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Presenilin-1 D257A and D385A Mutants Fail to Cleave Notch in Their Endoproteolyzed Forms, but Only Presenilin-1 D385A Mutant Can Restore Its γ-Secretase Activity with the Compensatory Overexpression of Normal C-terminal Fragment

2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês

10.1074/jbc.m502769200

1083-351X

Hangun Kim, Hyunkyoung Ki, Hee-Sae Park, Kwonseop Kim,

Amyloidosis: Diagnosis, Treatment, Outcomes

The enzyme γ-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the γ-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other γ-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of γ-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their γ-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349–467). However, the defective PS-1 D257A mutant could not restore their γ-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the γ-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation. The enzyme γ-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the γ-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other γ-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of γ-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their γ-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349–467). However, the defective PS-1 D257A mutant could not restore their γ-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the γ-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation. Alzheimer disease (AD) 1The abbreviations used are: AD, Alzheimer disease; Ab, antibody; CTF, C-terminal fragment; FAD, familial Alzheimer disease; GFP, green fluorescent protein; HA, hemagglutinin; HMW, high molecular weight; MEF, mouse embryonic fibroblast; NICD, Notch intracellular domain; NTF, N-terminal fragment; PS, presenilin; wt, wild type. is the most common cause of dementia, producing an impairment of memory and cognitive abilities which is gradual in onset but relentless in progression. AD is observed in more than 10% of individuals over the age of 65, but its exact pathogenic mechanism is elusive. AD is associated with massive accumulation of fibrillary aggregates in various cortical and subcortical regions of the brain, which include intracellular neurofibrillary tangles and extracellular amyloid plaques (1Rubinsztein D.C. Prog. Neurobiol. 1997; 52: 447-454Crossref PubMed Scopus (42) Google Scholar, 2Czech C. Tremp G. Pradier L. Prog. Neurobiol. 2000; 60: 363-384Crossref PubMed Scopus (126) Google Scholar). Familial Alzheimer disease (FAD) accounts for 5–10% of AD cases, and ∼50% of these have been definitely linked to mutations in the three genes that encode the amyloid precursor protein, presenilin 1 (PS-1), and presenilin 2 (PS-2). To date, more than 150 different FAD-linked PS-1 mutations have been identified and account for the vast majority of FAD-related mutations (2Czech C. Tremp G. Pradier L. Prog. Neurobiol. 2000; 60: 363-384Crossref PubMed Scopus (126) Google Scholar, 3Cruts M. Van Broeckhoven C. Hum. 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Studies with artificial and spontaneously occurring PS-1 mutants reveal that the activity of γ-secretase is critically dependent on the presence of two intact aspartate residues, Asp-257 and Asp-385, and on the endoproteolysis of PS-1. However, it is still unclear whether the defective PS-1 mutants, D257A and D385A, result from the changes in the catalytic domain or from the lack of their own endoproteolysis (12Yu G. Chen F. Nishimura M. Steiner H. Tandon A. Kawarai T. Arawaka S. Supala A. Song Y.Q. Rogaeva E. Holmes E. Zhang D.M. Milman P. Fraser P. Haass C. St. George-Hyslop P. J. Biol. Chem. 2000; 275: 27348-27353Abstract Full Text Full Text PDF PubMed Google Scholar, 13Levitan D. Lee J. Song L. Manning R. Wong G. Parker E. Zhang L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12186-12190Crossref PubMed Scopus (73) Google Scholar, 14Xia X. Wang P. Sun X. Soriano S. Shum W.K. Yamaguchi H. Trumbauer M.E. Takashima A. Koo E.H. Zheng H. Proc. Natl. Acad. Sci. U. S. 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Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (756) Google Scholar), and Notch (32Struhl G. Greenwald I. Nature. 