Familial Alzheimer’s disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of ≥45 residues

Abstract

Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated. Production of amyloid β-protein (Aβ) is carried out by the membrane-embedded γ-secretase complex. Mutations in the transmembrane domain of amyloid β-protein precursor (APP) associated with early-onset familial Alzheimer's disease (FAD) can alter the ratio of aggregation-prone 42-residue Aβ (Aβ42) to 40-residue Aβ (Aβ40). However, APP substrate is proteolyzed processively by γ-secretase along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38. Effects of FAD mutations on each proteolytic step are unknown, largely due to difficulties in detecting and quantifying longer Aβ peptides. To address this, we carried out systematic and quantitative analyses of all tri- and tetrapeptide coproducts from proteolysis of wild-type and 14 FAD-mutant APP substrates by purified γ-secretase. These small peptides, including FAD-mutant forms, were detected by tandem mass spectrometry and quantified by establishing concentration curves for each of 32 standards. APP intracellular domain (AICD) coproducts were quantified by immunoblot, and the ratio of AICD products corresponding to Aβ48 and Aβ49 was determined by mass spectrometry. Levels of individual Aβ peptides were determined by subtracting levels of peptide coproducts associated with degradation from those associated with production. This method was validated for Aβ40 and Aβ42 by specific ELISAs and production of equimolar levels of Aβ and AICD. Not all mutant substrates led to increased Aβ42/40. However, all 14 disease-causing mutations led to inefficient processing of longer forms of Aβ ≥ 45 residues. In addition, the effects of certain mutations provided insight into the mechanism of processive proteolysis: intermediate Aβ peptides apparently remain bound for subsequent trimming and are not released and reassociated. Cerebral plaques composed of the amyloid β-protein (Aβ) are a defining pathological feature of Alzheimer’s disease (1Querfurth H.W. LaFerla F.M. Alzheimer's disease.N. Engl. J. Med. 2010; 362: 329-344Crossref PubMed Scopus (3463) Google Scholar). Aβ is produced from the amyloid β-protein precursor (APP) through sequential proteolysis, by β-secretase shedding the ectodomain (2Cole S.L. Vassar R. The role of APP processing by BACE1, the β-secretase, in Alzheimer's disease pathophysiology.J. Biol. Chem. 2008; 283: 29621-29625Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) followed by γ-secretase cutting within the transmembrane domain (TMD) of the remnant 99-residue C-terminal fragment (C99) (3Wolfe M.S. Structure and function of the γ-secretase complex.Biochemistry. 2019; 58: 2953-2966Crossref PubMed Scopus (39) Google Scholar). Aβ peptides of 38 to 43 residues are secreted, with the aggregation-prone 42-residue form (Aβ42) being predominantly and disproportionally deposited in AD plaques (4Iwatsubo T. Odaka A. Suzuki N. Mizusawa H. Nukina N. Ihara Y. Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ monoclonals: Evidence that an initially deposited species is Aβ42(43).Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1504) Google Scholar). A pathogenic role for Aβ42 was strongly supported by the discovery of dominant missense mutations in APP and presenilins—the catalytic component of the γ-secretase complex—that cause early-onset familial Alzheimer’s disease (FAD) (5Tanzi R.E. The genetics of Alzheimer disease.Cold Spring Harb. Perspect. Med. 2012; 2a006296Crossref PubMed Scopus (425) Google Scholar). These mutations were found to elevate the ratio of Aβ42 to Aβ40, thereby increasing Aβ42 aggregation. Inconsistencies with the hypothesis that Aβ42 is the pathogenic variant in FAD emerged recently with a report on Aβ40 and Aβ42 production from 138 different FAD-mutant forms of the presenilin-1/γ-secretase complex, showing that many disease-causing mutations did not elevate Aβ42/Aβ40 (6Sun L. Zhou R. Yang G. Shi Y. Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E476-E485Crossref PubMed Scopus (164) Google Scholar). Yet the involvement of Aβ in FAD seems inescapable: after more than 30 years of searching, the only mutations associated with FAD are found in the substrate and enzyme that produces Aβ. Solving this puzzle requires recognition that processing of the APP TMD by the membrane-embedded γ-secretase complex occurs processively. The enzyme first cleaves near the cytosolic end of the APP TMD at the ε site to give either Aβ48 or Aβ49 and release the corresponding APP intracellular domain (AICD) composed of C99 residues 49 to 99 or 50 to 99 (7Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. Distinct intramembrane cleavage of the β-amyloid precursor protein family resembling γ-secretase-like cleavage of Notch.J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar) (Fig. 1). Carboxypeptidase cleavage generally every three amino acids then produces secreted Aβ peptides along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38 (8Takami M. Nagashima Y. Sano Y. Ishihara S. Morishima-Kawashima M. Funamoto S. Ihara Y. γ-Secretase: Successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment.J. Neurosci. 2009; 29: 13042-13052Crossref PubMed Scopus (341) Google Scholar). Understanding how FAD mutations initiate the disease process requires a comprehensive and quantitative analysis of all the proteolytic steps carried out by γ-secretase on APP. Such analysis is challenging, as Aβ peptides of 45 residues and longer are difficult to detect and quantify by mass spectrometry (MS) and have no specific antibodies for ELISA. We have previously shown that five different FAD-mutant γ-secretase complexes are deficient in carboxypeptidase function (9Fernandez M.A. Klutkowski J.A. Freret T. Wolfe M.S. Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid β-peptides (Aβ) by γ-secretase to increase 42-to-40-residue Aβ.J. Biol. Chem. 2014; 289: 31043-31052Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and increase the proportion of Aβ that is 45 residues and longer (10Quintero-Monzon O. Martin M.M. Fernandez M.A. Cappello C.A. Krzysiak A.J. Osenkowski P. Wolfe M.S. Dissociation between the processivity and total activity of γ-secretase: Implications for the mechanism of Alzheimer's disease-causing presenilin mutations.Biochemistry. 2011; 50: 9023-9035Crossref PubMed Scopus (89) Google Scholar). However, this limited study was accomplished using a long, hand-cast urea-PAGE system and western blotting, which is technically challenging, difficult to quantify, and insufficient for separating through we to tandem mass spectrometry as a method to quantify the tri- and tetrapeptide coproducts to the levels of each Aβ peptide produced by purified γ-secretase from wild-type and 14 different FAD-mutant APP of mutations and not all increased the Aβ42/Aβ40 In we that all 14 FAD mutations processive proteolysis by γ-secretase to elevate levels of Aβ peptides of 45 residues and we that long Aβ are not released and with the enzyme for subsequent remain bound for We first the effects of the APP FAD mutations on the production of Aβ42 and Aβ40 peptides and the ratio of Aβ42 to Aβ40 peptides. we a substrate with an and a C-terminal We and have this substrate in γ-secretase as C99 processing by the complex M. J. Shi J.A. is with γ-secretase activity in the Natl. Acad. Sci. U. S. A. PubMed Scopus Google Wolfe M.S. The tripeptide cleavage mechanism of PubMed Scopus Google Scholar). We and purified along with 14 different FAD-mutant (Fig. We γ-secretase in using a all and P. T. L. Yang G. Y. Zhou R. Shi Y. of 2014; PubMed Scopus Google Scholar) and then purified the complex to The and FAD-mutant substrates were to proteolysis by 30 of γ-secretase of in two different in or in These two were in to the give Aβ40 Aβ42 production that are to that of the more of as the of the with subsequent analysis of small peptide Levels of Aβ40 and Aβ42 from proteolysis of the substrates were quantified by specific ELISAs (Fig. and The produced more Aβ40 and Aβ42 peptides. However, the effects of the FAD mutations with substrate on Aβ40 and Aβ42 are between the two the of Aβ42 to Aβ40 are for all the substrate between the two different