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• W2032232498 abstract "A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, γ-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment γ (CTFγ) derivatives are dependent on PS expression and inhibited by a highly selective and potent γ-secretase inhibitor. Unexpectedly, the APP-CTFγ derivative is generated by processing between Leu-645 and Val-646 (of APP695), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid β protein peptides. Although the relationship of APP-CTFγ to the production of amyloid β protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent γ-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain. A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, γ-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment γ (CTFγ) derivatives are dependent on PS expression and inhibited by a highly selective and potent γ-secretase inhibitor. Unexpectedly, the APP-CTFγ derivative is generated by processing between Leu-645 and Val-646 (of APP695), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid β protein peptides. Although the relationship of APP-CTFγ to the production of amyloid β protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent γ-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain. presenilin amyloid β protein amyloid precursor protein carboxyl-terminal fragment matrix-assisted laser desorption/ionization time-of-flight mass spectrometer Notch intracellular domain polyacrylamide gel electrophoresis transmembrane Notch extracellular truncation Presenilin 1 and 2 (PS1 and PS2)1 are polytopic membrane proteins that are mutated in the majority of pedigrees with early-onset FAD. Compelling evidence has emerged supporting a role for PS in intramembranous γ-secretase processing of the type I membrane proteins, including APP (1De 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 (1544) Google Scholar, 2Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) and Notch1 (3De 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 (1792) Google Scholar). γ-Secretase processing of a set of membrane-tethered APP-CTFs results in the production of Aβ peptides that are secreted and subsequently deposited in brains of patients with Alzheimer's disease. On the other hand, γ-secretase processing of the Notch1 derivative, termed S2/NEXT, releases the intracellular domain (S3/NICD), a transcriptional coactivator that associates with CBF1/Su(H)/Lag1 (4Selkoe D.J. Curr. Opin. Neurobiol. 2000; 10: 50-57Crossref PubMed Scopus (67) Google Scholar, 5Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1350) Google Scholar, 6Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). The observation that Aβ and S3/NICD production are completely eliminated in cells with compound deletions of PS1 and PS2 (7Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 8Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (358) Google Scholar), and the demonstration that PS1 and PS2 can be photocross-linked to γ-secretase inhibitors (9Li 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 (861) Google Scholar), have led to the conclusion that PS are the molecules that execute intramembranous cleavage of APP and Notch1. Furthermore, PS shares limited homology with the prepilin peptidases, a family of bacterial aspartyl proteases (10Steiner H. Kostka M. Romig H. Basset G. Pesold B. Hardy J. Capell A. Meyn L. Grim M.L. Baumeister R. Fechteler K. Haass C. Nat. Cell Biol. 2000; 2: 848-851Crossref PubMed Scopus (251) Google Scholar), and PS binds to pepstatin, an aspartyl protease inhibitor (11Evin G. Sharples R.A. Weidemann A. Reinhard F.B. Carbone V. Culvenor J.G. Holsinger R.M. Sernee M.F. Beyreuther K. Masters C.L. Biochemistry. 