The Pro Domain of β-Secretase Does Not Confer Strict Zymogen-like Properties but Does Assist Proper Folding of the Protease Domain

Abstract

β-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid β peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4′ Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. Thekcat/Km of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4′ Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain. β-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid β peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4′ Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. Thekcat/Km of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4′ Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain. amyloid precursor protein conditioned medium β-site APP cleaving enzyme 492, acetyl-TTRPGSGLTNIK(6-(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVNLDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid high pressure liquid chromatography polyacrylamide gel electrophoresis L-405, 525, acetyl TTRPGSGLTNIK(6(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVKMDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide The amyloid precursor protein (APP)1 is cleaved sequentially by two proteolytic activities, β- and γ-secretase, to generate the N and C termini, respectively, of the amyloid β peptides. Aberrant production or compromised clearance of the amyloid β peptides, which give rise to neuritic plaques in brain parenchyma, may be a root cause of Alzheimer's disease (1Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1520) Google Scholar). β-Secretase (BACE; Asp2; memapsin 2) was recently cloned (2Vassar 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 (3271) Google Scholar, 3Sinha 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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 (1329) Google Scholar, 5Hussain 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. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 6Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (738) Google Scholar) and shown to be a novel type I transmembrane aspartyl protease. The cDNA encoding BACE predicts a precursor protein with a modular structure containing Pre, Pro, protease, transmembrane, and cytosolic tail domains (see Fig.1). Pro domains are commonly found in protease precursors where they have been shown to virtually abolish catalytic activity (7Khan A.R. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (378) Google Scholar) and assist in protein folding (8Baker D. Shiau A.K. Agard D.A. Curr. Opin. Cell Biol. 1993; 5: 966-970Crossref PubMed Scopus (151) Google Scholar). The Pro domain is typically cleaved from the protease precursor to generate the mature active protease. Edman degradation of purified human brain BACE revealed a protein that the protease domain 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar) is mature BACE from human cells that with the BACE cDNA an that the protease only the Pro 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar). of which was expressed in revealed proteolytic the between the Pro and protease domains R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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 (1329) Google Scholar). These results that of BACE to the Pro and generate mature BACE is in the expressed in insect cells a soluble of the BACE precursor that is truncated the between the protease and transmembrane regions A that an that to was purified from the conditioned medium The show that the Pro domain of than catalytic activity as in the of to facilitate the proper folding of the BACE protease domain. The BACE cDNA was cloned by of human brain using that from the BACE cDNA (2Vassar 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 (3271) Google Scholar, 3Sinha 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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 (1329) Google Scholar, 5Hussain 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. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (997) Google Scholar, 6Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (738) Google Scholar). the of the and the the in the from the The was to the BACE The of the BACE cDNA was by The cDNA encoding truncated was from using a containing a of the and a containing a the by an The was the of a to generate The of the BACE cDNA was by was insect cells with to generate to the was and as in cells in insect to and infected with BACE460 a of of and the was by and A peptide to the Pro of BACE was to and as for production of peptide that is the protease domain of BACE was similarly as an for production of The was purified by the an containing the immobilized from cells infected by BACE460 purified or purified BACE460 with acetyl-TTRPGSGLTNIK(6-(7-amino-4-methylcoumarin-3-acetyl)aminohexanoyl)TEEISEVNLDAEFRHDSGK(6-(biotinamido)hexanoyl)-amide a polypeptide that is from the acid of APP in the of the that the are the in the and respectively, that are in the Alzheimer's disease M. F. K. H. L. L. PubMed Scopus Google Scholar). that was to purified BACE460 and ProBACE460 is to that has the and respectively, which are the that are in the type APP in the presence of acid BACE in the presence of the BACE 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google and for and by the for by using a and substrate by with and of and The from BACE460 cells was with and and to a concanavalin of The was with A and and with A and was to the from the with a was to the and and The was to an containing immobilized P10-P4′ Stat(Val) 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar). The was sequentially with C and and The P10-P4′ Stat(Val) was with and and for BACE activity with activity and as ProBACE460 or BACE460 with in and and activity was with The