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• W1987016766 abstract "Membrane-bound BACE1 naturally cleaves its transmembrane substrate amyloid precursor protein (APP) at the two adjacent β- and β′-sites. Cleavage at these two sites generates the heterogeneous N-terminal end of APP C-terminal fragments that are further processed by γ-secretase to release Aβ-(1-40/42) or Aβ-(11-40/42). The significance underlying Aβ-(11-40/42) in Alzheimer's disease pathogenesis has remained to be experimentally elucidated, but increased production of Aβ-(1-40/42) has been broadly demonstrated to contribute to amyloid depositions in senile plaques. In this study, we show that the cleavage of APP at the β-site by BACE1 is readily disrupted through limited structural twists, whereas the β′-site is relatively better positioned to gain access to the BACE1 catalytic cavity. Radical insertion or deletion of residues between β- and β′-site also favors cleavage of APP at the β′-site. On the other hand, either lengthening or shortening the loop region of BACE1 has a minor impact on the selective cleavage of APP at these two adjacent sites, but significantly shortening the loop region impairs the ability of BACE1 to process APP at both sites. Thus, processing of APP by BACE1 is clearly dependent on a mutual structural compatibility in addition to the sequence feature. The knowledge gained from this study will potentially offer an opportunity for rational design of small molecule drugs to block the cleavage of APP specifically at the β-site while not disturbing the functions of other cellular aspartyl proteases. Membrane-bound BACE1 naturally cleaves its transmembrane substrate amyloid precursor protein (APP) at the two adjacent β- and β′-sites. Cleavage at these two sites generates the heterogeneous N-terminal end of APP C-terminal fragments that are further processed by γ-secretase to release Aβ-(1-40/42) or Aβ-(11-40/42). The significance underlying Aβ-(11-40/42) in Alzheimer's disease pathogenesis has remained to be experimentally elucidated, but increased production of Aβ-(1-40/42) has been broadly demonstrated to contribute to amyloid depositions in senile plaques. In this study, we show that the cleavage of APP at the β-site by BACE1 is readily disrupted through limited structural twists, whereas the β′-site is relatively better positioned to gain access to the BACE1 catalytic cavity. Radical insertion or deletion of residues between β- and β′-site also favors cleavage of APP at the β′-site. On the other hand, either lengthening or shortening the loop region of BACE1 has a minor impact on the selective cleavage of APP at these two adjacent sites, but significantly shortening the loop region impairs the ability of BACE1 to process APP at both sites. Thus, processing of APP by BACE1 is clearly dependent on a mutual structural compatibility in addition to the sequence feature. The knowledge gained from this study will potentially offer an opportunity for rational design of small molecule drugs to block the cleavage of APP specifically at the β-site while not disturbing the functions of other cellular aspartyl proteases. Amyloid peptides (Aβ), 1The abbreviations used are: Aβ, amyloid peptide; APP, amyloid precursor protein; HA, an epitope tag from human influenza hemagglutinin protein; HM cell, a HEK-293 cell derived cell line stably expressing HA-tagged BACE1; CTF, C-terminal fragment; BFA, brefeldin A; WT, wild type.1The abbreviations used are: Aβ, amyloid peptide; APP, amyloid precursor protein; HA, an epitope tag from human influenza hemagglutinin protein; HM cell, a HEK-293 cell derived cell line stably expressing HA-tagged BACE1; CTF, C-terminal fragment; BFA, brefeldin A; WT, wild type. the major components of the amyloid depositions found in senile plaques, are excised from amyloid precursor protein (APP) through sequential cleavage by two endopeptidases: β- and γ-secretases. Elevated levels of Aβ in human brains have been shown to correlate with cognitive decline (1Näslund J. Haroutunian V. Mohs R. Davis K.L. Davies P. Greengard P. Buxbaum J.D. J. Am. Med. Assoc. 2000; 283: 1571-1577Crossref PubMed Scopus (1102) Google Scholar). Genetic, pathological, and biochemical evidence has proclaimed Aβ as one of the potential etiological factors for Alzheimer's disease (2Hardy J. Selkoe D. Science. 