1999; 398: 522-525Crossref PubMed Scopus (702) Google Scholar, 33Ye Y. Lukinova N. Fortini M.E. Nature. 1999; 398: 525-529Crossref PubMed Scopus (461) Google Scholar, 34De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1800) Google Scholar). Among these, Notch is the receptor for Jagged and Delta, which are critically required for a variety of signaling events both during embryogenesis and in adulthood (35Fortini M.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 673-684Crossref PubMed Scopus (343) Google Scholar). PSs are involved in the proteolytic processing (S3 cleavage) of Notch, which results in the release of its intracellular domain (NICD), which in turn acts as a transcription factor in the nucleus after associating with either a member of the CSL family of DNA-binding proteins or with LEF-1 (36De Strooper B. Annaert W. J. Cell Biol. 2001; 152: F17-F20Crossref PubMed Google Scholar, 37Ross D. Kadesch T. Mol. Cell. Biol. 2001; 21: 7537-7544Crossref PubMed Scopus (93) Google Scholar). The Notch ligands, Delta1 and Jagged, are sequentially cleaved by an ADAM protease and γ-secretase, indicating that γ-secretase plays a pivotal role in the Delta1-Notch signaling pathway (38Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israel A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar, 39LaVoie M. Selkoe D.J. J. Biol. Chem. 2003; 278: 34427-34437Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). In addition to its role in neuronal cell differentiation, Notch 1 is known to compete with amyloid precursor protein for γ-secretase and down-regulates PS-1 gene expression (40Lleo A. Berezovska O. Ramdya P. Fukumoto H. Raju S. Shah T. Hyman B.T. J. Biol. Chem. 2003; 278: 47370-47375Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 41, Deleted in proofGoogle Scholar). However, the exact role of Notch 1 in the progression of AD remains to be demonstrated. More than 150 FAD-linked PS-1 mutations have been identified to date and are suggested to affect γ-secretase activity to different extents according to substrate levels. In this report, we used four different mouse embryonic fibroblast (MEF) cell lines with different PS-1 and -2 levels, as well as several PS-1 mutants to investigate the effect of exogenous PS-1 on the cleavage of Notch. Because the presence of endogenous PS-1 or -2 was sufficient to cleave Notch, PS-1/2-/- MEFs were used. We demonstrated that the cleavage of Notch depends on the mutation site in PS-1. With the use of NTF and CTF fragments, we clearly showed that the defects in PS-1 D257A and D385A are caused by changes in their catalytic domains rather than by the lack of endoproteolysis. The defects in PS-1 D385A mutants could be restored by the compensatory overexpression of its normal counterpart, wild type (wt) CTF. In contrast to D385A, defects in PS-1 D257A could not be restored by wt NTF. The minimal domain of wt CTF required to maintain γ-secretase activity is located between residues 349 and 467. Our results indicate that NTF and CTF showed different HMW aggregate compositions and displayed different restoring effects on PS-1 D257A and D385A, as well as on different PS-1 mutants, suggesting that their regulation or function can be different. Plasmids—The wt and mutant PS-1s (Y115H, M146V, M146/D257A, D257A, L286V, D385A, C410Y), PS-1 fragments (NTF: 1–298, 1–131, 1–200, 1–260, 131–298; CTF: 299–467, 299–349, 299–418, 349–418, 349–467, 418–487 amino acids), and PS-1 D257A mutated NTF and PS-1 D385A mutated CTF and its fragments (299–349, 299–418, 349–418, 349–468, 418–487) in pEGFP-C1 were generated by PCR amplification and cloned into pEGFP-C1 vector (Clontech). The wt PS-1 in pEGFP-N3 was generated by PCR amplification and cloned into pEGFP-N3 vector (Clontech). After construction, the sequences were confirmed by DNA sequencing and found to be in-frame. The Notch constitutive active mutant was a kind gift from Dr. Raphael Kopan (Washington University, St. Louis). The Nicastrin construct was generated by PCR and cloned into pcDNA3-V5 (Invitrogen) vector. A HA-tagged hPen-2 in pcDNA3.1D/V5-His-TOPO (Invitrogen) was a kind gift from Dr. Takeshi Iwatsubo (University of Tokyo). Antibodies—Peptide antibody (Ab) specific for green fluorescent protein (GFP 1:100) was purchased