production of BACE460 by of ProBACE460 was by using the Pro or protease regions of from the by using a the of BACE activity was with the of of ProBACE460 was with in for The was BACE460 was purified by chromatography with immobilized P10-P4′ Stat(Val) as that was from the from the BACE460 cells was with or and immobilized protein of a with the and the by with or and assayed for BACE activity using of the purified ProBACE460 and BACE460 was similarly that and The with and assayed for BACE activity using ProBACE460 and BACE460 for with guanidine in in the or presence of BACE Pro The by of to a of guanidine and for ProBACE460 and BACE460 to BACE activity to in an of and assayed for BACE activity using of the or ProBACE460 and BACE460 by using the of ProBACE460 with and by guanidine denaturation/renaturation a using an chromatography The gel was with and guanidine The acid of with a of the using containing in for of purified protein was by Edman degradation using an protein of by liquid was with an liquid to a A truncated BACE precursor that the Pre, Pro, and protease domains (PreProBACE460) was produced in cells using a This was to the of a soluble of BACE from the of from cells that infected with BACE460 revealed the presence of a protein that with the Pro domain 2) or protease domain not was with from cells infected with when similarly using the Pro domain or protease domain not of from BACE460 cells with the the Pro of in the presence of immobilized protein the that with the and the a less that with the and in the of ProBACE460 is from the of These that but not of the protein in the the Pro BACE activity in the was using the polypeptide derivative from the APP Alzheimer's disease in the of the The from cells infected with BACE460 the of A of on the of was BACE activity was not with from cells that infected with that of was the not The of the protease and not BACE activity in the from BACE460 the BACE activity in the was by the BACE inhibitor, P10-P4′ Stat(Val) from cells infected with the BACE460 that was of the Pro of BACE an of BACE activity when with This the that a Pro of BACE activity. The of BACE activity in by the with was when the Pro was The presence of BACE activity and not of ProBACE460 in the that a BACE in the BACE activity. The from BACE460 cells was with and by chromatography using immobilized concanavalin A. The BACE activity was and with and The containing the BACE activity and to chromatography using immobilized P10-P4′ Stat(Val) 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar). The BACE activity was and The recovery of BACE activity was The BACE that was from the of BACE460 cells a when to and by using the the Pro or protease regions of BACE with and The purified is a as by by immobilized concanavalin A and during with not Edman degradation of purified which was from revealed an N terminus that the Pro of BACE to ProBACE460 in cells by baculovirus-mediated using in an of ProBACE460 and mature BACE460 by concanavalin A and ProBACE460 was purified to from by chromatography with that is to protein the Protein The ProBACE460 was from the using The ProBACE460 that was purified in was as by activity that was similar to which was purified and as in the with ProBACE460 with of mature BACE460 as that a or of ProBACE460 with BACE460 be The of the ProBACE460 was by the of BACE using the or the ProBACE460 was using the Pro The protease that to mature BACE was recently shown to be B.D. Denis P. M. Teplow D.B. Kahn S. Louis J.-C. Citron R. J. Biol. 2000; PubMed Scopus Google Scholar). The for G. J. Biol. PubMed Google is the C terminus of the Pro of purified ProBACE460 with and to for The of ProBACE460 with the is and as the with is the the with the was that cleaves ProBACE460 the between the Pro and protease domains to BACE460 A of BACE460 was for by ProBACE460 with and the on the P10-P4′ Stat(Val) The protein from the was shown to be BACE460 from to with by and not in to the ProBACE460 of BACE460 with immobilized to the as by using the and The purified ProBACE460 activity the proteolytic activity of ProBACE460 of the purified ProBACE460 with immobilized in the of of the BACE activity not of the containing ProBACE460 to the immobilized revealed BACE activity not The results of that ProBACE460 and not a is for the activity in the ProBACE460 of ProBACE460 to BACE460 by is by a in BACE activity which the in that was by was an in BACE activity a with an of ProBACE460 with for and BACE activity by and of BACE460 with not BACE activity of with not cause the of not of by purified ProBACE460 and BACE460 a on substrate to substrate The of is in with the results from a BACE that a polypeptide substrate of similar 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar). The of ProBACE460 and BACE460 for are and ProBACE460 to of the activity of BACE460 for that is cleaved the by purified ProBACE460 and BACE460 not ProBACE460 and BACE460 a substrate that is to that the and respectively, which are the that are found in the type APP was for of enzyme to of that of the not Thekcat/Km of ProBACE460 for is of the by the of ProBACE460 and BACE460 with are not as was with the of ProBACE460 and BACE460 for are and respectively, of the for This is with that BACE a for the the and of the substrate (2Vassar 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 (3271) Google Scholar, 3Sinha 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler 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 (1329) Google Scholar). ProBACE460 and BACE460 similar to by P10-P4′ Stat(Val) The of P10-P4′ Stat(Val) for ProBACE460 and BACE460 are and The of P10-P4′ to between ProBACE460 and BACE460 is with the virtually of BACE during chromatography with immobilized not ProBACE460 and BACE460 are to by The of ProBACE460 and BACE460 in the presence of and respectively, of