2002; 297: 353-356Crossref PubMed Scopus (10791) Google Scholar, 3Sisodia S.S. St. George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Crossref PubMed Scopus (485) Google Scholar). Therapeutic interventions strategically targeted to either block Aβ production or enhance its clearance have begun to show promise in animal models (4Selkoe D.J. Shenk D. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 545-584Crossref PubMed Scopus (737) Google Scholar).A type I transmembrane aspartyl protease has been identified to be the β-secretase called BACE1, an acronym for β-site APP cleaving enzyme (5Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3255) Google Scholar, 6Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar, 7Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1472) Google Scholar, 8Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 9Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (737) Google Scholar). Mammalian aspartyl proteases are typically translated as zymogens, but BACE1 possesses its catalytic activity although its pro domain is still retained (10Ermolieff J. Loy J.A. Koelsch G. Tang J. Biochemistry. 2000; 39: 12450-12456Crossref PubMed Scopus (133) Google Scholar, 11Shi X.P. Chen E. Yin K.C. Na S. Garsky V.M. Lai M.T. Li Y.M. Platchek M. Register R.B. Sardana M.K. Tang M.J. Thiebeau J. Wood T. Shafer J.A. Gardell S.J. J. Biol. Chem. 2001; 276: 10366-10373Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chrietien M. Siedah N.G. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Full maturation of BACE1 occurs in the Golgi where its pro domain is removed by furin or furin-like convertases (12Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chrietien M. Siedah N.G. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 14Bennett B.D. Denis P. Haniu M. Teplow D.B. Kahn S. Louis J.C. Citron M. Vassar R.A. J. Biol. Chem. 2000; 275: 37712-37717Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15Pinnix I. Council J.E. Roseberry B. Onstead L. Mallender W. Sucic J. Sambamurti K. FASEB J. 2001; 15: 1810-1812Crossref PubMed Scopus (27) Google Scholar) and a complex sugar moiety is attached. Noticeably, BACE1 activity is independent of these posttranslational modifications. All these features are consistent with the findings that BACE1 is also active in the endoplasmic reticulum compartments where immature BACE1 is translated and properly folded (12Benjannet S. Elagoz A. Wickham L. Mamarbachi M. Munzer J.S. Basak A. Lazure C. Cromlish J.A. Sisodia S. Checler F. Chrietien M. Siedah N.G. J. Biol. Chem. 2001; 276: 10879-10887Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 13Creemers J.W. Dominguez D.I. Plets E. Serneels L. Taylor N.A. Multhaup G. Craessaerts K. Annaert W. De Strooper B. J. Biol. Chem. 2001; 276: 4211-4217Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 16Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 17Yan R. Han P. Miao H. Greengard P. Xu H. J. Biol. Chem. 2001; 276: 36788-36796Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 18Walter J. Fluhrer R. Hartung B. Willem M. Kaether C. Capell A. Lammich S. Multhaup G. Haass C. J. Biol. Chem. 2001; 276: 14634-14641Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Initially, BACE1 is expected to cleave APP only at the β-site for releasing Aβ-(1-40/42). When BACE1 is overexpressed in cells, an alternative processing of APP at the β′-site (between Tyr10-Glu11 within the Aβ region) becomes apparent (5Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3255) Google Scholar). Cleavage of APP at the β′-site seems more robust in rodent species than in humans because more Aβ-(11-40/42) in rodents has been reported previously (19Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 20Buxbaum J.D. Thinakaran G. Koliatsos V. O'Callahan J. Slunt H.H. Price D.L. Sisodia S.S. J. Neurosci. 1998; 18: 9629-9637Crossref PubMed Google Scholar). Mouse deficiency in BACE1 not only dramatically reduces production of Aβ-(1-40/42) but also Aβ-(11-40/42) (21Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (942) Google Scholar), suggesting that processing of APP at the β′-site occurs naturally. Recent studies have demonstrated that processing of APP at the β′-site by BACE1 is independent of the initial cleavage of APP at the β-site (23Liu K. Doms R.W. Lee V.M. Biochemistry. 