Xiao‐Ping Shi

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

We describe the development of cell-permeable beta-secretase inhibitors that demonstratively inhibit the production of the secreted amino terminal fragment of an artificial amyloid precursor protein in cell culture. In addition to potent inhibition in a cell-based assay (IC50 < 100 nM), these inhibitors display impressive selectivity against other biologically relevant aspartyl proteases.

Presenilin-1 (PS1) and presenilin 2 (PS2) are proposed to be transmembrane aspartyl proteases that cleave amyloid precursor protein and Notch. PS1- and PS2-mediated activities were individually characterized using blastocyst-derived (BD) cells and membranes from PS1+/–-PS2–/– and PS1–/–PS2+/+ mice, respectively. The relative amounts of PS1 and PS2 in the various BD cells were determined from the intensities of the anti-PS1 and anti-PS2 immunoblot signals by comparison with standard curves using radiolabeled PS1 and PS2 standards produced by in vitro transcription and translation. Cellular membranes from wild type, PS1–/–PS2+/+, and PS1+/–-PS2–/– but not PS1–/–PS2–/– BD cells generated the Aβ40 and Aβ42 products from the C100FLAG substrate. PS1-associated γ-secretase displays considerably higher specific activity than PS2-associated γ-secretase. Moreover, the PS1+/–PS2–/– BD cells and corresponding membranes exhibited much higher γ-secretase activity as compared with other BD cells and membranes. The PS1-mediated γ-secretase activity correlated better with the amount of PS1 that is modifiable by a photoactivated active site-directed γ-secretase inhibitor rather than total PS1; hence, only a small portion (<14%) of the PS1 in wild-type membranes appears to be engaged in an active γ-secretase complex. This finding suggests that PS1 may serve other biological functions in addition to that associated with its γ-secretase activity. Furthermore, the PS1 γ-secretase complex and the PS2 γ-secretase complex activities can be discriminated on the basis of their susceptibility to inhibition by a potent γ-secretase inhibitor. The distinct yet overlapping enzymatic properties of the PS1 γ-secretase complex and the PS2 γ-secretase complex imply that these two putative aspartyl class proteases may contribute to different biological processes. Presenilin-1 (PS1) and presenilin 2 (PS2) are proposed to be transmembrane aspartyl proteases that cleave amyloid precursor protein and Notch. PS1- and PS2-mediated activities were individually characterized using blastocyst-derived (BD) cells and membranes from PS1+/–-PS2–/– and PS1–/–PS2+/+ mice, respectively. The relative amounts of PS1 and PS2 in the various BD cells were determined from the intensities of the anti-PS1 and anti-PS2 immunoblot signals by comparison with standard curves using radiolabeled PS1 and PS2 standards produced by in vitro transcription and translation. Cellular membranes from wild type, PS1–/–PS2+/+, and PS1+/–-PS2–/– but not PS1–/–PS2–/– BD cells generated the Aβ40 and Aβ42 products from the C100FLAG substrate. PS1-associated γ-secretase displays considerably higher specific activity than PS2-associated γ-secretase. Moreover, the PS1+/–PS2–/– BD cells and corresponding membranes exhibited much higher γ-secretase activity as compared with other BD cells and membranes. The PS1-mediated γ-secretase activity correlated better with the amount of PS1 that is modifiable by a photoactivated active site-directed γ-secretase inhibitor rather than total PS1; hence, only a small portion (<14%) of the PS1 in wild-type membranes appears to be engaged in an active γ-secretase complex. This finding suggests that PS1 may serve other biological functions in addition to that associated with its γ-secretase activity. Furthermore, the PS1 γ-secretase complex and the PS2 γ-secretase complex activities can be discriminated on the basis of their susceptibility to inhibition by a potent γ-secretase inhibitor. The distinct yet overlapping enzymatic properties of the PS1 γ-secretase complex and the PS2 γ-secretase complex imply that these two putative aspartyl class proteases may contribute to different biological processes. Presenilin-1 (PS1) 1The abbreviations used are: PS1, presenilin-1; PS2, presenilin-2; BD, blastocyst-derived; AD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, β-amyloid; TNT, transcription/translation; PVDF, polyvinylidene difluoride; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; NTF, N-terminal fragment; CTF, C-terminal fragment. and presenilin-2 (PS2) are homologous (67% identity) polytopic membrane-spanning proteins that are endoproteolytically processed to form heterodimers. PS1 and PS2 were identified by their association with early onset Alzheimer's disease (AD) in humans. Autosomal dominant inheritance of mutations in the PS1 gene is the most common cause of familial early onset Alzheimer's disease (1Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. et al.Nature. 