scopus (22) Google Scholar, 11Bon S. Massoulié J. J. Biol. Chem. 1997; 272: 3007-3015Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 12Rosenberry T.L. Richardson J.M. Biochemistry. 1977; 16: 3550-3558Crossref PubMed Scopus (154) Google Scholar, 13Lee S.L. Heinemann S. Taylor P. J. Biol. Chem. 1982; 257: 12282-12291PubMed Google Scholar, 14Inestrosa N.C. Roberts W.L. Marshall T.L. Rosenberry T.L. J. Biol. Chem. 1987; 262: 4441-4444Abstract Full Text PDF PubMed Google Scholar, 15Gennari K. Brunner J. Brodbeck U. J. Neurochem. 1987; 49: 12-18Crossref PubMed Scopus (70) Google Scholar, 16Velan B. Grosfeld H. Kronman C. Leitner M. Gozes Y. Lazar A. Flashner Y. Marcus D. Cohen S. Shafferman A. J. Biol. Chem. 1991; 266: 23977-23984Abstract Full Text PDF PubMed Google Scholar). The formation of AChET dimers depends on contact between subunits at the level of two α-helices from each catalytic domain (α7,8 and α10), forming the “four-helix bundle” (17Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2480) Google Scholar), and on the formation of a disulfide bridge between the C-terminal cysteines of two t peptides (18Morel N. Leroy J. Ayon A. Massoulié J. Bon S. J. Biol. Chem. 2001; 276: 37379-37389Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), but it does not require the aromatic residues of the t peptide. It has been shown that rat AChET subunits, which carry a mutation in the α10 helix (F535A, 9 amino acids upstream of the t peptide), are deficient in dimerization despite the of Cys37 (18Morel N. Leroy J. Ayon A. Massoulié J. Bon S. J. Biol. Chem. 2001; 276: 37379-37389Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In the formation of or PRAD-linked tetramers depends on the cluster of aromatic residues. It is by disulfide but in their (9Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar). The of a t peptide induces an degradation of AChET subunits, which is increased with the mutation assemble with a of forming PRAD-linked degradation the degradation pathway. It by and is by indicating that are in its S. Massoulié J. Bon S. EMBO J. PubMed Scopus Google Scholar). the t peptide to a in protein showed that of the last two residues increased the secretion of that its C terminus behaves as a retention the of may due to the of the C-terminal with the endoplasmic reticulum retention (9Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar, B. Kronman C. Flashner Y. Shafferman A. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). In this mutations to the of the cluster of aromatic residues in the and of the aromatic hydrophobic of these residues in rat AChET We the and interaction with a PRAD for wild type and mutants with and without the C-terminal We showed that of the t peptide these different and the AChET subunit of rat AChE and an of were in the S., and S. Scholar). The of the protein is S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Abstract Full Text Full Text PDF PubMed Scopus (121) Google signal peptide is not the PRAD and the two cysteines are are in was with the of Roberts 1987; PubMed Scopus Google Scholar). The was by of of the C-terminal peptide from the of AChE, a cysteine at of the catalytic domain F. Ayon A. A. Leroy J. Massoulié J. Bon S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). were in with the as N. Massoulié J. Bon S. J. Biol. PubMed Scopus Google Scholar), with of The cellular and were or of AChE by of of cellular or AChE activity were with and of for at The AChE activity was with the Biochem. Pharmacol. PubMed Scopus Google Scholar). and the of AChE, of or were with the for which the cellular the of AChE activity at was by at The secretion of AChE was and of the were and replaced with AChE was to the of to protein and was to and and was for at in a by for at at The the was to activity was at with the of a was in or Na+ as S. K. Massoulié J. J. Neurochem. PubMed Scopus Google Scholar). of was in a for activity was on the by the of and L. J. PubMed Scopus Google Scholar). and of were and for in a without by a with and cysteine the were in a the was and the were at different The and the were with a AChE D. J. M. Massoulié J. J. Neurochem. PubMed Scopus Google and at on protein The was from the in a and and on for the were and AChE protein was with a of of t peptides was to B. 266: PubMed Google in the Mutations in the t mutations in the t peptides of rat AChET and of the the mutation in the α10 helix as in We the cluster of aromatic residues of the t peptide in The these residues was in the and the seven aromatic residues were replaced by in the and two residues and were replaced by in the In the the of the seven aromatic residues were with of residues that the helix In the the of the t