2001; 40: 8359-8368Crossref PubMed Scopus (43) Google Scholar). Although compelling, several observations have questioned the veracity of this model. First, and despite reports showing PS1 to be resident in late compartments, including endosomes and plasma membranes (12Ray W.J. Yao M. Mumm J. Schroeter E.H. Saftig P. Wolfe M. Selkoe D.J. Kopan R. Goate A.M. J. Biol. Chem. 1999; 274: 36801-36807Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 13Lah J.J. Levey A.I. Mol. Cell. Neurosci. 2000; 16: 111-126Crossref PubMed Scopus (80) Google Scholar, 14Schwarzman A.L. Singh N. Tsiper M. Gregori L. Dranovsky A. Vitek M.P. Glabe C.G. St. George-Hyslop P.H. Goldgaber D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7932-7937Crossref PubMed Scopus (63) Google Scholar), we and others have revealed that PS are preponderantly localized in the endoplasmic reticulum and associated compartments, sites that are incompatible with the known cellular sites for γ-secretase cleavage of APP and Notch1 that include the plasma membrane, Golgi, and endosomes (15Annaert W.G. Levesque L. Craessaerts K. Dierinck I. Snellings G. Westaway D. George-Hyslop P.S. Cordell B. Fraser P. De Strooper B. J. Cell Biol. 1999; 147: 277-294Crossref PubMed Scopus (276) Google Scholar, 16Cupers P. Bentahir M. Craessaerts K. Orlans I. Vanderstichele H. Saftig P. De Strooper B. Annaert W. J. Cell Biol. 2001; 154: 731-740Crossref PubMed Scopus (137) Google Scholar). Second, endocytosis and recycling of APP-CTFs is a major pathway for generating Aβ (17Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar), but production of the Notch S3/NICD does not require endocytic trafficking of S2/NEXT (18Struhl G. Adachi A. Mol. Cell. 2000; 6: 625-636Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Third, γ-secretase has contrasting substrate specificities for processing within the APP (19Murphy M.P. Hickman L.J. Eckman C.B. Uljon S.N. Wang R. Golde T.E. J. Biol. Chem. 1999; 274: 11914-11923Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar,20Lichtenthaler S.F. Wang R. Grimm H. Uljon S.N. Masters C.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3053-3058Crossref PubMed Scopus (192) Google Scholar) and Notch (5Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1350) Google Scholar) TM domains, and γ-secretase processing of the APP TM domain occurs at heterogeneous sites (19Murphy M.P. Hickman L.J. Eckman C.B. Uljon S.N. Wang R. Golde T.E. J. Biol. Chem. 1999; 274: 11914-11923Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Lichtenthaler S.F. Wang R. Grimm H. Uljon S.N. Masters C.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3053-3058Crossref PubMed Scopus (192) Google Scholar) whereas γ-secretase cleavage of Notch1 appears to generate a single S3/NICD species (5Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1350) Google Scholar). In the present report, we examine the production of γ-secretase-generated APP and Notch1 derivatives. We now document, both in living cells and cytosol-free membrane preparations, the generation of APP-CTFγ derived from membrane-tethered APP-CTFs. In parallel, we document the production of the Notch S3/NICD in these preparations. The production of the APP-CTFγ and S3/NICD derivatives are dependent on PS1 expression and inhibited by a γ-secretase inhibitor. Unexpectedly, the APP-CTFγ derivative produced in these reactions is generated by endoproteolytic processing at a site several amino acids carboxyl-terminal to the scissile sites for production of Aβ peptides but interestingly, occurs at a site immediately proximal to the analogous site for production of S3/NICD. Although the relationship of APP-CTFγ to the production of Aβ peptides is not known, we conclude that in contrast to the highly selective PS-dependent proteolysis of the Notch TM domain, the PS-dependent γ-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP TM domain. Cell culture, transfection, and immunoblot were performed as described (21Martys-Zage J.L. Kim S.H. Berechid B. Bingham S.J. Chu S. Sklar J. Nye J. Sisodia S.S. J. Mol. Neurosci. 2000; 15: 189-204Crossref PubMed Scopus (43) Google Scholar, 22Sisodia S.S. Koo E.H. Hoffman P.N. Perry G. Price D.L. J. Neurosci. 