Patrick C. Fraering

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.

The γ-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the β-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the γ-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of γ-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature γ-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease. The γ-secretase complex is an unusual multimeric protease responsible for the intramembrane cleavage of a variety of type 1 transmembrane proteins, including the β-amyloid precursor protein and Notch. Genetic and biochemical data have revealed that this protease consists of the presenilin heterodimer, a highly glycosylated form of nicastrin, and the recently identified gene products, Aph-1 and Pen-2. Whereas current evidence supports the notion that presenilin comprises the active site of the protease and that the other three components are members of the active complex required for proteolytic activity, the individual roles of the three co-factors remain unclear. Here, we demonstrate that endogenous Aph-1 interacts with an immature species of nicastrin, forming a stable intermediate early in the assembly of the γ-secretase complex, prior to the addition of presenilin and Pen-2. Our data suggest 1) that Aph-1 is involved in the early stages of γ-secretase assembly through the stabilization and perhaps glycosylation of nicastrin and by scaffolding nicastrin to the immature γ-secretase complex, and 2) that presenilin, and later Pen-2, bind to this intermediate during the formation of the mature protease. The γ-secretase complex is an unusual aspartyl protease responsible for the intramembranous cleavage of a variety of type 1 integral membrane proteins. This proteolytic activity is essential for the proper functioning of the Notch receptor, a critical signaling factor in metazoan development (1Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (850) Google Scholar). In fact, there is a growing list of substrates that require γ-secretase-mediated processing to release their signaling moieties (2Ebinu J.O. Yankner B.A. Neuron. 2002; 34: 499-502Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This intramembrane cleavage process and its implication in normal cell signaling have been termed regulated intramembrane proteolysis (3Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). In addition to its physiological function, γ-secretase is central to the pathogenesis of Alzheimer's disease, because the amyloid β-protein is generated by the γ-secretase-mediated cleavage of the β-amyloid precursor protein (APP). 1The abbreviations used are: APP, β-amyloid precursor protein; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; NCT, nicastrin; mNCT, mature NCT; iNCT, immature NCT; IP, immunoprecipitation; CHO, Chinese hamster ovary; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; BN, blue native; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HEK, human embryonic kidney; siRNA, small interfering RNA; DDM, n-dodecyl-β-d-maltoside; Big CHAP, N,N-bis-(3-O-gluconamidopropyl)deoxycholamide. Whereas recent studies have identified the gene products that comprise γ-secretase and established that their physical interaction is required for γ-secretase activity (see below), little is known about their specific roles in intramembrane proteolysis or the nature of the individual protein-protein interactions within the active complex. The first member of the γ-secretase complex to be identified was presenilin (PS), and extensive work during the last 5 years has suggested that it serves as the active site of the protease complex (4Wolfe M.S. Selkoe D.J. Science. 2002; 296: 2156-2157Crossref PubMed Scopus (56) Google Scholar). PS is initially expressed as an unstable holoprotein of about 50 kDa, with a half-life of ∼1 h (5Zhang J. Kang D.E. Xia W. Okochi M. Mori H. Selkoe D.J. Koo E.H. J. Biol. Chem. 1998; 273: 12436-12442Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 6Ratovitski T. Slunt H.H. Thinakaran G. Price D.L. Sisodia S.S. Borchelt D.R. J. Biol. Chem. 1997; 272: 24536-24541Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Through what appears to be an autoproteolysis, PS holoprotein undergoes cleavage to yield an N-terminal fragment (NTF; ∼28 kDa) and a C-terminal fragment (CTF; ∼22 kDa) that remain associated (6Ratovitski T. Slunt H.H. Thinakaran G. Price D.L. Sisodia S.S. Borchelt D.R. J. Biol. Chem. 1997; 272: 24536-24541Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 7Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar). This PS heterodimer is significantly more stable than the PS holoprotein, having a half-life of ∼30 h, and is thought to be the biologically active form of the protein (7Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. Hardy J. Levey A.I. Gandy S.E. Jenkins N.A. Copeland N.G. Price D.L. Sisodia S.S. Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 8Podlisny M.B. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E.H.M. Seubert P. St. George-Hyslop P. Teplow D.B. Selkoe D.J. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (274) Google Scholar, 9Capell A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 10Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu M. George-Hyslop Fraser J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). intramembranous within and of amyloid β-protein and PS M.S. Xia W. Diehl Selkoe D.J. PubMed Scopus Google Scholar). This with the that γ-secretase be to and Xu M. J.L. J. T. C. A. R.B. M.K. M.S. 2000; PubMed Scopus Google Scholar, Diehl T. Xia W. Selkoe D.J. M.S. Biol. 2000; PubMed Scopus Google that the PS heterodimer comprises the active site of it is the PS holoprotein, that A. Grunberg J. Pesold B. Diehlmann A. Citron M. Nixon R. Beyreuther K. Selkoe D.J. Haass C. J. Biol. Chem. 1998; 273: 3205-3211Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 10Yu G. Chen F. Levesque G. Nishimura M. Zhang D.M. Levesque L. Rogaeva E. Xu M. George-Hyslop Fraser J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and bind to an in an Ye W. Diehl Selkoe D.J. M.S. A. 2002; PubMed Scopus Google the holoprotein is an immature PS are components of the active The member of the γ-secretase complex to be identified was nicastrin initially through a in C. M. A. 2002; PubMed Scopus Google and later a G. Nishimura M. Zhang L. A. Rogaeva E. Chen F. T. A. Levesque L. H. E. P. Zhang D.M. Xu C. E. M. C. Zhang R. A. Fraser P. St. George-Hyslop P. 2000; PubMed Scopus Google Scholar). is a type 1 membrane protein that glycosylation within its studies have established that three of in the protein an glycosylated species and a kDa) that is the P. M. Xu H. Thinakaran G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. R. T. 2002; PubMed Scopus Google Scholar, Ye W. M.S. Selkoe D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. J. A. H. Haass C. J. Biol. 2002; PubMed Scopus Google Scholar). is this that is associated with active γ-secretase Ye W. M.S. Selkoe D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and PS is required for the of this species P. M. Xu H. Thinakaran G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. R. T. 2002; PubMed Scopus Google Scholar, Ye W. M.S. Selkoe D.J. J. Biol. 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A. 100: PubMed Scopus Google Scholar). the specific protein-protein interactions responsible for this multimeric protease complex and their of assembly remain we have that γ-secretase is associated with a of kDa, to the of PS, NCT, and Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar). we an intermediate complex in and Aph-1 a of kDa, with the of in cell expressed as as endogenous Aph-1 a Aph-1 and The interaction and Aph-1 was the PS heterodimer and the of the mature γ-secretase complex a Aph-1 and NCT, of the of PS and Pen-2. data the that Aph-1 and early in γ-secretase forming a stable intermediate prior to the of PS and Pen-2, PS and the glycosylation of NCT, of are known to with active and hamster and in and human of P. St. in with and as G. Nishimura M. Zhang L. A. Rogaeva E. Chen F. T. A. Levesque L. H. E. P. Zhang D.M. Xu C. E. M. C. Zhang R. A. Fraser P. St. George-Hyslop P. 2000; PubMed Scopus Google Scholar). The stable cell human and human and and human and in with or as Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar). of as suggested by the prior to as H. G. PubMed Scopus Google Scholar, H. G. PubMed Scopus Google Scholar). blue cell in and for kDa) in for and in with to and as and cell in CHAPSO, a protease and for 1 h, and the as Ye W. Diehl Selkoe D.J. M.S. A. 2002; PubMed Scopus Google and in or a protein and of protein in with for h, three for in and in and and is a to the N-terminal of presenilin M.B. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. Koo E.H.M. Seubert P. St. George-Hyslop P. Teplow D.B. Selkoe D.J. Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (274) Google and is a to the of for PS holoprotein and with of or for PS with of for with of and P. or for with or and 50 for with and for endogenous Aph-1 with the and and with with in with and in and and by as and used for of Aph-1 and in human and cell revealed an γ-secretase complex in Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar). This complex mature of of the known γ-secretase components NTF, CTF, NCT, and this we an intermediate complex blue that was for and Aph-1 and this be for an assembly intermediate of the of kDa) and this with Aph-1 for endogenous and evidence of other than within the active γ-secretase complex that this was an of or a of the γ-secretase complex, we with a protein and for the the of this complex. that for and Aph-1 in this within the first h of the has a half-life of ∼30 h, that this was a this we of the and the of and that to the of a The in the in in to protein and the of γ-secretase components and to the glycosylation of the in This revealed that the species was of In the species in the in the than the species the that this form is in for and that the PS with the mNCT, with an mature γ-secretase