The that three FAD and did not to in Aβ42/Aβ40 in the and did not increased Aβ42/Aβ40 in a increase in this The for the and that proteolytic processing of substrates by γ-secretase in the in a to occurs within a that Aβ42/Aβ40 from substrate is than occurs for C99 in in vitro were substrate L. Yang substrate concentration the Aβ42/Aβ40 Implications for Alzheimer's Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in Aβ42/Aβ40 between and FAD-mutant substrates can determined with the the Aβ42/Aβ40 as Wolfe M.S. The tripeptide cleavage mechanism of PubMed Scopus Google Scholar). The that increased Aβ42/Aβ40 is not for the of as not all FAD mutations in the APP TMD to we to and quantify all proteolytic products APP TMD proteolysis by γ-secretase. of small peptide coproducts and trimming is an to quantify the production of and Aβ40 along the Aβ40 and the production of and along the Aβ42 The substrate in the Aβ40 and and in the Aβ42 (Fig. 1). method was on (8Takami M. Nagashima Y. Sano Y. Ishihara S. Morishima-Kawashima M. Funamoto S. Ihara Y. γ-Secretase: Successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment.J. Neurosci. 2009; 29: 13042-13052Crossref PubMed Scopus (341) Google to and quantify the coproducts from the trimming of substrate by γ-secretase. the from the system and from the system were to analysis to detect small peptides. small peptides to were detected in the system (Fig. A and However, the method to detect peptides from the system not due to of the products from the as from the in as as by the levels of of curves of each peptide were by the concentration of peptide the of the three from The curves from to for and peptides, from to (Fig. all curves each peptide in the γ-secretase cleavage with was and quantified after and small peptides from substrate were in a with to (Fig. with the of the carboxypeptidase trimming Aβ40 peptides were with and Aβ42 peptides were with we quantified small peptides by γ-secretase from all 14 FAD-mutant of the 14 mutant substrates two small peptides from the Aβ40 and from the Aβ42 are different from the small peptides from trimming of the substrate due to within the Aβ the substrate is to the of small peptides from the curves for all the coproducts from processive proteolysis of all 14 FAD-mutant substrates were and from to (Fig. each small peptide proteolytic trimming for and all 14 FAD-mutant substrates was quantified (Fig. the Aβ40 levels of produced from the trimming step the production of and degradation of with all of the FAD-mutant substrates for with substrate (Fig. Levels of trimming products from of production of and degradation of and from production of Aβ40 and degradation of for for which were no with (Fig. and the Aβ42 levels of produced from the trimming step of and degradation of were with substrate with mutant substrates and and with and (Fig. Levels of the trimming from Aβ42 of Aβ42 and degradation of were increased in and with and in and (Fig. Levels of the trimming from of and degradation of increased from substrate and with and from and (Fig. to this production and degradation of all Aβ peptides were quantified by coproducts by for production of Aβ48 and To quantify Aβ48 and Aβ49 by ε cleavage of APP levels of coproducts AICD 49 to 99 and AICD 50 to 99 peptides were determined using a of and quantitative western AICD species in the system were with antibodies and by (Fig. The of corresponding to AICD 49 to 99 to AICD 50 to 99 were and for enzyme with and all mutant substrates (Fig. The ratio of AICD 49 to 99 to AICD 50 to 99 from substrate was and this ratio is increased with all the mutant substrates for and for which is This that of the FAD mutations ε cleavage in of Aβ48 with with T. Dohmae N. Y. N. Misonou H. R. H. Takio K. Morishima-Kawashima M. S. Ihara Y. between amyloid β-protein and C-terminal fragment of β-amyloid precursor Biol. Chem. Full Text Full Text PDF PubMed Scopus Google M. T. R. A. J. M. Alzheimer's disease mutations in APP not γ-secretase AICD PubMed Scopus Google Scholar). In the and mutations ε cleavage Aβ49 Wolfe M.S. The tripeptide cleavage mechanism of PubMed