from Clontech, BD Biosciences. Polyclonal Ab (anti-cleaved Notch 1 (Val-1744) or anti-NICD) recognizing cleaved Notch 1 (Val-1744) only when cleaved between Gly-1743 and Val-1744, but not full-length Notch 1 or Notch 1 cleaved at other positions, was purchased from Cell Signaling Technology, Inc. The HA epitope was detected with media from 12CA5 hybridoma. The V5 epitope was detected with anti-V5 Ab (Invitrogen). The FLAG epitope was detected with anti-FLAG M2 Ab (Sigma). For the quantification of protein loading, anti-tubulin Ab was used (Sigma). For the detection of the C-terminal fragment (CTF) of PS-1, Presenilin 1 (C-20) was employed (Santa Cruz Biotechnology, Inc.). Cell Culture and Transfection—MEF cell lines, PS-1-/- (deficient in PS-1), PS-2-/- (deficient in PS-2), PS-1/2-/- (deficient in both PS-1 and PS-2), and PS-1/2+/+ (expressing both PS-1 and PS-2), were obtained from Dr. De Strooper and cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. Cells were transfected with the use of Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. Immunoprecipitations—Immunoprecipitations were performed on lysates from transfected cells. Cells were lysed in lysis buffer containing 10 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.2 mm sodium orthovanadate, 0.2 mm phenylmethysulfonyl fluoride, and protease inhibitor mixture. Lysates were incubated with primary antibodies for 16 h at 4 °C and pulled down with protein G-Sepharose (Amersham Biosciences). Immunoprecipitated proteins were eluted at 95 °C for 2 min with 50 μl of 2 sample buffer (0.1 m Tris-HCl, pH 6.8, 0.2 m dithiothreitol, 4% SDS, 20% glycerol, 0.2% bromphenol blue, 1.43 m β-mercaptoethanol) and analyzed by immunoblotting as described below. Immunoblotting—Cells were harvested with radioimmune precipitation assay lysis buffer (1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 150 mm NaCl, 50 mm Tris-Cl, 2 mm EDTA, 1 mm sodium orthovanadate, 10 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mm phenylmethysulfonyl fluoride, protease inhibitor mixture). Cell lysates were quantified using the BCA assay kit (Pierce) and electrophoresed using 8% Tris-glycine gels (Novex). Proteins were transferred to a polyvinylidene difluoride membrane (Amersham Biosciences) that was subsequently probed with antibodies. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies followed by visualization with enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Biosciences). Even loadings on each lane were confirmed using direct blue 71 staining kits (EZBiopaQ, Co., Ltd.). For the NICD/PS-1 and NICD/tubulin ratio, a quantification of the protein bands was performed with a HP scanjet 3570c scanner (Hewlett-Packard) interfaced to an IBM computer, and image analysis was performed using the TINA 2.09 software program (Raytest). Notch Cleavage Depends on the Presence of Endogenous PS-1 and PS-2 and on the Type of PS-1 Mutants—Small amounts of endogenous PS-1 and PS-2 can interfere with the estimation of exogenously expressed PS-1. We used four different cell lines to investigate the effects of endogenous PS-1 and PS-2 on the cleavage of Notch. In the PS-1/2+/+ MEFs that express both endogenous PS-1 and PS-2 and in the PS-1-/- and PS-2-/- MEFs that express only endogenous PS-2 and PS-1, respectively, Notch cleavage occurred regardless of the presence of exogenous PS-1 (Fig. 1A). Furthermore, NICD, the cleaved form of Notch, was still generated in PS-1/2+/+ cell lines even after the overexpression of PS-1 D385A, the catalytically inactive form of PS-1 (data not shown). However, in PS-1/2-/- MEFs having no endogenous PS-1 and PS-2, Notch cleavage occurred only when exogenous PS-1 was overexpressed (Fig. 1A). Therefore, we concluded that PS-1/2-/- MEF cell lines having no endogenous PSs should be employed to investigate the effect of exogenously overexpressed PS-1. Using PS-1/2-/- cell lines and various mutant PS-1 constructs tagged with GFP, we observed different Notch cleavage patterns (top panel, Fig. 1B). The overexpressed wild types and mutant PS-1s were localized mainly in the endoplasmic reticulum and Golgi but were also found throughout the cytoplasm (data not shown). We analyzed the expression levels of different PS variants (middle