complex we a small of that in the first and was to the of the in the this of the complex with PS holoprotein, the with PS NTF, that a immature and a mature γ-secretase complex a The of the immature complex than the mature complex be for by the of by the in of and mNCT, or by a of The of this immature complex is with the complex that has been data of that the of In to the interaction we a of for we are human and we to a form of we that human we that human this of mature γ-secretase components and protease activity Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar). of and with an The with NCT, NTF, and The revealed or PS and little of PS and PS holoprotein in the of the In the we and PS holoprotein in the little or mNCT, NTF, or that the of γ-secretase components in of the protein the endogenous complex Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google we stable Aph-1 and that and associated with the expressed small of and associated with the was the other of PS holoprotein in the of the iNCT, immature PS with in the of of the γ-secretase components and in of the has been to Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google we of and with Aph-1 in have more mature γ-secretase and of its immature components in Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Aph-1 to PS holoprotein and more of PS than the and of are with a Aph-1 and that is more of γ-secretase components is in this with for Aph-1 Pen-2, we with that human and with the Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google of the endogenous γ-secretase complex. the other in the stable revealed of to NTF, CTF, and of with a active γ-secretase complex Ye W. Diehl Selkoe D.J. M.S. A. 2002; PubMed Scopus Google Scholar, Ye W. M.S. Selkoe D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of PS holoprotein with with the of in PS T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of in the the to appears specific for the glycosylation of has been to require PS T. R. R. T. 2002; PubMed Scopus Google Scholar, Ye W. M.S. Selkoe D.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. J. A. H. Haass C. J. Biol. 2002; PubMed Scopus Google Scholar, A. Chen F. G. H. H. M. Nixon Gandy S.E. St. George-Hyslop P. Fraser J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google data suggest that PS and are to with the has was cell to the in we of and iNCT, PS holoprotein, and PS and associated with as in the This that the of with components of the γ-secretase complex is by is in the in the cell Aph-1 and a of the γ-secretase complex is to the in it has been Ye W. Diehl Selkoe D.J. M.S. A. 2002; PubMed Scopus Google Scholar, Xu M. J. M.K. A. 2000; PubMed Scopus Google Scholar). Whereas have been to γ-secretase and protease activity, including CHAPSO, CHAPS, Big CHAP, and other in the of γ-secretase activity or the and of the complex and Ye W. Diehl Selkoe D.J. M.S. A. 2002; PubMed Scopus Google Scholar). we a variety of for their to γ-secretase as by of the we the cell and of in three DDM, with an the the of the expressed cell of mNCT, iNCT, PS holoprotein, PS NTF, PS CTF, and the in CHAPSO, a with γ-secretase activity Xu M. J. M.K. A. 2000; PubMed Scopus Google known components of the γ-secretase complex by of the γ-secretase components in the with the 5 and the in DDM, with of and iNCT, there was PS holoprotein, PS NTF, PS CTF, or and the γ-secretase components that in the than the and the in was the γ-secretase member by the with other components in the data demonstrate that members of the γ-secretase complex are to Aph-1 in the of and are it in a of the mature complex that and the PS a of and This is with the of a stable complex and the in of the in in an cell we with and with and the and membrane for and for and endogenous and three the The of three the of the we identified in the and the the mature γ-secretase complex Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar). that the protein was in the and there in the as that this was an of we the for endogenous Aph-1 to and that endogenous Aph-1 was in the intermediate or the γ-secretase complex The of endogenous Aph-1 in the γ-secretase complex that expressed endogenous Aph-1 is a member of the active γ-secretase complex Chen F. T. H. M. W. A. A. Fraser St. George-Hyslop P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with evidence that of Aph-1 in the endogenous γ-secretase complex Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google of cell of that the Aph-1 with the the mature complex that the intermediate the was in a of Aph-1 and and that the was iNCT, we the of the complex by in The addition of the cell a of the γ-secretase complex and the intermediate the that the of is to their with that is by This of the in the is with its in In the the complex a than the that was in the The for the in of the intermediate in cell is we have in the of this intermediate the of it be to in the of to and the for γ-secretase a that was for the cell the of a stable of Aph-1 and endogenous proteins, we by for endogenous and in