1993; 13: 3136-3142Crossref PubMed Google Scholar, 23Lamb B.T. Bardel K.A. Kulnane L.S. Anderson J.J. Holtz G. Wagner S.L. Sisodia S.S. Hoeger E.J. Nat. Neurosci. 1999; 2: 695-697Crossref PubMed Scopus (90) Google Scholar). Myc-tagged Notch derivatives were detected using 9E10 (21Martys-Zage J.L. Kim S.H. Berechid B. Bingham S.J. Chu S. Sklar J. Nye J. Sisodia S.S. J. Mol. Neurosci. 2000; 15: 189-204Crossref PubMed Scopus (43) Google Scholar). Full-length APP and APP-CTFs were detected by CT15 (22Sisodia S.S. Koo E.H. Hoffman P.N. Perry G. Price D.L. J. Neurosci. 1993; 13: 3136-3142Crossref PubMed Google Scholar). For detection of Aβ, conditioned medium was immunoprecipitated with 26D6 antibody (23Lamb B.T. Bardel K.A. Kulnane L.S. Anderson J.J. Holtz G. Wagner S.L. Sisodia S.S. Hoeger E.J. Nat. Neurosci. 1999; 2: 695-697Crossref PubMed Scopus (90) Google Scholar) and immunoblotted with 26D6. N2aWT.7 or D385A.16 cells were labeled for 15 min with 750 μCi/ml [35S]methionine (PerkinElmer Life Sciences). Cells were either harvested after pulse or chased for 45 min at 37 °C in the presence of 0.5 mm cold l-methionine (Life Technologies, Inc.). Conditioned media were collected, centrifuged briefly, and immunoprecipitated with 4G8. Cell pellets were treated with 250 μl of 3% SDS in phosphate-buffered saline containing 10 μl/ml β-mercaptoethanol and subjected to vortexing and heating at 95 °C for 10 min, followed by sonication and centrifugation at 100,000 × g for 10 min. The supernatants were diluted 1:4, adjusted to a final concentration of 2% Triton X-100 and 190 mm NaCl, 20 mm Tris-Cl, pH 8.8, and 2 mm EDTA, and subjected to immunoprecipitation with 369 or 4G8. Purification of membrane fractions from cultured cells was performed as described (24Smart E.J. Ying Y.S. Mineo C. Anderson R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar). Briefly, cell pellets from ten 10-cm dishes were resuspended in Buffer A (250 mm sucrose, 20 mm HEPES, pH 7.4) containing protease inhibitor mixture. Post-nuclear supernatant was layered onto 30% Percoll solution and subjected to high speed centrifugation. Cellular membranes was resuspended in 14% iodixanol and overlaid with 12 and 6% iodixanol. Following centrifugation at 52,000 × g for 90 min, membranes were collected from each interface. Collected membranes were incubated at 4 or 37 °C for 60 min, and the reactions were terminated by the addition of Laemmli sample buffer. In the γ-secretase inhibitor experiments, the reaction tubes were preincubated at 4 °C for 10 min with 100 nm L-685,458 (25Shearman M.S. Beher D. Clarke E.E. Lewis H.D. Harrison T. Hunt P. Nadin A. Smith A.L. Stevenson G. Castro J.L. Biochemistry. 2000; 39: 8698-8704Crossref PubMed Scopus (365) Google Scholar) prepared in Me2SO, or an equivalent concentration of Me2SO, and then incubated at 37 °C for 60 min. The reaction samples were centrifuged at 100,000 × g for 1 h to separate the membrane-bound and soluble APP-CTFs. CTFγ from the soluble fraction was immunoprecipitated with CT15 and protein A-agarose and analyzed by MALDI-TOF MS (26Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). We demonstrated previously that a Notch6-myc-green fluorescent protein chimera is subject to PS1-dependent γ-secretase processing; this processing event is stimulated either by addition of extracellular ligand or by depletion of extracellular calcium (21Martys-Zage J.L. Kim S.H. Berechid B. Bingham S.J. Chu S. Sklar J. Nye J. Sisodia S.S. J. Mol. Neurosci. 