panel, Fig. 1B), which were quantified and related to the amounts of NICD formed (bottom panel, Fig. 1B). Each of four spontaneous PS-1 mutants cleaved Notch to a different extent. As shown in Fig. 1B, PS-1 with a point mutation at M146V cleaved Notch slightly more than wt PS-1 did, whereas PS-1 with a mutation at Y115H and L286V cleaved Notch to a lesser extent than did wt PS-1. Interestingly, the spontaneous PS-1 C410Y mutant had defects in Notch cleavage. When the PS-1 putative catalytic site residues, Asp-257 and Asp-385, were artificially point mutated to Ala, the resulting PS-1 mutants could not cleave Notch. Likewise, M146V/D257A double mutated PS-1 did not cleave Notch, indicating that the presence of both Asp-257 and Asp-385 is required for Notch cleavage. When this set of experiments was performed in PS-1/2+/+ cells, all PS-1 mutants that were defective in cleaving Notch in PS-1/2-/- MEFs generated NICD regardless of the PS-1 mutant type (data not shown), supporting the notion that PS-1/2-/- MEFs should be used to investigate the effect of exogenous PS-1 constructs. Both PS-1 D257A and D385A Mutants Fail to Cleave Notch in Their Endoproteolyzed Forms, but the PS-1 D385A Mutant Can Restore Its γ-Secretase Activity with the Compensatory Overexpression of Normal CTF—Both aspartate residues, Asp-257 and Asp-385, are required for γ-secretase activity. There are controversial reports debating whether the defects of D257A and D385A mutants are the result of a change in their catalytic domains or a lack of endoproteolytic activity (12Yu G. Chen F. Nishimura M. Steiner H. Tandon A. Kawarai T. Arawaka S. Supala A. Song Y.Q. Rogaeva E. Holmes E. Zhang D.M. Milman P. Fraser P. Haass C. St. George-Hyslop P. J. Biol. Chem. 2000; 275: 27348-27353Abstract Full Text Full Text PDF PubMed Google Scholar, 13Levitan D. Lee J. Song L. Manning R. Wong G. Parker E. Zhang L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12186-12190Crossref PubMed Scopus (73) Google Scholar, 14Xia X. Wang P. Sun X. Soriano S. Shum W.K. Yamaguchi H. Trumbauer M.E. Takashima A. Koo E.H. Zheng H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8760-8765Crossref PubMed Scopus (31) Google Scholar, 15Nyabi 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, 16Capell A. Steiner H. Romig H. Keck S. Baader M. Grim M.G. Baumeister R. Haass C. Nat. Cell Biol. 2000; 2: 205-211Crossref PubMed Scopus (139) Google Scholar, 17Edbauer D. Winkler E. Regula J.T. Pesold B. Steiner H. Haass C. Nat. Cell Biol. 2003; 5: 486-488Crossref PubMed Scopus (776) Google Scholar, 18Schroeter E.H. Ilagan M.X.G. Brunkan A.L. Hecimovic S. Li Y.M. Xu M. Lewis H.D. Saxena M.T. De Strooper B. Coonrod A. Tomita T. Iwatsubo T. Moore C.L. Goate A. Wolfe M.S. Shearman M. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13075-13080Crossref PubMed Scopus (181) Google Scholar). To demonstrate directly that the defects of PS-1 D257A and D385A mutants are caused by changes in their catalytic sites and also to analyze the different effects of the PS-1 NTF and CTF, we constructed normal and mutant NTFs and CTFs tagged with GFP at the N terminus as follows: wt NTF (1–298 amino acids), wt CTF (299–467 amino acids), D257A NTF (1–298 amino acids in which the aspartate in residue 257 is point mutated to alanine), D385A CTF (299–467 amino acids in which the aspartate in residue 385 is point mutated to alanine). The overexpression of either wt NTF or wt CTF alone (Fig. 2A, lanes 4 and 5, respectively) was not sufficient to cleave Notch, but overexpression of wt NTF and wt CTF together (Fig. 2A, lane 6) cleaved Notch to the same extent as full-length PS-1 (Fig. 2A, lane 3), indicating that the NTF and CTF constructs functioned as expected. When we combined D257A NTF and D385A CTF with their normal counterparts, wt CTF and wt NTF, respectively (Fig. 2A, lanes 7 and 8), Notch was not cleaved, indicating that the defects of full-length PS-1 D257A and D385A are caused by changes in their catalytic domains rather than the lack of endoproteolysis. Interestingly, the defects of full-length PS-1 D385A could be restored by overexpressing its normal counterpart, wt CTF (Fig. 2A, lane 10), whereas the defect of D257A could not be restored by wt NTF (Fig. 2A, lane 9). Along