Contribution of Presenilin Transmembrane Domains 6 and 7 to a Water-containing Cavity in the γ-Secretase Complex
2006; Elsevier BV; Volume: 281; Issue: 37 Linguagem: Inglês
10.1074/jbc.m604997200
ISSN1083-351X
AutoresAlexandra Tolia, Lucía Chávez‐Gutiérrez, Bart De Strooper,
Tópico(s)Prion Diseases and Protein Misfolding
Resumoγ-Secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of γ-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective γ-secretase inhibitors could be designed. γ-Secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of γ-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective γ-secretase inhibitors could be designed. The Presenilin (PS) 2The abbreviations used are: PS, Presenilin; APP, amyloid precursor protein; Aβ, amyloid β peptide; NICD, Notch intracellular domain; TM, transmembrane domain; NEM, N-ethylmaleimide; mAb, monoclonal antibody; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Bicine, N,N-bis(2-hydroxyethyl)glycine; ELISA, enzyme-linked immunosorbent assay; NTF, N - terminal fragment; CTF, C - terminal fragment. proteins are the prototypic members of a group of aspartic proteases involved in regulated intramembrane proteolysis, a mechanism responsible for cleavage of peptide bonds within the lipid bilayer (1Urban S. Freeman M. Curr. Opin. Genet. Dev. 2002; 12: 512-518Crossref PubMed Scopus (131) Google Scholar, 2Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). More than 150 mutations in Presenilin 1 and 10 mutations in Presenilin 2 have been associated with Alzheimer disease (for a list of the mutations, see molgen.ua.ac.be/ADMutations), demonstrating their pivotal role in the pathogenesis of the disease. Presenilins are critical for the γ-secretase cleavage of the amyloid precursor protein (APP) that generates the amyloid β peptide (Aβ) (3De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1560) Google Scholar). They are also responsible for the intramembrane proteolysis of several other type I transmembrane proteins (reviewed in Ref. 4Kopan R. Ilagan M.X. Nat. Rev. Mol. Cell Biol. 2004; 5: 499-504Crossref PubMed Scopus (499) Google Scholar), including the S3 cleavage of Notch that releases the Notch intracellular domain (NICD), a major regulator of gene transcription (5De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. 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Mutation of two conserved aspartates, Asp-257 and Asp-385, located in transmembrane domains (TMs) 6 and 7 respectively, abolishes activity as well as binding to the transition state inhibitors of γ-secretase (14Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1699) Google Scholar, 15Esler W.P. Kimberly W.T. Ostaszewski B.L. Diehl T.S. Moore C.L. Tsai J.Y. Rahmati T. Xia W. Selkoe D.J. Wolfe M.S. Nat. Cell Biol. 2000; 2: 428-434Crossref PubMed Scopus (508) Google Scholar, 16Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. Register R.B. Sardana M.K. Shearman M.S. Smith A.L. Shi X.P. Yin K.C. Shafer J.A. Gardell S.J. Nature. 2000; 405: 689-694Crossref PubMed Scopus (867) Google Scholar), supporting the hypothesis that these residues constitute the catalytic site of the protein. Furthermore, inhibitor profiling studies have provided evidence for at least one additional substrate binding site on Presenilin, distinct from, but in close proximity to, the catalytic site (17Annaert W.G. Esselens C. Baert V. Boeve C. Snellings G. Cupers P. Craessaerts K. De Strooper B. Neuron. 2001; 32: 579-589Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 18Esler W.P. Kimberly W.T. Ostaszewski B.L. Ye W. Diehl T.S. Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2720-2725Crossref PubMed Scopus (345) Google Scholar, 19Kornilova A.Y. Bihel F. Das C. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3230-3235Crossref PubMed Scopus (183) Google Scholar). Structure-function studies of the γ-secretase complex are not easy to perform because of its hydrophobic nature and its sensitivity to membrane lipid composition and detergent extraction procedures. This explains why most of our knowledge of the catalytic activity of the complex is based on indirect evidence and assumptions. For instance, hydrolysis of peptide bonds requires that the active catalytic site of the protease have access to water within the lipid bilayer, but no formal proof for this assumption has been provided in the case of Presenilin. To probe experimentally the microenvironment of the catalytic site of Presenilin, we employed cysteine-scanning mutagenesis, a method widely used to investigate the structural features of polytopic membrane proteins (reviewed in Refs. 20 and 21). The principle involves substitution of amino