2002; 41: 3128-3136Crossref PubMed Scopus (92) Google Scholar, 24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar). In fact, the preferential cleavage at either site largely relies on the sequence feature (24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar). In the case of wild type human APP (APPWT), in vitro assays with synthetic peptide substrates indicate that the β-site is slightly superior to the β′-site (24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar). More Aβ-(1-40/42) is therefore detected by mass spectroscopy than Aβ-(11-40/42) in cells expressing APPWT, and this phenomenon is more evident in cells expressing Swedish mutant APP (APPSW) for its far better cleavable sequence at the β-site than β′-site. When the protein levels of BACE1 are greatly increased, processing of APP at the β′-site is enhanced, probably due to the sequential processing of both APP and CTF99 substrates by an increased amount of BACE1 (22Huse J.T. Liu K. Pijak D.S. Carlin D. Lee V.M. Doms R.W. J. Biol. Chem. 2002; 277: 16278-16284Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 23Liu K. Doms R.W. Lee V.M. Biochemistry. 2002; 41: 3128-3136Crossref PubMed Scopus (92) Google Scholar, 24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar).With the knowledge that BACE1 is capable of cleaving APP at two adjacent cleavable sites, we then ask whether the structural flexibility between the APP β- and β′-site would contribute to the selective cleavage of a site without an optimal sequence feature. The knowledge gained from this study may potentially be applicable to the discovery of small molecules that potentially block processing at the β-site. In this report, we found that the distal space of the cleavage sites from the membrane is one important factor for cleavage at the β-site by BACE1. However, the structural compatibility between BACE1 and its APP substrate is more important. Cleavage at the β-site by BACE1 is easily disrupted upon twisting the secondary structure near the β-site. These results have revealed important spatial requirements for BACE1 to process APP at the β-site. Hence, spatial interactions between BACE1 and APP should be an alternative consideration for disrupting production of amyloidogenic peptides.MATERIALS AND METHODSCell Lines—Human HEK-293 cells were maintained at 37 °C in a humidified, 5% CO2 controlled atmosphere in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 IU/ml penicillin, 50 μg/ml streptomycin and glutamine. HEK-293 cells were used to generate a stable cell line (HM) expressing BACE1 under the selection of hygromycin B (100 μg/ml).Transfection—Transfection was performed using the Lipofect-AMINE 2000 reagent (Invitrogen). A total of 20 μg of DNA were transfected into cells plated in 10-cm dishes using 80 μl of LipofectAMINE 2000 reagent. DNA and LipofectAMINE solutions were made in a total of 2 ml of Opti-MEMI media and added to each dish containing 8 ml of antibiotic-free Dulbecco's modified Eagle's medium.Western Blot Analysis—Cell lysates were prepared according to Yan et al. (6Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1328) Google Scholar). Equal amounts of cellular extracts were loaded onto a 4-12% Nupage gel or 16% Tricine gel (Invitrogen), and the proteins were separated by electrophoresis. The protein was then transferred to a nitrocellulose membrane according to standard procedures followed by Western analysis. The primary antibody C8 recognizes the C-terminal 15 amino acids of APP; 6E10 recognizes the sequence 1-16 within the Aβ region; and 22C11 recognizes the N terminus of APP. Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), immunoreactivity was detected by chemiluminescence using the SuperSignal West PICO reagent (Pierce).Site-directed Mutagenesis—Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, Palo Alto, CA) according to the standard procedure. APPWT was initially mutated to APPSW (K670N/M671L) or APPISYEV (V669I/K670S/M671Y/D672E/A673V). ISY was incorporated into the β′-site (S679I/G680S) to generate APPWT/ISY and APPISY/ISY. Two Pro substitutions (F675P/R676P) were incorporated into APPISY/ISY to generate APPISY/PP/ISY, and amino acids R676 and H677 were deleted in APPWTΔRH, APPISYΔRH, and APPISY/ΔRH/ISY. One and two Ala residues were inserted between R676 and H677 in APPISY/+A/ISY and APPISY/+2A/ISY, respectively. One and two additional Ala residues were inserted between A692 and E693 to generate APPISY/+2A/ISY/21A and APPISY/+2A/ISY/21A2 or APPISY/ISY/21A and APPISY/ISY/21A2, respectively. Four Ala residues were inserted between R676 and H677 in APPWT/+4AWT, APPISY/+4A/WT, and APPISY/+4A/ISY, and an uncleavable sequence (Y681K/E682K) was subsequently added to APPWT/+4A/KK and APPISY/+4A/KK. In