the that the activity of ProBACE460 from to BACE460 during with substrate was on ProBACE460 with substrate which of the substrate was to The acid the N terminus of Pro of ProBACE460 to BACE460 of the presence of substrate or to does not appear to using the to show of ProBACE460 with substrate not the of the Pro domain in protein ProBACE460 and BACE460 with for and by a renaturation of the to BACE containing the The guanidine during the BACE has effect on the activity of ProBACE460 or BACE460. BACE activity is when ProBACE460 or BACE460 is to the an renaturation of ProBACE460 to the to recovery of BACE activity. of the ProBACE460 activity is a denaturation/renaturation the recovery of activity from the BACE460 is only of the ProBACE460 and BACE460 revealed similar of when with the the recovery of BACE activity of BACE460 with guanidine and renaturation is not to to protein The protein in the ProBACE460 that BACE activity during gel the as ProBACE460 not The active ProBACE460 only recovery of protein on and with the of the activity of the ProBACE460 that during gel as a is from that of ProBACE460 not The to of the BACE activity denaturation/renaturation of ProBACE460 the of ProBACE460 The presence of the BACE Pro peptide during the denaturation/renaturation of BACE460 to the recovery of BACE activity to but the Pro peptide has effect on the recovery of activity from ProBACE460 with the effect of the Pro domain on protein folding and not of the BACE Pro peptide has only a effect on the activity of BACE460 not Pro for aspartyl as and human are and that the activity of M. M. J. M. PubMed Scopus Google Scholar). of the acid of BACE with of the aspartyl protease revealed the of a polypeptide that is between the and protease domains (2Vassar 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 (3271) Google Scholar, 3Sinha 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar, 4Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashler J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. 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Nature. 1999; 402: 533-537Crossref PubMed Scopus (1329) Google Scholar). A of the Pro in aspartyl is to on the precursor (7Khan A.R. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (378) Google Scholar). show that the Pro of has a inhibitory effect on BACE but does appear to a in the proper folding of the protease domain. The activity of ProBACE460 was not from that of BACE460 when assayed with polypeptide to the type and APP and of the BACE460 The of the ProBACE460 that was by and to ProBACE460 as for the BACE activity. with than of the BACE activity was from the ProBACE460 by using the the from the BACE catalytic activity. of ProBACE460 to BACE460 during with substrate was by and using the The of 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. Zhao J. McConlogue L. 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These results are with the of ProBACE460 to BACE460 by the The finding that ProBACE460 activity on the as to where is to in the to the of activity the of BACE (2Vassar 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 (3271) Google Scholar, 3Sinha 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. Zhao J. McConlogue L. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar) or is to the in the B.D. D.G. J. Biol. Scholar) to A. H. M. H. C. J. S. G. C. J. Biol. 2000; PubMed Scopus Google the in the J. S. Biol. 2000; 7: PubMed Scopus Google Scholar) does not appear to be to that is in the but is with in the J.P. J. G. F. P. H. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google which is with the of BACE activity. that BACE is and to J. Biol. 2000; PubMed Scopus Google which is an A. H. M. H. C. J. S. G. C. J. Biol. 2000; PubMed Scopus Google Scholar) that be for J. S. Biol. 2000; 7: PubMed Scopus Google Scholar). is to that of in the of with that BACE activity A. H. M. H. C. J. S. G. C. J. Biol. 2000; PubMed Scopus Google the activity by may not be the are to the of activity. the Pro in does not strict on does appear to a in the proper folding of the protease domain. The of the BACE Pro peptide to assist folding of mature protease domain in is of results with protease Agard D.A. Nature. PubMed Scopus Google Scholar) and J. M. A.R. 1993; PubMed Scopus Google Scholar). that the Pro peptide with BACE460 during and may as a that of The of the Pro peptide to BACE460 activity to the activity of to BACE460 to a between the Pro domain and the active in The that the BACE Pro folding but is not a of the protease is but not A that the inhibitory and of the Pro peptide are not X. M. U. J. Biol. 2000; PubMed Scopus Google Scholar). be that are to the that is a strict zymogen with to to membrane-bound the of a that a soluble of the transmembrane derivative of 2) expressed in catalytic activity J. Koelsch G. Tang J. 2000; PubMed Scopus Google Scholar). was not cleaved by a finding that from that of and and B.D. Denis P. M. Teplow D.B. Kahn S. Louis J.-C. Citron R. J. Biol. 2000; PubMed Scopus Google Scholar). The of to may in the structure of BACE a acid of the of the N terminus for in cells (2Vassar 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 (3271) Google Scholar). the of protein on the E. by These two BACE of memapsin and which a and respectively, as with BACE J. Koelsch G. Tang J. 2000; PubMed Scopus Google Scholar). The on activity of from the structure of the mature BACE to be novel that the catalytic activity of a protease that a in the of Alzheimer's The that ProBACE460 activity to BACE460 aspartyl protease from of protease from a the of the Pro in of BACE activity the may be of of activity. the Pro domain in is not to to the proper folding of the protease domain. the activity by ProBACE460 that the of to BACE have little or on the production of the amyloid β peptide.