acid residues of interest with cysteine, which is average in size and thus normally quite well tolerated and amenable to highly specific modification with sulfhydryl-directed reagents. Combination of membrane-permeable and -impermeable reagents can provide valuable information about the extracellular or cytosolic position of a cysteine (22Feramisco J.D. Goldstein J.L. Brown M.S. J. Biol. Chem. 2004; 279: 8487-8496Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 23Kimura-Someya T. Iwaki S. Konishi S. Tamura N. Kubo Y. Yamaguchi A. J. Biol. Chem. 2000; 275: 18692-18697Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), whereas cysteines embedded in the membrane, unless exposed to a water-containing cavity, are not reactive with these reagents (24Slotboom D.J. Konings W.N. Lolkema J.S. J. Biol. Chem. 2001; 276: 10775-10781Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 25Kuwabara N. Inoue H. Tsuboi Y. Nakamura N. Kanazawa H. J. Biol. Chem. 2004; 279: 40567-40575Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 26Mueckler M. Makepeace C. J. Biol. Chem. 2005; 280: 39562-39568Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This makes the technique a valuable tool for topological studies and even allows detection of conformational changes (27Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 28Huang W. Osman R. Gershengorn M.C. Biochemistry. 2005; 44: 2419-2431Crossref PubMed Scopus (23) Google Scholar). In addition, in combination with disulfide cross-linking strategies, domains can be identified that are remote in the primary structure but in close proximity in the tertiary structure of the protein (29Lynch B.A. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10402-10406Crossref PubMed Scopus (124) Google Scholar, 30Loo T.W. Bartlett M.C. Clarke D.M. J. Biol. Chem. 2004; 279: 7692-7697Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Taking advantage of these unique properties of cysteine-scanning mutagenesis, we have studied here the contribution of TMs 6 and 7 to a potential hydrophilic pocket in the γ-secretase complex. Site-directed Mutagenesis and Generation of Stable Cell Lines—All mouse PS1 mutants were constructed using the multisite-directed mutagenesis kit (Stratagene). Immortalized mouse embryonic fibroblasts (MEFs) derived from PS1/PS2-deficient mice were cultured in Dulbecco's modified Eagle's medium/F-12 containing 10% fetal bovine serum (Sigma). At 30-40% confluency, the MEFs were transduced using a replication-defective recombinant retroviral expression system (Clontech) with either wild-type or mutant PS1. Cell lines stably expressing the desired proteins were selected based on their acquired resistance to 5 μg/ml puromycin. Antibodies—Polyclonal antibodies against mouse PS1 NTF (B19.3) and CTF (B32.2), APH-1a (B80.2), PEN-2 (B96.2), and APP C terminus (B63.3), and monoclonal 9C3 against the C terminus of Nicastrin have been described previously (17Annaert W.G. Esselens C. Baert V. Boeve C. Snellings G. Cupers P. Craessaerts K. De Strooper B. Neuron. 2001; 32: 579-589Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 32Esselens C. Oorschot V. Baert V. Raemaekers T. Spittaels K. Serneels L. Zheng H. Saftig P. De Strooper B. Klumperman J. Annaert W. J. Cell Biol. 2004; 166: 1041-1054Crossref PubMed Scopus (153) Google Scholar). Other antibodies, as follows, were purchased: anti-N-cadherin from BD Biosciences, anti-NICD (cleaved Notch1 Val-1744) from Cell Signaling Technologies, mAb WO2 from Abeta GmbH, (Heidelberg, Germany), mAb 9E10 (Sanver Tech), and MAB5232 against PS1 CTF (Chemicon). Preparation of Cell Lysates and Immunoblotting—Total cell extracts were prepared in lysis buffer containing 250 mm sucrose, 5 mm Tris-HCl (pH 7.4), 1 mm EGTA, 1% Triton X-100, and Complete protease inhibitors (Roche Applied Science). After centrifugation at 13000 × g for 15 min at 4 °C, 20 μg of protein from the post-nuclear extracts were separated on 4-12% BisTris gels (Invitrogen). Proteins were transferred to nitrocellulose membranes and then blocked and probed with antibodies as indicated. For detection, horseradish peroxidase-coupled secondary antibodies (Bio-Rad) were used followed by chemiluminescence detection with Renaissance (PerkinElmer Life Sciences). Quantifications were performed by means of densitometry. Labeling with Thiol-specific Reagents—MEFs in 100-mm plates were washed three times in cold phosphate-buffered saline (pH 7.4) followed by incubation with 200 μm EZ-Link biotin-HPDP (N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide (Pierce) for 45 min at 4 °C. After three washes with phosphate-buffered saline, cells were lysed and 500 μg of protein were incubated