Scopus Google Scholar). the substrate the led to ε cleavage at three AICD to 99 along with AICD 49 to 99 and AICD 50 to analysis of AICD products from the system and with antibodies (Fig. that the system γ-secretase cleavage of APP substrate as occurs within a of AICD 49 to 99 and AICD 50 to 99 levels by as of specific AICD that we did not the were to quantitative western using antibodies (Fig. of were in to a with concentration of protein (Fig. this the concentration of total in the enzyme can quantified (Fig. AICD by γ-secretase from substrate was a of with previously J. A. R. H. proteolysis of β-amyloid precursor protein by γ-secretase is an J. Full Text Full Text PDF PubMed Scopus Google Y. Wolfe M.S. to by substrate transmembrane Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and of the of by intramembrane S. S. the is a not by substrate Full Text Full Text PDF PubMed Scopus Google Scholar). of the total AICD levels for each mutant substrate increased ε cleavage with substrate for mutations and and ε cleavage for mutations and mutant Mutations and no in total AICD production with that with The concentration of each AICD and AICD then using the total AICD determined by quantitative western and the ratio of AICD 49 to 99 to AICD 50 to 99 determined from (Fig. The of AICD 49 to 99 and AICD 50 to 99 thereby the of production of coproducts Aβ48 and In the of of AICD to 99 of the of produced through ε cleavage of this mutant The of each species of Aβ by γ-secretase from each APP substrate was from the quantified coproducts peptides and AICD levels of Aβ peptides were determined by subtracting the concentration of small peptide of degradation from the concentration of peptide of production (Fig. The concentration of each Aβ peptide species for and mutant substrates are and in with with substrate in and in Aβ40 and Aβ42 levels determined by this method are to that determined from the enzyme by specific ELISAs by by by by This of the method for the of each Aβ processing of all FAD mutations in the APP TMD led to increased levels in or more Aβ peptides of 45 to 49 residues in The led to the of and The Aβ48 and the increased and mutations led to increased and the peptide is with study showing that amino acids as are not in the Wolfe M.S. The tripeptide cleavage mechanism of PubMed Scopus Google The in the for the trimming thereby this proteolytic and mutations increased Aβ49 and The mutant and and in increased and The mutant Aβ49 and and increased mutations Aβ42 and and the peptide T. T. N. K. Funamoto S. N. Y. K. P. J. M. N. Ihara Y. and of Neurosci. 2011; PubMed Scopus Google J. A. J. A. T. H. and of by the presenilin-1 mutant the presenilin hypothesis of Alzheimer's Med. PubMed Scopus Google S. T. L. Alzheimer's disease mutations in presenilin Aβ peptide Full Text Full Text PDF PubMed Scopus Google Scholar) with by mutations and did not increase of long Aβ peptides from each of the 14 FAD-mutant APP substrates by γ-secretase are due to in the first carboxypeptidase trimming as the of Aβ peptide produced that is carried through the processing step (Fig. Aβ42 long to the pathogenic Aβ variant in Alzheimer’s This Aβ variant is the component of the amyloid plaques that the and FAD mutations in APP and the can elevate the ratio of Aβ42 to However, of pathogenic of aggregation-prone Aβ42 and of have The that many presenilin-1 FAD mutations not elevate Aβ42/Aβ40 (6Sun L. Zhou R. Yang G. Shi Y. Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E476-E485Crossref PubMed Scopus (164) Google Scholar) Aβ42 as an of Understanding how FAD mutations alter APP processing by γ-secretase requires a and quantitative analysis of all proteolytic to this not for FAD we a study of 14 different FAD missense mutations in the APP TMD and that all mutations led to levels of Aβ peptides of to in Such are due to deficient first trimming steps by γ-secretase. The of long Aβ peptides is challenging, as are no specific antibodies for of and are difficult to detect and quantify by We previously in a limited study that five FAD-mutant presenilin-1/γ-secretase complexes were deficient in