the same line, it is worth noting that the defects in cleaving Notch by the C410Y mutant was also restored by overexpressing wt CTF but not wt NTF (Fig. 2A, lanes 13 and 12, respectively). Our results suggest that the defects of NTF and CTF regional mutations can be restored differentially by overexpressing their normal PS-1 fragments. Endoproteolysis of PS-1 D257A and D385A Mutants Depends on the Presence of Endogenous PS-1/2—To measure the level of endoproteolysis of D257A and D385A mutants, we transiently transfected both mutants to PS-1/2-/- MEF cell lines that had neither endogenous PS-1 nor PS-2. In PS-1/2-/- cell lines, a generated endoproteolyzed form of PS-1 was detected with a specific antibody against their CTF. The endoproteolyzed form of PS-1 D257A and D385A was barely detectable compared with that of wt PS-1 (Fig. 2B, lanes 1, 2, and 5). The compensatory overexpression of NTF or CTF did not induce any significant increase in the endoproteolysis of PS-1 D257A and D385A (Fig. 2B, lanes 3, 4, 6, and 7). Therefore, the restoration of γ-secretase activity in PS-1 D385A by the compensatory overexpression of normal CTF may result from the direct binding to the Asp-257 NTF portion of uncleaved PS-1 D385A rather than from increased endoproteolysis. The weak endoproteolysis of PS-1 D257A and D385A mutants in PS-1/2-/- MEF cells was more apparent in PS-1/2+/+ cells, suggesting that the degree of endoproteolysis of PS-1 D257A and D385A mutants might be affected by the presence of functional γ-secretase (Fig. 2C). The Minimal Domain of Wild Type CTF Maintaining the γ-Secretase Activity Is Located between Residues 349 and 467, and Notch Cleavage Can Be Abolished with D385A CTF and Its 349–467 Fragment in a Dose-dependent Manner—As shown above, PS-1 is processed endoproteolytically, generating NTF and CTF. Laudon and co-workers (42Laudon H. Mathews P.M. Karlstrom H. Bergman A. Farmery M.R. Nixon R.A. Winblad B. Gandy S.E. Lendahl U. Lundkvist J. Naslund J. J. Neurochem. 2004; 89: 44-53Crossref PubMed Scopus (47) Google Scholar) have reported that the overexpression of both wt NTF and CTF, but not necessarily full-length PS-1, is sufficient to cleave Notch, which is in agreement with our current observations. To understand the roles of NTF and CTF more precisely and map the minimal regions responsible for γ-secretase activity, we constructed several deleted NTFs and CTFs tagged with GFP at their N termini, as illustrated in Fig. 3A. We confirmed the expression of each construct by Western blot analysis (Fig. 3B). Wild type CTF and deleted NTF and CTF fragments migrated as we expected, but wt NTF was incorporated mainly into HMW aggregates. Fragments B and E as well as CTF fragment A were slightly heavier than we expected, possibly because of the phosphorylation of residue 346, a known target site of protein kinase C (43Fluhrer R. Friedlein A. Haass C. Walter J. J. Biol. Chem. 2004; 279: 1585-1593Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). When we coexpressed one of the deleted NTFs with wt CTF in PS-1/2-/- MEFs, none of the deleted NTFs showed Notch cleavage, suggesting that an almost full conformation of wt NTF is required for γ-secretase activity (Fig. 3C). Nevertheless, we found that the minimal domain of wt CTF responsible for generating γ-secretase activity is located between residues 349 and 467 of PS-1 (Fig. 3D, F fragment). Neither fragment E (residues 299–417) nor fragment D (residues 418–467) was sufficient to cleave Notch. To define the factors affecting the PS-1/γ-secretase activity in CTF, especially fragments E and F, we transfected wt NTF and a different CTF fragment in PS-1/2-/- MEFs and performed immunoblot analysis as shown in Fig. 4. Noticeable decreases in Nicastrin and Notch binding to PS-1 were observed in cells transfected with wt NTF and fragment E (residues 299–417), whereas no decrease in binding in cells transfected with wt NTF and fragment F (residues 349–467) was seen, indicating that the C terminus of PS-1 (amino acids 418–467) might be important for Nicastrin and/or Notch binding (Fig. 4A). The binding of Nicastrin and Notch in wt NTF and D385A CTF-transfected cells was observed (second lane), indicating once more that the defects of PS-1 D385