with 35 μl of immobilized NeutrAvidin protein beads (Pierce) at 4 °C overnight. After extensive washing, the proteins were eluted from the beads by boiling in Nu-Page sample buffer. For cross-linking experiments, membrane aliquots were treated for 20 min at 4 °C with 200 μm 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate or 1,2-ethanediyl bismethanethiosulfonate (in 10 mm Tris-HCl (pH 7.4), 150 mm NaCl, Complete EDTA-free protease inhibitors) with or without pretreatment with 10 mm NEM/10 mm EDTA. The cross-linking reaction was quenched with NEM, and extracts were prepared in Nu-Page sample buffer without β-mercaptoethanol and separated on a 7% Tris acetate gel. Statistical Analysis—Data from three independent experiments were used for calculations (S.E. values of the mean are indicated), which were then subjected to one-way analysis of variance with a Bonferroni correction to determine their significance. Transduction with APP Adenovirus-Urea Gel Electrophoresis—Subconfluent stable MEF cell lines were transduced with the recombinant adenovirus Ad5/cytomegalovirus-APP bearing human APP-695 (33Yuan H. Zhai P. Anderson L.M. Pan J. Thimmapaya B. Koo E.H. Marquez-Sterling N.R. J. Neurosci. Methods. 1999; 88: 45-54Crossref PubMed Scopus (11) Google Scholar) for 7 h at 37 °C, after which they were kept overnight at 37 °C in Dulbecco's modified Eagle's medium supplemented with 0.2% fetal bovine serum. 24 h post-infection, the conditioned medium (1000 μl total volume) was collected and cleared by centrifugation, and 10 μl were assayed immediately for the production of Aβ40 and Aβ42 by specific ELISA (see below). A similar volume sample was used for the determination of the amount of secreted APP fragments (APPs) by SDS-PAGE and direct Western blotting with the polyclonal antibody 22C11 (Chemicon). The remaining conditioned medium (equal volumes) was immunoprecipitated overnight with mAb B7/8 raised against the N terminus of the Aβ sequence (34De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (162) Google Scholar) and 30 μl of protein G-Sepharose (Amersham Biosciences). After extensive washing, the bound material was eluted from the beads by boiling for 10 min in sample buffer (0.74 m BisTris, 0.32 m Bicine, 0.88 m sucrose, 2% SDS, and 0.015% bromphenol blue) and was loaded on a 12%T/5%C (%T, percentage (w/v) of total acrylamide monomer; %C, percentage (w/v) of bisacrylamide/total acrylamide monomer) Bicine/Tris SDS-PAGE gel containing 8 m urea (35Wiltfang J. Esselmann H. Cupers P. Neumann M. Kretzschmar H. Beyermann M. Schleuder D. Jahn H. Ruther E. Kornhuber J. Annaert W. De Strooper B. Saftig P. J. Biol. Chem. 2001; 276: 42645-42657Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Separation was allowed to proceed at room temperature for 2 h at a constant current of 24 mA/gel and was followed by Western blotting and visualization of the Aβ species with mAb WO2. ELISA—For the measurement of secreted Aβ40 and Aβ42, specific ELISA kits (The Genetics Company) were used according to the manufacturer's protocol. Analysis of Notch Processing—Subconfluent MEF cell lines were infected with the Ad5/dE1dE2a/cytomegalovirus Myc-tagged Notch ΔE adenovirus (36Michiels F. van Es H. van Rompaey L. Merchiers P. Francken B. Pittois K. van der Schueren J. Brys R. Vandersmissen J. Beirinckx F. Herman S. Dokic K. Klaassen H. Narinx E. Hagers A. Laenen W. Piest I. Pavliska H. Rombout Y. Langemeijer E. Ma L. Schipper C. Raeymaeker M.D. Schweicher S. Jans M. van Beeck K. Tsang I.R. van de Stolpe O. Tomme P. Arts G.J. Donker J. Nat. Biotechnol. 2002; 20: 1154-1157Crossref PubMed Scopus (89) Google Scholar) for 24 h, and after treatment with the proteasomal inhibitor lactacystin (Calbiochem) for 4 h at 37 °C, cell extracts were prepared. Samples of 10 μg of total protein were separated on a 7% Tris acetate gel, transferred to nitrocellulose membranes, and probed with the appropriate antibodies to assess the levels of Notch ΔE infection and NICD production, respectively. Blue Native Gel Electrophoresis—This method was performed as described previously (37Nyabi O. Bentahir M. Horre K. Herreman A. Gottardi-Littell N. Van Broeckhoven C. Merchiers P. Spittaels K. Annaert W. De Strooper B. J. Biol. Chem. 2003; 278: 43430-43436Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), with the exception that the samples (20 μg) were separated on a 5-16% polyacrylamide gradient at a constant voltage of 200 for 3.5 h. Generation of a Cysteine-less PS1—Mouse PS1 contains five endogenous cysteines (Fig. 1A), which we replaced with alanine using site-directed mutagenesis. The resulting “cysteine-less” (Cys-less) PS1 was able to rescue the