carboxypeptidase activity (9Fernandez M.A. Klutkowski J.A. Freret T. Wolfe M.S. Alzheimer presenilin-1 mutations dramatically reduce trimming of long amyloid β-peptides (Aβ) by γ-secretase to increase 42-to-40-residue Aβ.J. Biol. Chem. 2014; 289: 31043-31052Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) and increased the proportion of long Aβ peptides (10Quintero-Monzon O. Martin M.M. Fernandez M.A. Cappello C.A. Krzysiak A.J. Osenkowski P. Wolfe M.S. Dissociation between the processivity and total activity of γ-secretase: Implications for the mechanism of Alzheimer's disease-causing presenilin mutations.Biochemistry. 2011; 50: 9023-9035Crossref PubMed Scopus (89) Google Scholar). However, detecting through Aβ49 long, hand-cast urea-PAGE and western that were difficult to quantify and did not Aβ peptides of residues and many FAD mutations in the APP TMD to mutant forms of long Aβ peptides that on M.A. G. R. Wolfe M.S. substrate for γ-secretase processing of APP PubMed Scopus Google Scholar). The to quantify the small peptide coproducts by a method by the Ihara (8Takami M. Nagashima Y. Sano Y. Ishihara S. Morishima-Kawashima M. Funamoto S. Ihara Y. γ-Secretase: Successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment.J. Neurosci. 2009; 29: 13042-13052Crossref PubMed Scopus (341) Google an of quantifying each Aβ by subtracting the of small peptide of degradation from the of small peptide of APP substrate and γ-secretase in a we found that levels of Aβ40 and Aβ42 determined by the method were to levels determined by specific These that Aβ40 and Aβ42 are produced by γ-secretase along the of Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38 and that the of Aβ peptides is that production of Aβ49 and Aβ48 was by a different method of AICD by western in with of the ratio of AICD the of Aβ peptide levels is to the total AICD as N. Funamoto S. S. M. S. Dohmae N. Ihara Y. production of amyloid β-protein and amyloid precursor protein intracellular domain from fragment by Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of Aβ peptides along either the or is to the of the corresponding AICD 1). This in the of the of Aβ levels from and with levels of AICD and quantified by and produced through ε proteolysis of the mutant substrate was as this of was not produced through ε proteolysis that AICD 50 to produced through ε proteolysis of the mutant substrate was as this of was not produced through ε proteolysis that AICD 50 to in a comprehensive analysis of γ-secretase processing of 14 different FAD-mutant APP substrates with we to the as this provided more Aβ40 and Aβ42 than the the system the by the system of levels of with small peptide and effects of the 14 FAD-mutant substrates on Aβ40 and Aβ42 levels with substrate in the two were as were effects on the ratio of AICD 50 to 99 to AICD 49 to that the system that are of occurs within a In not all of the 14 FAD-mutant substrates to from a analysis of 138 FAD-mutant complexes (6Sun L. Zhou R. Yang G. Shi Y. Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E476-E485Crossref PubMed Scopus (164) Google Scholar). with the hypothesis that increased Aβ42/Aβ40 is for in FAD is to APP of all the small peptide products from γ-secretase processing of the 14 FAD-mutant substrates by curves for each small including mutant of each small peptide in In this the of each carboxypeptidase trimming step from quantitative western of total AICD production and of the ratio of the two AICD quantitative of the production of Aβ48 and Aβ49 from each mutant substrate as all in the of individual Aβ production and levels of each of the different Aβ peptides from γ-secretase processing of each of the 14 FAD-mutant APP substrates determined and with that for The effects of FAD mutations on Aβ40 and Aβ42 and Aβ42/Aβ40 were generally to those determined by ELISA. five of the 14 mutations Aβ42 and two of the mutations and did not increase with for every mutant the of Aβ peptides was to the total AICD and the of Aβ peptides along the or was to levels of the AICD 1). The two were the of Aβ peptides along the from substrate and the of Aβ peptides along the from In the of the the of a tripeptide trimming of with the tripeptide by the trimming of This the for the Aβ48 with this mutant more is produced than is from the of Aβ48 production. The in the with the mutant substrate is apparently was produced by ε cleavage and not the produced is the of the levels of Aβ peptides in the is to the determined of AICD 50 to all the levels the corresponding AICD that the of each Aβ species is This is that all the mutant substrates mutations into Aβ The with the substrate insight into the of processive This mutant substrate is to Aβ48 Aβ48 is the wild-type is to In Aβ48 produced from substrate is This that the Aβ not from the enzyme and then for as in a the trimming of Aβ48 the this peptide was from or for the trimming from the substrate is that the AICD not from the enzyme as as AICD. Dissociation of AICD to the processing The is that the of substrate with γ-secretase and subsequent ε cleavage to Aβ48 the enzyme in a different that a of for out the processing the processing of from the substrate is the that produced from this mutant is the wild-type These to by which FAD mutations specific trimming steps to elevate long Aβ peptides. is the that each of the 14 different FAD mutations in the APP TMD to trimming of Aβ peptides from 45 to 49 residues in These peptides of the APP TMD and are and not secreted Y. Morishima-Kawashima M. Y. G. N. Y. M. R. Ihara Y. forms of amyloid Implications for the mechanism of intramembrane cleavage by Neurosci. PubMed Scopus Google Scholar) (Fig. is or pathological for the long, Aβ peptides, and have largely only as to the secreted a role of peptides in the of AD was by Ihara and in report on the discovery of Y. Morishima-Kawashima M. Y. G. N. Y. M. R. Ihara Y. forms of amyloid Implications for the mechanism of intramembrane cleavage by Neurosci. PubMed Scopus Google Scholar). to long Aβ peptides as at in peptides FAD or can only into and or and with In the of γ-secretase to process Aβ peptides of 45 residues and longer to to the of FAD mutations in the in the M. J. Shi J.A. is with γ-secretase activity in the Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) were by The wild-type and FAD-mutant were then into were in at with to were with and for were in of and and by The was and with for at The were then with and protein with with and with of each substrate was by with and by western with was carried out using a the of the complex Wolfe M.S. The tripeptide cleavage mechanism of PubMed Scopus Google P. T. L. Yang G. Y. Zhou R. Shi Y. of 2014; PubMed Scopus Google Scholar). and were carried out as previously G. A. R. Wolfe M.S. and of the γ-secretase complex.Biochemistry. PubMed Scopus Google P. H. L. Wolfe M.S. H. of purified γ-secretase at Biol. 2009; PubMed Scopus Google Wolfe M.S. for intramembrane 2017; PubMed Scopus Google Scholar). for the 30 γ-secretase was for 30 at in composed of 50 and with and were by of purified substrate concentration and at for The were by in and at the 30 purified γ-secretase was into total in 50 as Wolfe M.S. for intramembrane 2017; PubMed Scopus Google Scholar). The was with for at for the of then were and the was with concentration of The was by at and carried out for The proteolytic products from enzyme from were by immunoblot, and mass spectrometry as of γ-secretase peptides were with in for at The were with two peptides were then from the with with proteolytic products were detected using a mass from γ-secretase and were to on to and to were then with for at with antibodies for at then and with antibodies for at were and for and were by peptides were using an mass by of the and of the peptides were in a and with a step of and a The three from were by tandem for each small To a peptide the from the three were using an mass of were in are in the and The that have no of with the of this We Shi of in for the the of the γ-secretase complex. We of the at for S. and T. the S. T. and M. S. the and the S. and M. S. the This was by and from the of to M. S. The is the of the and not the of the of

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