subscript designations such as in APPWT/ISY, the WT refers to the wild type sequence at the β-site, and the ISY refers to the P3-P1 residues at the β′-site (see Fig. 1A). Amino acids 447-451 and 444-453 were deleted from BACE1 to generate BACE1Δ L5 and BACE1Δ L10, respectively. A repeat sequence (ESTLMTLDMEDCGYNESTLMTLGHGRLWLQRVNPHALDMEDCGYN) was inserted between N446 and E452 to generate BACE1Δ L5+L45 (see Fig. 5A). All constructs were validated by double strand DNA sequencing.Fig. 5Shifted cleavage to the β′-site in mouse APP variants. A, both APPWT and APPISY/ISY were mutated to mouse counterparts as denoted. B, HM cells were transfected with indicated constructs for 48 h, and lysates were examined by Western blot with antibody C8. Lysates from HEK-293 cells transfected with APPWT were loaded as a control lane for locating CTFα.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSAbolished Cleavage of the APP β-Site by BACE1 upon Local Conformational Change—Our previous studies with a mutant APP construct termed APPISY/ISY containing the optimal ISY-EV sequence at both β- and β′-sites have demonstrated that both β- and β′-sites are cleavable by BACE1 (24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar). This has also been confirmed with a pulse-chase experiment (data not shown). Two possible scenarios may account for the simultaneous cleavage of two adjacent APP sites by BACE1: 1) the APP structure overlapping the β- and β′-site is sufficiently flexible like a random coil so that BACE1-mediated hydrolysis at either site can be accommodated, and 2) the secondary structure spanning this region is relatively rigid, either in a β-turn or α-helix, but allows both the β- and β′-site to be positioned in proximity to the BACE1 catalytic cleft. Because the structure of APP has not been resolved, we intend to differentiate between these possibilities by a mutagenesis approach to distort conformation near the BACE1 cleavage sites. We first used APPISY/ISY as a template and generated additional APP variants by either insertion or deletion of residues between the β- and β′-site as outlined in Fig. 1A. We reasoned that either addition or deletion of residues between these two sites should cause a shift of the conformation in this region. As shown in Fig. 1B, the patterns of BACE1-cleaved C-terminal fragments were variable in cells expressing different APP constructs. Insertion of either one or two Ala residues did not noticeably affect the cleavage of APP at these two sites, whereas insertion of four Ala residues almost completely shifted the cleavage to the β′-site. Interestingly, when two residues (Arg-His) in the center of this region were deleted, the cleavage at the β′-site was also predominant (Fig. 2B). Thus, to either shorten or extend the length between β- and β′-site would favor BACE1 to cleave APP at the β′-site.Fig. 2Balancing of an optimal BACE1 β-site with a structural twist. A, both APPWT and APPISY were mutated by either insertion of Ala or deletion of Arg-His residues between the β- and β′-site. B, HM cells were transfected with the indicated constructs for 48 h, and the lysates were analyzed by Western blot with antibody C8.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A Pro residue is known to typically break a α-helical or β-sheeted structure. Theoretically, the substitution (F4P/R5P) in the Aβ region would cause conformational changes in this region. To test further which cleavable subsite will be more sensitive to a structural bending, we generated such a construct named APPISY/PP/ISY (Fig. 1A). Transfection of this construct in HM cells showed that CTFβ′ was again a preferred product that migrated at the expected size on a gel (Fig. 1B). To our surprise, we also observed a fragment that migrated obviously slower than the expected CTFβ (denoted with an arrowhead in Fig. 1, B and C). We referred to this fragment as CTFx because it has an undefined N-terminal end. Sequence inspection of a BACE1 cleavable site suggests that an upstream EIS-EV is a potential processing site for generating this CTFx (24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar and 25Turner III, R.T. Koelsch G. Hong L. Castenheira P. Ghosh A. Tang J. Biochemistry. 