1995; 375: 754-760Crossref PubMed Scopus (3585) Google Scholar). Familial early onset Alzheimer's disease-associated mutations are also present in PS2, although they are far less prevalent than in PS1 (2Levy Lahad E. Wasco W. Poorkaj P. Romano D.M. Oshima J. Pettingell W.H. Yu C.E. Jondro P.D. Schmidt S.D. Wang K. et al.Science. 1995; 269: 973-977Crossref PubMed Scopus (2230) Google Scholar). In addition to their purported role in the regulation of amyloid precursor protein (APP) processing, the presenilins have been implicated in Notch signaling, Wnt/β-catenin signaling, apoptosis, capacitative calcium entry (3De Strooper B. Annaert W. Nat. Cell Biol. 2001; 3: E221-E225Crossref PubMed Scopus (48) Google Scholar), and ErbB-4-mediated signaling (4Ni C.Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (756) Google Scholar). Moreover, PS1 and PS2 are closely related to the Caenorhabditis elegans protein, sel-12, which is involved in cell fate decisions (5Levitan D. Doyle T.G. Brousseau D. Lee M.K. Thinakaran G. Slunt H.H. Sisodia S.S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (343) Google Scholar). APP is processed by two proteases, β-secretase and γ-secretase, that generate the N and C termini of the Aβ-peptides, respectively (6Li Y.-M. Mol. Intervent. 2001; 1: 198-207PubMed Google Scholar). The Aβ-peptides are generally 40 or 42 amino acids in length (Aβ40 and Aβ42, respectively). Aβ42 is more prone to aggregation and is believed to be a pivotal player in the etiology of AD. There is considerable evidence showing that the presenilins are closely associated with γ-secretase-mediated cleavage. Firstly, familial early onset Alzheimer's disease mutations in PS1 and PS2 cause increased production of Aβ42 in transfected cells or transgenic mice. Secondly, isolated cultured neurons derived from PS1-deficient mice accumulate APP fragments that fail to be processed by γ-secretase (7De 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 (1552) Google Scholar, 8Naruse 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 (333) Google Scholar). Thirdly, γ-secretase activity is abolished in cultured cells derived from mice that are deficient in both PS1 and PS2 (9Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 10Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). Fourthly, mutagenesis of two conserved aspartates in the transmembrane regions of PS1 significantly reduces γ-secretase activity (11Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar). Thus, it was proposed that PS1 is either a unique diaspartyl cofactor for γ-secretase or is itself γ-secretase, an intramembranous aspartyl protease. Finally, biochemical evidence that PS1 and PS2 contain the active sites of γ-secretase was obtained from studies in which presenilins were covalently modified by active site-directed γ-secretase inhibitors (12Li 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. M.K. M.S. Nature. 2000; PubMed Scopus Google Scholar, Kimberly W.T. Ostaszewski B.L. Diehl T.S. T. Xia W. Selkoe D.J. M.S. Nat. Cell Biol. 2000; 2: PubMed Scopus Google both PS1 PS2 are for the γ-secretase-mediated the different of the PS1-deficient and mice that these proteins The PS1-deficient mice and J. Xia W. Selkoe D.J. S. Full Text Full Text PDF PubMed Scopus Google Scholar, P.C. H. H. D.J. Price D.L. Van Sisodia S.S. Nature. PubMed Scopus Google Scholar), the mice are and and only and with A. D. Annaert W. Saftig P. Craessaerts K. Serneels L. L. F. Vanderstichele H. R. P. D. A. Van Leuven F. De Strooper B. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). PS1 and PS2 are present in a of the of significantly and and M.K. Slunt H.H. Thinakaran G. G. M. E. Price D.L. Sisodia S.S. J. 1996; PubMed Google Scholar). The of the putative in PS1 and PS2 to with different overlapping with PS1 and PS2 to in protein G. F. Levesque G. M. D.M. Levesque L. Rogaeva E. Xu D. Liang Y. M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). PS1 and PS2 is from the of either protein to the of C. elegans (5Levitan D. Doyle T.G. Brousseau D. Lee M.K. Thinakaran G. Slunt H.H. Sisodia S.S. Greenwald I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14940-14944Crossref PubMed Scopus (343) Google Scholar). the of the PS1 and PS2-mediated γ-secretase activities have yet to be In to the γ-secretase activities by PS1 and (BD) cells from PS1–/–PS2+/+, and PS1–/–PS2–/– mice Ikeda M. H. Bernstein A. 1999; PubMed Scopus Google were used to membranes that were with an in vitro γ-secretase Y.M. Lai M.T. Xu M. Huang Q. DiMuzio Mower J. M.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). In these BD cell were transfected with an the C-terminal of APP and the of were the γ-secretase activities associated with the PS1+/–PS2–/– BD cells and PS1–/–PS2+/+ BD cells are by the PS1 γ-secretase complex and the PS2 γ-secretase respectively. This is by the of γ-secretase activity associated with the PS1 and PS2 deficient BD In and of the of and PS1 and PS2 were the and 2 of the PS1 and PS2 was for and on The was to 40 of 2 of and of membranes The were for and in a for The was with of and in of The products were by and to membranes. The was on the for and with a The amounts of the presenilins from the signals were for the of in the PS1 and PS2 the was the was and by using PS1 and signals were with the for the the amounts of PS1 and PS2 with the intensities of the corresponding was used to the relative of the activities of the PS1 and PS2 immunoblot and Cell cells from PS1–/–PS2+/+, and PS1–/–PS2–/– were obtained as Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). were cultured in modified and BD cell membranes were as Y.M. Lai M.T. Xu M. Huang Q. DiMuzio Mower J. M.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google with the that the cells were with a was determined using a protein with as a protein of the BD Cell membranes PS1 and PS2 were by and to membranes. The membranes were for the of PS1 and PS2 with and respectively. signals were using the for the as for in vitro γ-secretase and of proteins were as (12Li 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. M.K. M.S. Nature. 2000; PubMed Scopus Google Scholar, Y.M. Lai M.T. Xu M. Huang Q. DiMuzio Mower J. M.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). PS2 in the Y.M. Lai M.T. Xu M. Huang Q. DiMuzio Mower J. M.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google that anti-PS1 γ-secretase activity from cell membranes. of the PS1 that PS2 is not present two of with anti-PS1 is PS1 in the the PS2 is not as compared with the that PS2 is not engaged in the complex that displays γ-secretase activity. of and with more PS1- and PS2-mediated γ-secretase to the relative amounts of PS1 and PS2 in the different BD cell PS1 and PS2 standards were produced by in vitro of the corresponding presenilin in the of The radiolabeled products were by and to membranes. the of membranes amounts of the radiolabeled products generated with the PS1 and PS2 PS1 and PS2 contain and respectively. of signals for the different of in PS1 and is of amounts of The the products were also with the or The for the immunoblot was much than the for the was hence, the immunoblot is not by the The intensities of the and immunoblot signals as a of the amounts of PS1 and PS2, are in that the immunoblot for of is more than the corresponding immunoblot for of of PS1 and PS2 in the BD in the various BD cells was characterized by using and PS1 and PS2 were in membranes and respectively). The relative amounts of PS1 and PS2 in the membranes were by with the corresponding PS1 or PS2 standard curves the amount of PS2 in cell membranes appears to be of the amount of PS1 but not PS2 was in PS1+/–PS2–/– membranes 2 and respectively). PS2 but not PS1 was in PS1–/–PS2+/+ cell membranes and respectively). from PS1–/–PS2–/– BD cells not protein that with either the or and respectively). The PS1 in PS1+/–PS2–/– cell membranes was only of that with wild-type cell membranes the higher protein in 2 compared with The of PS1 in the PS1+/–PS2–/– cell membranes as compared with the cell membranes was by with the of PS2 in and cell membranes and a of PS2 in the PS1-deficient In by BD Cell γ-secretase activities by the various BD cell membranes were with the in vitro γ-secretase using the Y.M. Lai M.T. Xu M. Huang Q. DiMuzio Mower J. M.