2000; 15: 189-204Crossref PubMed Scopus (43) Google Scholar). To assess the role for PS1 in the production of an APP carboxyl-terminal derivative, termed APP-CTFγ, that would be generated following intramembranous proteolysis by γ-secretase, we performed Western blot analysis of detergent lysates from PS1-deficient fibroblasts transiently cotransfected with cDNA encoding APPmyc and either wild type PS1 or a PS1 aspartate variant (D385A) that has been shown to reduce Aβ secretion (27Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1679) Google Scholar, 28Capell 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 (138) Google Scholar) (Fig.1 A). As expected, upon transfection of APPmyc cDNA, ∼12-kDa CTFmycα and ∼14-kDa CTFmycβ appear that are significantly elevated over the ∼10-kDa endogenous CTFα seen in untransfected cells (Fig. 1 A, compare lanes 1 and 2). On the other hand, coexpression of human wild type PS1 with APPmyc reduces the levels of these ∼12- and ∼14-kDa CTFmyc species, commensurate with the accumulation of a novel ∼7-kDa CTFmyc species, which we propose is CTFmycγ (Fig. 1 A, lane 3). In support of this notion, coexpression of the D385A PS1 variant and APPmyc restores the levels of accumulated CTFmycα and CTFmycβ species seen in APPmyc-transfected cells and most importantly, eliminates the production of the CTFmycγ (Fig. 1 A, lane 4). We confirmed these findings by showing that expression of APPmyc does not lead to appreciable levels of secreted Aβ but that coexpression of wild type PS1 restores Aβ secretion (Fig. 1 A, lanes 2′ and 3′). Moreover, the PS1 D385A variant inhibits Aβ production, as expected (Fig. 1 A, lane 4′). These results in transient transfection analyses were fully confirmed in biosynthetically labeled N2a cell lines that stably coexpress APPmyc and either wild type PS1 (WT.7) or the PS1 D385A variant (D385A.16). Pulse-labeling with [35S]methionine for 15 min and immunoprecipitation analysis of detergent lysates using antibody 369 revealed identical synthetic levels of APP in both lines and low but detectable levels of ∼12- and ∼14-kDa CTFmyc species (Fig.1 B, lanes 1 and 2); intracellular Aβ peptides are not detected at this time point (Fig. 1 B,lanes 1′ and 2′). After 45 min of chase, we observed increased levels of ∼12-kDa CTFmycα and ∼14-kDa CTFmycβ species and an ∼7-kDa CTFmycγ species in lysates of WT.7 line (Fig. 1 B, lane 3). In contrast, we observed very high levels of CTFmycα and CTFmycβ species in lysates of cells expressing the D385A PS1 variant (Fig. 1 B, lane 4). However, the level of ∼7-kDa CTFmycγ in cells expressing the D385A PS1 variant was markedly lower than the levels observed in cells expressing wild type PS1 (Fig. 1 B, compare lanes 3 and 4). Indeed, the levels of Aβ both in cell lysates and in the conditioned medium from cells expressing the D385A variant were substantially reduced compared with those from cells expressing wild type PS1 (see Fig. 1 B, compare lanes 3′ and 4′ and lanes 3“ and 4”; Aβ secretion is not detectable at the end of the pulse-labeling period). To develop a biochemically tractable system to examine γ-secretase processing of Notch and APP, we isolated membrane fractions from PS1±fibroblasts that express the Notch-green fluorescent protein chimera and examined EDTA-induced γ-secretase processing (Fig.2 A). We show that incubation of these membranes at 37 °C led to the production of the S3/NICD fragment (Fig. 2 A, lanes 1 and 2) and that this reaction was inhibited by addition of the γ-secretase inhibitor L-685,458 to 100 nm (Fig. 2 A,lane 3). In parallel, we assessed the production of a previously described ∼6-kDa CTFγ derived from γ-secretase cleavage of endogenous ∼10-kDa CTFα (29McLendon C. Xin T. Ziani-Cherif C. Murphy M.P. Findlay K.A. Lewis P.A. Pinnix I. Sambamurti K. Wang R. Fauq A. Golde T.E. FASEB J. 2000; 14: 2383-2386Crossref PubMed Scopus (105) Google Scholar, 30Pinnix I. Musunuru U. Tun H. Sridharan A. Golde T. Eckman C. Ziani-Cherif C. Onstead L. Sambamurti K. J. Biol. Chem. 2001; 276: 481-487Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). We detected the CTFγ only after incubation at 37 °C (Fig. 2 B,lanes 1 and 2), and this reaction was also inhibited by addition of the γ-secretase inhibitor (Fig.2 B, lane 3). In titration experiments using varying concentrations of L-685,458, we have demonstrated