maturation of Nicastrin and the stabilization of PEN-2, as well as the accumulation of unprocessed APP C-terminal fragments in PS1-/- PS2-/- fibroblasts (Fig. 1B). Further functional characterization of the Cys-less PS1 revealed that it has a similar rescuing activity to the wild-type PS protein in PS1-/- PS2-/- fibroblasts as far as it concerns the in vivo processing of three major γ-secretase substrates, APP (Fig. 1C), N-cadherin, and Notch (data not shown). Transmembrane domains 6 and 7 contain the putative catalytically active aspartates and therefore likely contribute structurally also to the catalytic site of PS1. Alignment of these domains from various species (supplemental Fig. S1) revealed, apart from the two catalytic aspartates, several other highly conserved amino acids that could be involved in substrate binding, in catalysis, or in delineating a hypothetical interior water-containing chamber in the complex (38Lazarov V.K. Fraering P.C. Ye W. Wolfe M.S. Selkoe D.J. Li H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6889-6894Crossref PubMed Scopus (151) Google Scholar). We, therefore, substituted these residues one by one with cysteines and generated stably transfected PS1-/- PS2-/- fibroblast cell lines with the mutants in a similar manner as the cysteine-less PS1 (Fig. 1C). Two mutations (G382C and K395C) resulted in impaired PS1 endoproteolysis, but all 22 mutants were nevertheless able to rescue the stabilization of PEN-2 and the maturation of Nicastrin. In addition, we also substituted the catalytic aspartic residues, both individually and in combination, without again perturbing the ability of PS1 to stabilize the other γ-secretase components (Fig. 1D). Water Accessibility of Introduced Cysteines and Disulfide Cross-linking—Recent electron microscopy studies have shown that an electrolucent channel is present in the interior of the γ-secretase complex. Our primary aim was to investigate biochemically whether such a postulated “hydrophilic pocket” (38Lazarov V.K. Fraering P.C. Ye W. Wolfe M.S. Selkoe D.J. Li H. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6889-6894Crossref PubMed Scopus (151) Google Scholar) indeed exists and to delineate the amino acid residues exposed to it. We used the membrane-permeable reagent EZ-link biotin-HPDP, which readily reacts via its pyridyldithiol moiety with free sulfhydryl (-SH) groups exposed to water. As shown in Fig. 2, A and B, of 10 different residues in TM6 tested, only W247C was labeled. In TM7, in contrast, 6 of 11 introduced cysteines showed reactivity with biotin-HPDP. In addition, PS1 D385C in TM7 was modified, whereas the D257C mutant in TM6, which represents the putative second catalytic aspartate of PS1, was not modified in our assay. However, in order for the aspartic residues to perform substrate cleavage, they should be in close proximity to each other and be exposed to a hydrophilic environment. To investigate this further, we used a specific disulfide cross-linking strategy in the cell line expressing the double mutant PS1 D257C/D385C. We chose two homobifunctional alkylthiosulfonates of the type depicted in Fig. 2C (3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate and 1,2-ethanediyl bismethanethiosulfonate with spacer arm lengths 13 and 5.2 Å, respectively), which react selectively with -SH groups, resulting in the formation of disulfide bridges between two cysteines and the spacer arm of the cross-linker. This reaction can only take place when the cysteines have free sulfhydryl groups accessible to water and are located at a maximum distance from each other equal to the length of the spacer arm of the cross-linker. Membrane extracts from PS1 D257C/D385C cells treated with either of the two cross-linkers at 4 °C (to reduce protein molecular motions) and separated in SDS-PAGE under non-reducing conditions displayed a band running at an apparent molecular weight close to full-length PS1 (Fig. 2D). Because this band could be stained with antibodies specific for both PS1 NTF and CTF, we propose that it reflects an intramolecular cross-linking product between the two cysteines replacing the aspartates in TMs 6 and 7. The disulfide bond causes an expected shift in mobility compared with the full-length unprocessed protein, because it prevents complete unfolding under non-reducing conditions. When the free sulfhydryls are blocked with the alkylating agent NEM prior to the cross-linking reaction (Fig. 2D, lanes labeled with + NEM) or when reducing conditions are applied (data not shown), this band is not observed confirming its specificity. Furthermore, no cross-linked products are observed with Cys-less PS1, single D257C and D385C PS1 