peptide and the of and residues were not to which may the of its The region is to form an α-helix in the of the and mutants as in the wild type t peptide In this to the wild type aromatic region of the t peptide or not with the mutation in the catalytic domain or and with mutations in the C-terminal which a cysteine or a at by C or for In the the last two residues (Asp-Leu) that the C terminus We replaced the last residues by the retention signal mutants the t peptide of the catalytic domain only or of the catalytic domain by a peptide a cysteine We that was of mutations in the aromatic-rich region on the of cellular and AChE The mutants a wild type or a catalytic domain as AChE and and Cys37 or not as The cellular and were of of in the in each were to for the wild type which was used as a at The are the of at that secretion was increased by the of the aromatic-rich region or to a by the the of AChE protein was the same for all of the mutants as shown in and all of the mutants the same catalytic activity not the the of the in this mutations in the C-terminal t peptide of rat AChE in the or of mutation and in the α helix of the catalytic dimerization through the In the t peptide was and in it was replaced by the seven residues from the C-terminal peptide of the splice a cysteine In this to the wild type aromatic-rich and of the of the wild type and The mutations were with mutations in the C-terminal of Cys37 by a as by an or of the last two residues with the mutation and of the last residues by the retention signal of the mutants We the of these mutants by their with an We that the catalytic was for cellular and and was not affected by the of the by the or of the t or by the different mutations that were in the t peptide not with or without on the the in for the different mutants of rat AChET subunits in with or without S. Coussen F. Massoulié J. J. Biol. Chem. 1997; 272: 3016-3021Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, cellular from wild type AChET subunits dimers and a of tetramers the of and the AChET subunits with the and tetramers The forms of AChE with the were similar to with the wild the formation of tetramers was this was with the formation of was The and dimers but or with was at a without or with the and dimers but It not form but a in the and The and mutants the of an aromatic cluster for dimerization was in all of the The subunits only from the wild type by the with of tetramers and the wild The and mutants only It is that the not form tetramers or in to the that disulfide to the of and was in the of the subunits the and mutations with or without with but at a level with the wild and these were not in cellular The similar to not form heteromeric In and the of AChE in the the of activity of the different as the and mutants were the WT, and The of and on a of in the t the of a DOC, on the of AChE forms in the of the The were to of the and which not possess the cluster of hydrophobic residues and The of amphiphilic as the wild type and dimers was in the of with by the a these the of in the and of as an of We similar for cellular and were by as their aromatic each and tetramers were not The wild type and mutants similar the mutants were to the and the mutants were not the amphiphilic depends on the of hydrophobic residues. It is that a amphiphilic which as of the on and AChE the of AChE activity in cellular and in the for the different mutants in to for the wild type AChET Cys37 was the cellular activity was increased to and for and the activity was to and the the cellular activity was to or for the activity was increased to was increased even for the and the same level as that for the which the t peptide, without a C-terminal or with cysteine near the of the catalytic dimerization The cellular of these mutants were increased for or decreased for In the of cysteine cellular and were an increased but the of the mutations were secretion was increased when the aromatic was or and to a when aromatic residues were replaced by In secretion was and an of the cellular activity when two were replaced by this effect was when the of aromatic residues was by their The cellular activity with the a wild type t peptide with or without Cys37 was wild and secretion was with that of the indicating a degradation as (18Morel N. Leroy J. Ayon A. Massoulié J. Bon S. J. Biol. Chem. 2001; 276: 37379-37389Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). the that the mutation not the cellular of the of the different mutations in the t peptide were similar to without the It is that the mutants and were in to and the wild type AChE and somewhat activity the that the of to cellular were the that the cysteine retention in with the that possessing