to the three in the 1 and the of the γ-secretase complex and the we in the with the of the in the we for endogenous Aph-1 the that to 1) the and 2) the mature γ-secretase complex the γ-secretase complex of endogenous by and that Aph-1 by with the to Aph-1 we to with of and PS in the mature γ-secretase complex. with the in the demonstrate that the of the in be for by the of γ-secretase complex the notion that this be an of membrane of the in glycosylated is in cell that we have for endogenous γ-secretase and and CHO, and cell J. P. C. B. L. W. W. T. M. and J. the mature form has been by to be a of this is cell of immature In and of cell of the because and are of form interacts with this we with cell in and the with an to the C-terminal of In we of the with a to the for NCT, NTF, and In and with of and and In of revealed a of of iNCT, with little or little heterodimer was with the The of with was to the in the cell endogenous NCT, we in Whereas the of in with with and with the revealed the with iNCT, the of more in the associated with the mature components of mNCT, NTF, and with the of an intermediate of and of a Aph-1 that the to endogenous Aph-1 in the of mNCT, NTF, and and are with and and that of Aph-1 is associated with and is associated with and PS in the active γ-secretase complex. Aph-1 are endogenous and of in the of and PS suggest that Aph-1 and form a prior to the assembly of the and active γ-secretase that the PS holoprotein with this the of Pen-2. the evidence for this complex we with NCT, Pen-2, or a The data that the in a of a in 5 and the as and of γ-secretase with The that the of and PS holoprotein is with that with Aph-1 in an stable γ-secretase three co-factors the addition of as the member of the complex and PS T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Here, we demonstrate three the biochemical and assembly of the γ-secretase complex. we a intermediate complex and we that this is of the immature form of that has glycosylation in the and that the prior to the of PS the complex. This interaction with is PS and bind in that bind to the complex a later of we evidence that PS holoprotein to this to form a prior to the addition of and complex we evidence that the physical interaction Aph-1 and in the mature γ-secretase complex be more to than the interactions Aph-1 and PS or Pen-2, supports a protein-protein interaction Aph-1 and that require γ-secretase data about the nature and of assembly the components of the γ-secretase complex. and of the γ-secretase complex revealed the of γ-secretase studies that used of γ-secretase the complex a of H. E. G. A. M. Haass C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google the of a of the known In we have an complex of the components in in cell and In this we as of kDa) and kDa) to the of and we the DDM, in an to the the of the γ-secretase complex and that a complex is Whereas we have a γ-secretase complex this the that γ-secretase in as a complex or be as work be required to this complex The active γ-secretase complex is thought to of the PS heterodimer, a highly glycosylated form of NCT, and H. E. G. A. M. Haass C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar). of glycosylated have been by is known what to the of kDa) the form it has been established that the of the PS heterodimer is required for the of to it has been that the active complex is associated the Here, we for the first that an intermediate appears to form early in γ-secretase assembly and is of Aph-1 and The of this stable intermediate be with endogenous and expressed components and and we have the interaction in cell with evidence that PS is required for the formation and stabilization of mNCT, we PS associated with the This that PS interacts with Aph-1 and of the are for the Aph-1 to and its glycosylation in the and remain associated with γ-secretase Aph-1 be to bind the has been to and Aph-1 in the require The we and evidence about the γ-secretase complex as Whereas Aph-1 with and with a for iNCT, appears to with This that interacts with the in the process of perhaps prior to or the glycosylation of is of a active γ-secretase data are with the recent that of in the unusual stabilization of the PS holoprotein T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This stabilization an half-life of the protein T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with its a stable complex T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar). studies suggest that the of PS holoprotein is to its with the members of the γ-secretase complex and and with this of endogenous Aph-1 or the of the PS holoprotein T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by T. M. M. Thinakaran G. T. PubMed Scopus Google that PS to and a stable complex, and Pen-2, for PS and the glycosylation of Our data are with this the suggest that PS with a intermediate and that PS bind to Aph-1 and Whereas a interaction Aph-1 and PS has been data of and demonstrate that PS holoprotein in a complex with and This of the is by the that of the endogenous