mutants, or a control mutant with two cysteines at remote positions in TMs 1 and 9. Note also that no band derived from intermolecular cross-linking of two different PS1 molecules was observed in any case under our experimental conditions. This experiment demonstrates that, in the tertiary structure of PS1, cysteines introduced at the positions of the catalytic aspartic residues are both accessible to water, facing each other with a maximal distance of ∼5.2 Å. Activity of the Mutants on APP Processing—We next investigated whether any of the cysteine substitutions influences the γ-secretase processing of APP. As shown in Fig. 3A, APP-CTF fragments generated by α-secretase from endogenously expressed APP (the direct substrate for γ-secretase) accumulate in PS knock-out fibroblasts. This phenotype could be completely rescued by reintroduction of wild-type or Cys-less PS1 in these cells, but not, as expected, by PS1 bearing either the D257C and D385C mutations or each of the substitutions G382C, G384C, F388C, Y389C, or K395C. Therefore, these residues seem to be particularly important for the activity of the protease. After transduction of the fibroblasts with full-length APP695Swe and direct quantification of the Aβ produced by ELISA (Fig. 3B), we observed that, indeed, three of the mutants (G382C, G384C, and K395C) were causing a total loss of function with regard to Aβ generation. Interestingly, the Y389C mutation displayed residual activity (∼10% Aβ40 production compared with the Cys-less PS1) but no detectable Aβ42 production. The remaining mutants can be divided into three categories: 1) the ones that reduce the production of both Aβ40 and Aβ42 (S254C, V261C), 2) those that cause a significant decrease in the levels of Aβ40 with minor effects on Aβ42 (T245C, Y256C, F388C), and 3) those that produce amounts of Aβ similar to the wild-type or Cys-less PS1 (all of the rest). Strikingly, none of these mutations seemed to severely affect the ratio of Aβ42/40 produced (Fig. 3B), with the exception of Y256C and F388C, which, due to a dramatic reduction in Aβ40, behaved like extreme “clinical” familial Alzheimer disease mutations (with 4.5- and 6-fold increase in the ratio, respectively, compared with Cys-less PS1). The effect observed for some of the mutations in the individual production of Aβ40 and Aβ42 was also independently confirmed by urea gel electrophoresis (supplemental Fig. S2). Effects of the Mutations on the Processing of Other Substrates—Next, we analyzed the behavior of the cysteine mutants in the processing of N-cadherin. Similar to APP, the C-terminal fragment of N-cadherin generated by metalloprotease cleavage accumulated in PS-deficient cells. This knock-out phenotype was not rescued by the T245C, S254C, Y256C, V261C, G382C, G384C, F388C, Y389C, K395C, and the catalytic D257C and D385C mutants (Fig. 4A). Finally, the production of NICD (Notch intracellular domain) from a membrane-tethered form of Notch (Myc-tagged Notch ΔE) was investigated (Fig. 4B). Similar to APP and N-cadherin, PS1 bearing the G382C, G384C, Y389C, or K395C mutations were unable to support any NICD production. Minimal activity was, however, also seen with the T245C, S254C, Y256C, and V261C mutants in contrast to what we observed for APP. In addition, three mutants appeared to increase Notch processing significantly (W247C, L250C, and L258C). Finally, and important in the context of the question of whether APP and Notch processing can be specifically modulated by γ-secretase, the F388C substitution, which caused a remarkable decrease in the levels of Aβ40, did not affect significantly the production of NICD. To verify that the observed effects on the processing of different substrates were not a consequence of deficient γ-secretase complex formation, we confirmed the integrity of the complexes by blue native gel electrophoresis (supplemental Fig. S3). In the present study, we have reported our efforts to investigate the catalytic site of PS1 by cysteine-scanning mutagenesis. A primary prerequisite for the use of this technique is the generation of a protein lacking all endogenous cysteines but retaining the structure and functional properties of the wild-type molecule. Although clinical mutations have been identified at three of the five endogenous cysteines in PS1 (C92S, C263F/R, C410Y) (39Tedde A. Nacmias B. Ciantelli M. Forleo P. Cellini E. Bagnoli S. Piccini C. Caffarra P. Ghidoni E. Paganini M. Bracco L. Sorbi S. Arch. Neurol. 2003; 60: 1541-1544Crossref PubMed Scopus (45) Google Scholar, 40Wasco W. Pettingell W.P. Jondro P.D. Schmidt S.D. Gurubhagavatula S. Rodes L. DiBlasi T. 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