that the in vitro generation of S3/NICD and APP-CTFγ are equally sensitive to the inhibitor with an IC50 of ∼50 pm, 2T. I. and S. S. S., submitted for publication. findings that suggest that these derivatives are generated by similar, if not identical, γ-secretase activities. As the levels of endogenous APP-CTFγ generated in the fibroblasts were extremely low, we chose to analyze the CTFγ fragment generated in membranes from a cell line (N2aWT.11) that stably expresses APPmyc and wild type PS1. Incubation of membranes at 37 °C for 60 min from N2aWT.11 cells leads to unchanged levels of full-length APP but a diminution in levels of the ∼12- and ∼14-kDa CTFmyc derivatives. Commensurate with reduction in levels of these CTFmyc species was the appearance of an ∼7-kDa CTFmycγ derivative, similar to the PS1-dependent CTFmycγ fragment observed in living cells (Fig.3 A, lanes 1 and 2; see Fig. 1 B). With the assumption that the CTFmycγ fragment might be extruded into the cytoplasmic compartment in a manner similar to the S3/NICD fragment of Notch, we prepared membrane and soluble fractions. The ∼12- and ∼14-kDa CTFmyc fragments were membrane-bound prior to, or after, incubation at 37 °C (Fig. 3 B, lanes 2 and 4), and as expected, the CTFmycγ fragment was found exclusively in the soluble fraction (Fig. 3 B, lane 6). To establish the identity of the CTFmycγ fragment in the soluble fraction, we immunoprecipitated the peptide with CT15 and subjected recovered material to MALDI-TOF MS (Fig. 3 C). We observed principal peptides of mr 7341.3 and 7110.4, which includes the mass of the 12-amino acid myc epitope tag; the prominent mr 7341.3 peptide is generated by endoproteolytic cleavage between Leu-645 and Val-646 (of APP695), whereas the mr 7110.4 peptide is generated by cleavage between Met-647 and Leu-648 (Fig.3 C). A series of genetic and pharmacological approaches have revealed that PS are required for γ-secretase processing of Notch1 and APP (31Selkoe D.J. Wolfe M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5690-5692Crossref PubMed Scopus (64) Google Scholar). In the case of Notch1, a membrane-tethered S2/NEXT is subject to processing by γ-secretase, leading to the generation of a cytoplasmic fragment, S3/NICD (4Selkoe D.J. Curr. Opin. Neurobiol. 2000; 10: 50-57Crossref PubMed Scopus (67) Google Scholar). APP is processed by α- and β-secretases to generate a set of membrane-tethered CTFs, and these derivatives are subsequently processed by γ-secretase to generate a spectrum of secreted Aβ peptides (see below). However, the residual CTFγ resulting from γ-secretase processing of the CTFα and -β, has been elusive. Very recently, McLendon et al. (29McLendon C. Xin T. Ziani-Cherif C. Murphy M.P. Findlay K.A. Lewis P.A. Pinnix I. Sambamurti K. Wang R. Fauq A. Golde T.E. FASEB J. 2000; 14: 2383-2386Crossref PubMed Scopus (105) Google Scholar) and Pinnix et al. (30Pinnix I. Musunuru U. Tun H. Sridharan A. Golde T. Eckman C. Ziani-Cherif C. Onstead L. Sambamurti K. J. Biol. Chem. 2001; 276: 481-487Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) reported on the generation of a CTFγ derivative in isolated membranes from brain or cultured cells. This derivative was not biochemically characterized, but its production was inhibited by high concentrations of pepstatin A, MG132, and a substrate-based difluoroketone (30Pinnix I. Musunuru U. Tun H. Sridharan A. Golde T. Eckman C. Ziani-Cherif C. Onstead L. Sambamurti K. J. Biol. Chem. 2001; 276: 481-487Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). To these latter observations, the present report offers several novel insights relevant to γ-secretase processing of the Notch S2/NEXT and the APP-CTFs. First, we document that a peptide corresponding to APP-CTFγ can be detected in lysates of transfected cells and that the production of this derivative is dependent on PS1 expression and largely eliminated by expression of the dominant negative D385A