the C terminus were with In the of the mutants the of to cellular was with a cysteine for WT, Scr, and but it was for and as as for and of the C-terminal of the t on mutants in which the cysteine was replaced by a with mutants the last two residues with and with mutants in which the last residues were replaced by the retention These C-terminal were with the different mutations of the aromatic-rich region as in The increased the cellular activity but only for with or without mutation It the activity in all of the mutants but only for The mutation increased the secretion of and but effect on or mutation not the cellular activity of WT, or and decreased it for and for The of to cellular a of these was decreased by in all but for The mutation increased it for WT, Scr, and but not for and of AChE and of AChE showed that all of the proteins were at the same as the and the were with the of a in the region the C-terminal t peptide. In of amino acids for a the of AChE protein by the was the same for all is in for and that it is not by the mutation or by the of the aromatic-rich region of the t peptide. The of the or 40 were the same was not of cellular AChE by a not We the of AChE activity its by two the the activity increased in the of that the of a t peptide decreased the of of AChE with mutants that t peptide or a t peptide from which the aromatic region was In the mutation in the dimerization of the catalytic domain all AChE proteins were at the same and all the same catalytic the that the of a t peptide the of AChE activity that of the AChE We the of the mutations that in the aromatic-rich region of the t peptide The of AChE activity was to similar for the WT, Scr, and peptides with the which this The in which all of the aromatic residues were replaced by leucines, the activity of all of the subunits assemble as tetramers in which the t peptides are the effect of with We that this not the of of AChE activity not of AChE and the secretion of a of with the cellular activity increased the of the secretion a of to the for the of proteins from the to the and its level The secretion was for the by and and wild type with the in with the of in the Degradation of AChE and the degradation of AChE in which a for the of protein a of several in the and in the of the mutants from the The and mutants not in the and the was In the and mutants were in with the of the degradation of AChE activity as as was by and secretion was by A. These were with mutants Cys37 with or without the and that AChE activity was for all of the mutants that the mutation with mutants without this mutation that the of protein was similar to that of activity the same In all of the the the wild type and the was The and mutants were similar to the wild type or were We similar results for mutants possessing a C-terminal without not studies that the t peptide of AChE an autonomous interaction domain that allows the formation of as as with the proteins and which possess a The t peptide 40 amino acids and forms an amphiphilic α-helix with a cluster of seven aromatic residues. its in the t peptide AChET subunits for degradation through the degradation when the catalytic domain the mutation in its dimerization In this the of the t peptide that the cellular fate of rat AChE degradation or this the aromatic-rich region of the t peptide of AChET subunits in the aromatic cluster by the and hydrophobic aromatic by the hydrophobic or the aromatic-rich region We the last which the Cys37 and two C-terminal residues of the retention and in the aromatic-rich region of AChE showed that the of aromatic residues the amphiphilic properties of AChET and dimers (10Bon S. Dufourcq J. Leroy J. Cornut I. Massoulié J. Eur. J. Biochem. 2004; 271: 33-47Crossref PubMed Scopus (22) Google Scholar). In this was conserved when the two were replaced by even these residues are or when the seven aromatic residues of the t peptide were replaced by In these dimers amphiphilic indicating a partial of the hydrophobic was when the aromatic-rich region was and but when the cluster of aromatic residues was by of these aromatic residues the helix without the amino In with a of of AChET subunits, that aromatic on one side of an amphiphilic are for the of or the of a cysteine (Cys37) is for the formation of dimers (9Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar). the mutants the to form or PRAD-linked tetramers but not the Scr, or We on the and secretion of the the of mutations in the aromatic-rich region were the same with or without the C-terminal cysteine or and with or without the which dimerization or the were not to dimerization. of with an of the showed that mutations in the t peptide not the catalytic of the as the catalytic