2001; 40: 10001-10006Crossref PubMed Scopus (195) Google Scholar). This suggested that a BACE1 inaccessible cleavage site in a normal circumstance could very well be aligned into the BACE1 active cleft upon a structural twist.To further understand these cleavages in subcellular compartments, we treated cells with either brefeldin A (BFA) or monensin. BFA arrests secretory proteins in the endoplasmic reticulum by blocking the exit of secretory vesicles from the endoplasmic reticulum, whereas monensin locks transport of secretory vesicles in the Golgi. As demonstrated previously (23Liu K. Doms R.W. Lee V.M. Biochemistry. 2002; 41: 3128-3136Crossref PubMed Scopus (92) Google Scholar, 24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar), processing of APP at the β-site is presumably favored in cells treated with BFA, whereas monensin treatments favor production of CTFβ′. We found that the patterns of aforementioned APP mutants processed by BACE1 in monensin-treated cells were generally similar to the patterns in the untreated transfected cells (data not shown). However, BFA treatment indeed accumulated more CTFβ than the untreated condition (comparing Fig. 1, B with C) consistent with that seen previously (23Liu K. Doms R.W. Lee V.M. Biochemistry. 2002; 41: 3128-3136Crossref PubMed Scopus (92) Google Scholar, 24Tomasselli A.G. Qahwash I. Emmons T.L. Lu Y. Leone J.W. Lull J.M. Fok K.F. Bannow C.A. Smith C.W. Bienkowski M.J. Heinrikson R.L. Yan R. J. Neurochem. 2002; 84: 1006-1017Crossref Scopus (44) Google Scholar). Interestingly, although production of CTFβ was detectable in BFA-treated cells expressing APPISY/PP/ISY, the anomalous fragment CTFx was still more robustly generated. Clearly, the cleavage of the β-site in APPISY/PP/ISY was suppressed whereas a totally new product was produced with a local conformational shift. Thus, conformational changes in the Aβ N-terminal region mainly disrupt the cleavage of APP at the β-site.Given the fact that the template used for the above study contains ISYEV at both β- and β′-sites, it is unclear whether similar insertions or deletions in either APPWT or APPISY would replicate the same selective cleavage. To resolve this, we generated more mutant APP as illustrated in Fig. 2A. Because the β-site in APPISY is much more superior to the wild type β′-site, we routinely observed CTFβ as a predominant cleavage product in APPISY-expressing cells (see lane APPISY in Fig. 2B). Remarkably, a significant shift of the processing to the β′-site still occurred in both APPISY insertion and deletion variants regardless of the fact that the β-site possesses a much more optimal cleavable sequence than the β′-site in APPISY/+4A/WT (Fig. 2B). The same shift was also confirmed to occur in variants with wild type cleavage sites (Fig. 2, A and B). Thus, a structurally perturbed superior β-site cannot compete against a well positioned suboptimal β′-site.Taken together, these data suggested that BACE1 cleavage at the β-site is more easily disrupted than the β′-site if there is a structural change in this region. Natural processing of APP at the β′-site is clearly caused by its favorable orientation together with the presence of the cleavable sequence.The Extended or Shortened Linker Region Does Not Disrupt Cleavage at the β-Site—Given the fact that a significant structural twist between the β- and β′-site (as caused in APPISY/PP/ISY) would suppress the cleavage at the β-site by BACE1, it was still unclear whether the similarly suppressed cleavage at the β-site in APP 4A-insertion constructs (APPISY/+4A/ISY, APPWT/+4A/WT, or APPISY/+4A/WT) was primarily caused by the structural or the distal changes of the β-site from the membrane. To differentiate between the changes, we inserted Ala residues between Ala21 and Glu22 in the APPISY/+2A/ISY template because it retains cleavage at both sites by BACE1. As a consequence, the distance of the β-site to the membrane in APPISY/+2A/ISY21A2 would mimic that in APPISY/+4A/ISY (Fig. 3A). Examinations of these new APP variants actually revealed no changes at both the β- and β′-site in HM cells transfected with APPISY/+2A/ISY21A or APPISY/+2A/ISY21A2 (Fig. 3B). Moreover, examination of another construct named APPISY/+2A/ISY21A4 that placed the β-site farther from the membrane by inserting four Ala residues at position 21 also did not indicate a noticeable shift (data not shown).Fig. 3Insertion of residues between the transmembrane domain and BACE1 cleavage sites. A, APPISY/+2A/ISY was used as a template, and additional mutants were generated by the insertion of Ala residue between Ala21 and Glu22. B and C, HM cells were transfected with the indicated constructs for 48 h followed by Western blot analysis with antibody C8.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also examined APP mutants with shortened linker regions spanning the BACE1 cleavage sites and the residues that anchor APP in the membrane. When four residues (Phe19-Phe20-Ala21-Glu22) were deleted in APPISY/ISY, patterns of cleaved products were not changed as shown by cells expressing APPISY/ISY/ΔFFAE (Fig. 3C). Therefore, insertion or deletion of a few residues outside the β- and β′-sites does not disrupt APP processing patterns unlike the mutants shown in Figs. 1 and 2. These results suggest that the distance of the β-site from the membrane is not a determining factor for BACE1 to cleave at the β-site, whereas the proper conformation in this region is more important.The Unfavorable β-Site Is Cleavable by BACE1 if the β′-Site Is Blocked—Having seen the inhibited cleavage at the β-site by BACE1 in APPISY/+4A/WT and similar mutants, it was intriguing to see whether this inhibition is attributable to an absolute inaccessibility of the mutant APP β-site to the BACE1 cleft after a structural twist. Therefore we examined another mutated APP protein named APPWT/+4A/KK with a Y10K/E11K substitution in APPWT/+4A/WT. Prior analysis of cleavable sequences suggests that the β′-site is blocked by such a substitution (data not shown) or a similar mutation (23Liu K. Doms R.W. Lee V.M. Biochemistry. 2002; 41: 3128-3136Crossref PubMed Scopus (92) Google Scholar), whereas cleavage at the wild type β-site is retained. Utilizing APPWT/+4A/KK would allow us to determine whether a structurally twisted β-site is actually accessible to the BACE1 active site. Examination of cells expressing APPWT/+4A/KK showed clear cleavage at the β-site, whereas no CTFβ′ was detectable (Fig. 4A). Moreover, the blocked β′-site was not processed by BACE1 even with monensin treatment, a condition known to favor the production of CTFβ′ (Fig. 4B). Thus, if a well positioned β′-site is not cleavable, BACE1 would continue to search for the structurally twisted β-site for processing. Again, these data support the idea that the cleavage of APP by BACE1 possesses elasticity despite the dockings of both BACE1 and APP in the membrane.Fig. 4A non-cleavable β′-site in structurally twisted APP variants. HM cells were transfected with indicated constructs for 48 h either in the absence (A) or presence (B)of 10 μm monensin. Cell lysates were then analyzed by Western blot with antibody C8.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mouse APP Is Better Aligned for Processing at the β′-Site by BACE1—In rodent species, cleavage of APP at the β′-site seems more robust as more Aβ-(11-40/42) is detected (19Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 20Buxbaum J.D. Thinakaran G. Koliatsos V. O'Callahan J. Slunt H.H. Price D.L. Sisodia S.S. J. Neurosci. 1998; 18: 9629-9637Crossref PubMed Google Scholar) and cleavage at the β′-site is likely species-specific (21Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C Nat. Neurosci. 2001; 4: 233-234Crossre" @default.
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• W1987016766 title "Processing Amyloid Precursor Protein at the β-Site Requires Proper Orientation to Be Accessed by BACE1" @default.
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• W1987016766 cites W1968703962 @default.
• W1987016766 cites W1971176785 @default.
• W1987016766 cites W1987452659 @default.
• W1987016766 cites W1987686583 @default.
• W1987016766 cites W1996736769 @default.
• W1987016766 cites W1997632705 @default.
• W1987016766 cites W2000706612 @default.
• W1987016766 cites W2003283600 @default.
• W1987016766 cites W2009285665 @default.
• W1987016766 cites W2026053103 @default.
• W1987016766 cites W2028627603 @default.
• W1987016766 cites W2029214965 @default.
• W1987016766 cites W2040252771 @default.
• W1987016766 cites W2045445527 @default.
• W1987016766 cites W2062283435 @default.
• W1987016766 cites W2079651671 @default.
• W1987016766 cites W2082269672 @default.
• W1987016766 cites W2087524018 @default.
• W1987016766 cites W2087932565 @default.
• W1987016766 cites W2090705035 @default.
• W1987016766 cites W2091959459 @default.
• W1987016766 cites W2096410114 @default.
• W1987016766 cites W2098510314 @default.
• W1987016766 cites W2108649886 @default.
• W1987016766 cites W2113690827 @default.
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• W1987016766 cites W2124135429 @default.
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