PS1 variant. Second, we establish that both the APP-CTFγ and S3/NICD can be generated in cytosol-free membranes and that the production of these derivatives is inhibited by a γ-secretase inhibitor. Third, and most surprising, is our demonstration using MALDI-TOF MS that the prominent in vitro-derived APP-CTFγ is generated by proteolysis between the 21st (Leu-645) and 22nd (Val-646) amino acids of the 24-amino acid APP TM domain. Notably, the P1′ Val-646 is one residue more carboxyl-terminal to the P1′ Val that is necessary for Notch processing (5Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1350) Google Scholar). Our studies are consistent with recent results from Sastre et al. (32Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO J. 2001; 2: 835-841Crossref Scopus (426) Google Scholar) and Gu et al. (33Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y.J. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Having established the identity of CTFγ, we are now faced with the conundrum that the processing sites for generation of this CTF are distinct from the scissile sites for Aβ40 or Aβ42 peptides, which occur between the 12th (Val-636) and 13th (Ile-637) or 14th (Ala-638) and 15th (Thr-639) amino acids, respectively. Taken together with our present data showing that CTFγ production is dependent on PS1 expression, and inhibited by a selective γ-secretase inhibitor that also blocks production of Aβ, we offer three mechanistic scenarios. The first of these is that CTFγ is derived by secondary endoproteolysis of the CTF57 or CTF59 derivatives that are the residual fragments from proteolysis at the scissile bonds that generate Aβ. We consider this unlikely, as we have detected neither the CTF57 nor CTF59 derivatives in in vivo pulse-chase studies nor in kinetic studies in cytosol-free membrane preparations. The second possibility is that proteolysis between Leu-645 and Val-646 is obligatory for subsequent endo- or exoproteolytic activity events necessary for generating Aβ40/42 peptides. This notion can be tested by assessing CTFγ and Aβ production in cells expressing APP variants with mutations surrounding the scissile site. In this regard, and in view of the finding that a V1744G substitution at the P1′ site in the Notch blocks NICD production and Notch activity (34Huppert S.S. Le A. Schroeter E.H. Mumm J.S. Saxena M.T. Milner L.A. Kopan R. Nature. 2000; 405: 966-970Crossref PubMed Scopus (284) Google Scholar), Sastre et al. (32Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO J. 2001; 2: 835-841Crossref Scopus (426) Google Scholar) have recently examined CTFγ production in membranes prepared from cells expressing APP with a V646G substitution. Surprisingly, the production of CTFγ was unimpaired. Clearly, a more rigorous mutational analysis is required before a conclusion can be drawn about the relationship between the generation of CTFγ and Aβ production. The third alternative is that proteolysis giving rise to Aβ does not require prior generation of the CTFγ or vice versa. In this scenario, the membrane-tethered APP-CTFs are randomly processed at multiple sites. In view of the multiplicity of Aβ-related peptides with termini at 34, 37, 38, 39, 42, and 43 that are invariably detected (19Murphy M.P. Hickman L.J. Eckman C.B. Uljon S.N. Wang R. Golde T.E. J. Biol. Chem. 1999; 274: 11914-11923Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Lichtenthaler S.F. Wang R. Grimm H. Uljon S.N. Masters C.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3053-3058Crossref PubMed Scopus (192) Google Scholar,26Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar), and the demonstration that extensive mutagenesis of the APP TM domain have little effect on the production of Aβ-related peptides (19Murphy M.P. Hickman L.J. Eckman C.B. Uljon S.N. Wang R. Golde T.E. J. Biol. Chem. 1999; 274: 11914-11923Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 20Lichtenthaler S.F. Wang R. Grimm H. Uljon S.N. Masters C.