domain and the t peptide are In the which dimerization of the catalytic on its catalytic the catalytic by the different mutants were to the of AChE We that mutations or in the t peptide with or without the mutation not the of of AChE proteins in in with the that the mutations were to a of the protein in the C-terminal the of AChE activity and AChE subunits was to the of a t peptide. of the the of AChE was not affected by the mutation but was increased by of the t peptide or of its aromatic-rich region with not the of of wild type despite the formation of the in which the t peptides are in a interaction with the PRAD in S. Massoulié J. J. Biol. Chem. 1997; 272: 3007-3015Abstract Full Text Full Text PDF PubMed Scopus (68) Google in a that their aromatic-rich are The of effect of on the of AChE that the of the this association has been shown to in the (9Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar). The of a t peptide therefore a fraction of AChE from their its hydrophobic aromatic-rich region to in the catalytic of the at the to an of the the t peptide are into catalytic a of to in the of a wild type t peptide. The Scr, or mutants the of effect was with the in which all seven aromatic residues were replaced by and when two were replaced by which are misfolding to from the of residues in the t peptide and does not depend on their aromatic or on their in an amphiphilic studies showed that degradation was increased by the mutation and by the of aromatic residues in the t peptide S. Massoulié J. Bon S. EMBO J. PubMed Scopus Google Scholar). degradation was for the dimerization deficient for the wild type catalytic domain the of mutations in the aromatic-rich region was similar in We similar degradation by the of cellular AChE activity or of AChE protein when secretion was by a retention signal or by C and suggested that and AChE were in the same and that not the degradation In the which the of was the wild type and the or mutants but the degradation is by the of hydrophobic residues but by aromatic residues by It does not depend on the of an amphiphilic α-helix as shown by the degradation of the It is to the between the mutation in the catalytic domain and the t peptide for of the C-terminal that the C-terminal residues of the t peptide with or without the cysteine or not the of AChE Cys37 does not secretion of The of to cellular activity was only when the of this cysteine was with the mutation or with the mutation in the catalytic domain and a t peptide aromatic residues Scr, or a “signals” in different of the The of the C-terminal of AChE as a retention signal is not and retention by an was by dimerization through Cys37 B. Kronman C. Flashner Y. Shafferman A. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). We showed that the of the last two residues of AChET subunits decreased the cellular activity and increased the activity (9Belbeoc'h S. Falasca C. Leroy J. Ayon A. Massoulié J. Bon S. Eur. J. Biochem. 2004; 271: 1476-1487Crossref PubMed Scopus (32) Google Scholar). a retention of rat AChET subunits, the of the C-terminal with that of the retention and the effect of the last two residues The a retention it secretion and increased the cellular It not the of cellular AChE with that in retention was in the which as with the indicating that the of a may by upstream It is that the C-terminal is of with the of the residues from increased the activity of the but effect on secretion of the and In of their cellular for it is not to that an as a retention signal but these that it may an effect that is by mutations in the aromatic-rich of the on the of of AChE activity and the of secretion of the cellular a of and its the and the of secretion into the is to the of of AChE activity in the the of the indicating that of the is In the of secretion to is for and and for Scr, indicating that the of aromatic residues the of when the cluster is disorganized. the hydrophobic residues not the from secretion is not by the of an amphiphilic helix but by aromatic residues. it is to the of the t peptide that the and secretion of AChE with a of the catalytic domain depends on not on the aromatic of or on their in a Degradation is with aromatic residues and is not to their The of aromatic residues the effect due to the mutation in the dimerization of the catalytic this is not due to the of catalytic for dimerization. was by by aromatic and this effect was when the of these residues was disorganized. The C-terminal a retention which is that of a signal and only in the of aromatic residues. The of the t peptide that are different from that degradation or AChE and its t peptide an for the of cellular of a with or without We and Leroy for their with was a of