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). PS1–/–PS2–/– cell membranes fail to the of the or products not that activity by BD cell membranes using the in vitro γ-secretase is to PS1 or the amount of PS1 present in the PS1+/–PS2–/– cell membranes of the in cell the amount of total generated by these membranes was of that produced by cell membranes membranes from the PS1–/–PS2+/+ BD which serve as a of PS2 γ-secretase the of the Aβ-peptides from total generated by PS1–/–PS2+/+ cell membranes is only of that produced by cell vitro and γ-secretase activity of BD cell of cell membranes cell were transfected with of The cell membranes The cell were transfected with of in a of the in vitro γ-secretase activities of the PS1+/–PS2–/– and PS1–/–PS2+/+ membranes with the of the relative of PS1 and PS2 in these for the enzymatic activities of the PS1 γ-secretase complex and the PS2 γ-secretase complex the that the relative activity of the PS1 γ-secretase complex in the PS1+/–PS2–/– cell is than that of the PS2 γ-secretase complex in the PS1–/–PS2+/+ cell with This on the that PS1 and PS2 are engaged in the putative γ-secretase complex in these membranes. The amount of Aβ42 generated with cell membranes was total PS1+/–PS2–/– and PS1–/–PS2+/+ cell membranes also the of the and from The amount of Aβ42 to total produced by the PS1- and membranes is and respectively. the PS1 γ-secretase complex may be more than the PS2 γ-secretase the Aβ42 The comparison of the activities of PS1 and PS2 suggests that the PS1 γ-secretase complex is for the of the in vitro γ-secretase activity by cell membranes. it is that and cell membranes generate amounts of total more PS1 in the cells compared with the The specific activity of the PS1 γ-secretase complex in the PS1+/–PS2–/– cell membranes appears to the specific activity of the PS1 γ-secretase complex in the cell membranes. the amount of the cell membranes in the in a in the amount of not the of PS1 in the cell membranes not to the of the in vitro to PS1 activity. by BD the C-terminal of various BD cells were transfected with an the APP that to the C-terminal produced by β-secretase cleavage. The BD cells were to the for was on the cells for an and the was and for Aβ40 and The BD cells were with a protein to for with to and cell The amount of the Aβ-peptides that in of the various transfected BD cells for on the protein is in I. The PS1–/–PS2–/– BD cells fail to Aβ40 or Aβ42 not which is an that is with (9Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 10Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). the of PS1 protein in PS1+/–PS2–/– cells is only of that in BD cells the amounts of total by the cells is of that by the The of PS2 protein in PS1–/–PS2+/+ BD cells is higher than that in BD the cells only produced by the The relative activity of the PS1 γ-secretase complex in PS1+/–PS2–/– BD appears to be than that of the PS2 γ-secretase complex in PS1–/–PS2+/+ BD are with the studies with the BD cell that PS1 higher relative activity in the substrate. Furthermore, it suggests that from cells is produced by PS1-mediated γ-secretase activity. The amount of Aβ42 to total by BD cells was The corresponding of Aβ42 by PS1+/–PS2–/– BD cells PS1 and PS1–/–PS2+/+ BD cells PS2 were and respectively. in to studies with the cell it appears that the PS2 γ-secretase complex in the may have a than the PS1 γ-secretase complex to the PS1-mediated with of the PS1 activity of PS1 in the BD cells from either specific activity of the PS1-associated γ-secretase complex or from PS1 not engaged in the active γ-secretase complex. The active site-directed γ-secretase which to the active form of (12Li 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. M.K. M.S. Nature. 2000; PubMed Scopus Google Scholar), was used as a to the of PS1 in the BD cell membranes. displays γ-secretase activity in membranes from and PS1+/–PS2–/– BD cells not amounts of protein from the different BD cell membranes were with and the were to of active γ-secretase. The proteins were isolated with and by using anti-PS1 and anti-PS2 were the PS1–/–PS2–/– cell membranes were not Moreover, the of the PS1 and PS2 were the was in the of not of the signals by more PS1 in cell membranes than in PS1+/–PS2–/– cell membranes. Moreover, is less PS2 in cell membranes than in PS1–/–PS2+/+ cell membranes. that γ-secretase activity better with the amount of PS1 rather than total amount of PS1 in and BD cell membranes. PS1- and PS2-mediated potent γ-secretase M.S. D. Harrison T. P. Nadin A. G. Castro J.L. 2000; PubMed Scopus Google Scholar), of by BD cell membranes The was from the that was with the from cell membranes Y.M. Lai M.T. Xu M. Huang Q. DiMuzio Mower J. M.K. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). The PS1-mediated γ-secretase activity by PS1+/–PS2–/– BD cell membranes and the PS2-mediated γ-secretase activity by PS1–/–PS2+/+ BD cell membranes were both by and respectively). the PS1 γ-secretase complex appears to be more to inhibition by than is the PS2 γ-secretase complex. This of to PS1 and PS2 was also with membranes from derived from and mice not the other γ-secretase more γ-secretase activities by PS1+/–PS2–/– and PS1–/–PS2+/+ BD cell membranes The of C for the PS1- and PS2-mediated γ-secretase activities were and respectively. is that the of the inhibitors determined with the cell membranes are more to the determined with PS1+/–PS2–/– BD membranes than to the determined with PS1–/–PS2+/+ cell membranes evidence that the complex is the of γ-secretase activity in BD cells and their corresponding membranes. The of a active site-directed γ-secretase inhibitor (12Li 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. M.K. M.S. Nature. 2000; PubMed Scopus Google to both PS1 and PS2 suggests that the presenilins have overlapping This is with studies both PS1 and PS2 in APP and Notch (9Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat Cell Biol. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 10Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar, A. D. Annaert W. Saftig P. Craessaerts K. Serneels L. L. F. Vanderstichele H. R. P. D. A. Van Leuven F. De Strooper B. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, Ikeda M. H. Bernstein A. 1999; PubMed Scopus Google Scholar). the of the presenilins (67% identity) suggests that PS1 and PS2 may with to their The exhibited by the PS1- and mice may J. Xia W. Selkoe D.J. S. Full Text Full Text PDF PubMed Scopus Google Scholar, P.C. H. H. D.J. Price D.L. Van Sisodia S.S. Nature. PubMed Scopus Google Scholar). the of the PS1 and PS2 in mice and cells from these are not of or but of the these PS1- and PS2-mediated activities were individually using BD cells from and PS1–/–PS2+/+ mice, respectively. a on of immunoblot signals to the relative of PS1 and PS2 proteins in the different BD cell The of PS1 and PS2 proteins in BD cells is The amount of PS1 protein in PS1+/–PS2–/– BD cells is only of that in which is far less than the The less amount of PS1 protein in PS1+/–PS2–/– BD cells more or of the PS1-associated the other the amount of PS2 in PS1–/–PS2+/+ cells is that the of the PS2 in The higher of PS2 in PS1-deficient cells of increased of that are by PS1 in wild-type that the PS1 γ-secretase complex displays activity than the PS2 γ-secretase complex both in the in vitro using the and the using the substrate. that PS1 is more active than PS2 the APP fragments that are generated by of biochemical evidence that PS1 a role in the of APP, a on studies with cells derived from PS1 or (7De 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 (1552) Google Scholar, A. D. Annaert W. Saftig P. Craessaerts K. Serneels L. L. F. Vanderstichele H. R. P. D. A. Van Leuven F. De Strooper B. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). The that PS1- and PS2-mediated activities different to γ-secretase inhibitors that PS1 and PS2 have unique active sites that PS1+/–PS2–/– and PS1–/–PS2+/+ BD cells and the corresponding cell membranes γ-secretase activities that generated both Aβ40 and The of Aβ42 total to be and only different for the PS1- and PS2-mediated γ-secretase PS1 generated more than PS2 in the in vitro the was in the The for the of Aβ40 and Aβ42 with the in vitro and is but it may the of or in the studies that Aβ42 is not by only of the two PS1 and PS2 are of The different PS1 in the and cells were not by a corresponding in their γ-secretase The PS1 and γ-secretase activity is not of a in in which the of is in the of a amount of active γ-secretase using a inhibitor to on the of the used in the is much higher than its the of the relative amount of the active in the membranes. The amount of PS1 in cell membranes is only than that in PS1+/–PS2–/– cell PS1 in wild cell membranes is more than that in cell membranes. the amount of the is more with the relative γ-secretase activity by the cell membranes. that PS1 in PS1+/–PS2–/– cell membranes is engaged in the active γ-secretase only total PS1 in the BD cells is engaged in the active γ-secretase complex. The of the γ-secretase complex that are for activity to be that the PS1 may be but not for γ-secretase activity. This is with studies in which of PS1 or of its and fragments not γ-secretase activity D. Lee J. Song L. R. Wong G. E. L. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). G. M. S. D. L. A. Song Rogaeva E. F. T. A. Levesque L. Yu H. E. P. Liang Y. D.M. Xu C. Rogaev E. M. C. Y. R. S. A. P. P. Nature. 2000; PubMed Scopus Google Scholar), C. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. G. J. M. J. M. M. B. Xu W. Li J. M. R. C. D. 3: Full Text Full Text PDF PubMed Scopus Google Scholar), and R. G. J. M. J. M. M. B. Xu W. Li J. M. R. C. D. 3: Full Text Full Text PDF PubMed Scopus Google are proteins that are engaged in the complex and may be for γ-secretase activity. putative proteins may be in the that to γ-secretase activity. The PS1 complex may other functions as and regulation of inhibitors serve as to the functions of the presenilins the other putative functions that only a small portion of the PS1 in membranes is involved in an active γ-secretase complex may to γ-secretase The of γ-secretase activity to the presenilins been the presenilins and the γ-secretase not to be to the P. M. Craessaerts K. I. Vanderstichele H. Saftig P. De Strooper B. Annaert W. J. Cell Biol. 2001; PubMed Scopus Google Scholar). in the early of the the γ-secretase Notch or the M.T. A. D.J. R. Mol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that only a small portion of PS1 is engaged in the active γ-secretase complex in with evidence for a small amount of presenilins on the cell M. J. Saftig P. M. Selkoe D.J. R. J. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google may have are also involved in the of Notch that the Notch which is involved in of transcription G. 2000; PubMed Scopus Google Scholar). studies P. M.S. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, D. M.S. R. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google that inhibition of γ-secretase activity by inhibitors the Notch activity and cell be to studies PS1 and PS2-mediated γ-secretase activities Notch the of PS1 and PS2 different and inhibitors to γ-secretase inhibitors for Moreover, it is to the of to presenilins by neurons and cells J. PubMed Scopus Google Scholar). γ-secretase activity is believed to in two in the M. X. J. 2000; PubMed Google and γ-secretase activity in neurons and cells may contribute to and in other In studies in the the presenilins and γ-secretase activity. were as the of the PS1 γ-secretase complex and the PS2 γ-secretase complex. other in the PS1 and γ-secretase activity were as the with to the activity of PS1 in wild-type BD The that is exhibited by the presenilins their in a of biological that to be C. and K. for with the and S. for the

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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John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1474) Google Scholar) and ProBACE460 to the P10-P4′ Stat(Val) is with the that is active only active of aspartyl are to to immobilized of aspartyl to immobilized J. PubMed Scopus Google Scholar, H. S. T. PubMed Scopus Google Scholar, R. J. Protein PubMed Scopus Google in the precursors for the immobilized which is not the with that purified ProBACE460 to BACE460 is with a that or a in cells is for the of B.D. Denis P. M. Teplow D.B. Kahn S. Louis J.-C. Citron R. J. Biol. 2000; PubMed Scopus Google Scholar). to using that a soluble BACE precursor in to the Pro domain B.D. Denis P. M. Teplow D.B. Kahn S. Louis J.-C. Citron R. J. Biol. 2000; PubMed Scopus Google Scholar). activity in insect in is in cells from Cell Biol. 14: PubMed Scopus Google Scholar). that are expressed in insect cells are typically not cleaved is in the cells F. 1998; PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). 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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.