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3053-3058Crossref PubMed Scopus (192) Google Scholar), we would argue that the PS-dependent γ-secretase activity is largely nonselective. We would offer the proposal that the APP TM domain is subject to processing by a presently unidentified membrane-resident, multicatalytic protease similar to the proteosome. We envision that the function of this protease would be to introduce nicks in TM segments of many membrane proteins, resulting in slippage of the residual TM segments into either the cytoplasmic or lumenal space wherein the entire domain is subject to the proteolytic activities resident within those compartments. In this vein, it is equally conceivable that regulated forms of this protease class would be responsible for generating cytosolic fragments that play critical roles in nuclear signaling events (35Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar). The recent demonstration of a potential nuclear signaling role for the cytoplasmic domain of APP (36Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1050) Google Scholar) underscores the importance of understanding the mechanisms responsible for intramembranous processing of APP. Despite the differences in the precise sites of proteolysis and sequence requirements within the APP and Notch TM domains, we have shown that L-685,458 is an equally potent inhibitor of the reaction(s) that generate Notch S3/NICD and CTFγ (see Ref. 21Martys-Zage J.L. Kim S.H. Berechid B. Bingham S.J. Chu S. Sklar J. Nye J. Sisodia S.S. J. Mol. Neurosci. 2000; 15: 189-204Crossref PubMed Scopus (43) Google Scholar and this study).2 These data would imply that if γ-secretase is a single entity, it must be highly unusual. In this regard, it is not clear how expression of PS1 harboring a D257A substitution is still capable of generating Aβ but fails to generate Notch S3/NICD (28Capell 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 (138) Google Scholar). Similarly, expression of FAD-linked L166P PS1 variant or the experimental L286E or L286R PS1 mutants leads to overproduction of Aβ42 but fails to generate S3/NICD (37Kulic L. Walter J. Multhaup G. Teplow D.B. Baumeister R. Romig H. Capell A. Steiner H. Haass C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5913-5918Crossref PubMed Scopus (77) Google Scholar). 3C. Haass, personal communication. Finally, recent studies have revealed that processing of APP and Notch1 can be discriminated by a JLK family of non-peptidic inhibitors (38Petit A. Bihel F. Alves da Costa C. Pourquie O. Checler F. Kraus J.L. Nat. Cell Biol. 2001; 3: 507-511Crossref PubMed Scopus (193) Google Scholar). These inhibitors block Aβ production but have very little, if any, effect on production of S3/NICD (38Petit A. Bihel F. Alves da Costa C. Pourquie O. Checler F. Kraus J.L. Nat. Cell Biol. 2001; 3: 507-511Crossref PubMed Scopus (193) Google Scholar). Collectively, these experiments prove unequivocally that γ-secretase processing of APP and Notch can be dissociated, leading us to conclude that the catalytic activities responsible for processing these substrates are not one in the same. Thus, whereas PS are critical for regulating γ-secretase activities, it is our view that these polypeptides are unlikely to be the sole effectors of intramembranous proteolysis of APP and Notch1. In the final analysis, it will be critical to develop in vitroreconstitution systems with purified components to establish the role(s) of PS and their interacting components in facilitating γ-secretase processing of APP and Notch1. We thank Drs. Mark S. Shearman and Yue-Ming Li (Merck Research Laboratories) for providing L-685,458." @default.
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• W2032232498 title "Characterization of a Presenilin-mediated Amyloid Precursor Protein Carboxyl-terminal Fragment γ" @default.
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