in was with endogenous Aph-1 that there be to with PS other than in this we the intermediate the of the γ-secretase complex in the of a that the interaction Aph-1 and in the mature complex require other co-factors for and Our data are with recent in that suggested a protein-protein interaction Aph-1 and NCT, it was suggested that form a stable we have J. Biol. PubMed Scopus Google Scholar). data the of T. M. M. Thinakaran G. T. PubMed Scopus Google suggested that of Pen-2, a complex of NCT, and PS Here, we evidence of this intermediate and that require the of we data that the of this intermediate the stabilization of PS holoprotein through its interaction with Aph-1 and to be an of T. M. M. Thinakaran G. T. PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google appears to of the physiological process of γ-secretase PS is thought to the site of of γ-secretase have the of co-factors to PS it is that γ-secretase co-factors and in the of The later of PS with of its three co-factors during assembly a function, in that the is are there has been stabilization of an the of γ-secretase substrates and the nature and activity of the mature γ-secretase complex, and perhaps be studies have that the of γ-secretase activity are by of other (6Ratovitski T. Slunt H.H. Thinakaran G. Price D.L. Sisodia S.S. Borchelt D.R. J. Biol. Chem. 1997; 272: 24536-24541Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, G. T. Davenport F. Slunt H.H. Price D.L. Borchelt D.R. Sisodia S.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). it has that of PS heterodimer, NCT, and is to the of γ-secretase activity and the of mature PS and Ye W. M.S. Selkoe D.J. A. 100: PubMed Scopus Google Scholar, H. H. Kim Lee Thinakaran G. Kim G. Xu H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and that of components in γ-secretase activity E. Pesold B. H. Haass C. Biol. PubMed Scopus Google Scholar). In the current we that are of the we endogenous with the we that was was there a of that was with that the assembly of the mature γ-secretase complex, of the intermediate are In demonstrate that Aph-1 and form a stable intermediate that later to PS holoprotein and during the assembly of the active γ-secretase complex, the intramembranous proteolysis of signaling proteins. remain to be as γ-secretase co-factors with and the protein-protein that The current data the of γ-secretase assembly are to Gandy for the PS and P. for the and Haass for the P. St. George-Hyslop for the cell and the Aph-1

Gamma-secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N- and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer's disease because it releases the neurotoxic amyloid beta-peptide (Abeta) from its precursor, APP. Gamma-secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2. Because the complex contains at least 18 transmembrane domains, crystallographic approaches to its structure are difficult and remote. We recently purified the human complex essentially to homogeneity from stably expressing mammalian cells. Here, we use EM and single-particle image analysis on the purified enzyme, which produces physiological ratios of Abeta40 and Abeta42, to obtain structural information on an intramembrane protease. The 3D EM structure revealed a large, cylindrical interior chamber of approximately 20-40 A in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, approximately 120-A-long complex within the membrane and revealed approximately 20-A pores at the top and bottom that provide potential exit ports for cleavage products to the extra- and intracellular compartments. Our reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments.

Gamma-secretase is a member of a new class of proteases with an intramembrane catalytic site and cleaves numerous type I membrane proteins, including the amyloid beta-protein precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: a heterodimeric form of presenilin (PS), composed of its N- and C-terminal fragments (PS-NTF and PS-CTF, respectively), a highly glycosylated, mature form of nicastrin (NCT), Aph-1, and Pen-2. However, it is unclear how these components interact physically with each other and assemble into functional complexes. We and others recently found that Aph-1 interacts with a less glycosylated, immature form of nicastrin as an intermediate toward full assembly of gamma-secretase. Here we show that (1) the detergent dodecyl beta-d-maltoside (DDM) mediates the dissociation and inactivation of active gamma-secretase in a concentration-dependent manner, (2) DDM-dependent dissociation of the active gamma-secretase complex generates two major inactive complexes (Pen-2-PS1-NTF and mNCT-Aph-1) and two minor inactive complexes (mNCT-Aph1-PS1-CTF and PS1-NTF-PS1-CTF), and (3) Pen-2 can also associate with the PS holoprotein in complexes devoid of NCT and Aph-1. Taken together, our results demonstrate that Pen-2 interacts with PS-NTF within active gamma-secretase and offer a model for how the components of active gamma-secretase interact physically with each other.