Secretase Complex Reveals Nicastrin and a Substrate

Activity-dependent isolation of the presenilin– ␥-secretase complex reveals nicastrin and a ␥ substrate

William P. Esler*†, W. Taylor Kimberly*†‡, Beth L. Ostaszewski*, Wenjuan Ye*, Thekla S. Diehl*, Dennis J. Selkoe*‡§, and Michael S. Wolfe*§

*Center for Neurologic Diseases, Brigham and Women’s Hospital and ‡Program in Neuroscience, Harvard Medical School, 77 Avenue Louis Pasteur, Boston MA, 02115

Edited by William J. Lennarz, State University of New York, Stony Brook, NY, and approved December 26, 2001 (received for review August 20, 2001) Presenilin heterodimers apparently contain the active site of embryonic development (21, 22). Knockout of PS1 in mice is ␥-secretase, a polytopic aspartyl protease involved in the trans- lethal in utero or soon after birth, with a phenotype similar to that membrane processing of both the Notch receptor and the seen after deletion of Notch1 (23, 24). Cultured cells from amyloid-␤ precursor protein. Although critical to embryonic devel- PS1͞PS2 double knockout mice have no ␥-secretase activity with opment and the pathogenesis of Alzheimer’s disease, this protease respect to either APP or Notch (8, 9), and mutation of either is difﬁcult to characterize, primarily because it is a multicomponent conserved PS aspartate (15, 25, 26) or treatment with ␥-secretase complex of integral membrane proteins. Here the functional inhibitors also prevents Notch proteolysis (20, 26). Moreover, ␥-secretase complex was isolated by using an immobilized active APP and Notch can compete with one another for ␥-secretase site-directed inhibitor of the protease. Presenilin heterodimers and processing (27), and swapping the Notch transmembrane domain nicastrin bound speciﬁcally to this inhibitor under conditions into APP allows efficient formation of A␤-like peptides (J. tightly correlating with protease activity, whereas several other Zhang and D.J.S., unpublished data). presenilin-interacting proteins (␤-catenin, calsenilin, and preseni- Although PSs seem to be the catalytic component of ␥-secre- lin-associated protein) did not bind. Moreover, anti-nicastrin anti- tase, these proteins alone do not show ␥-secretase activity, which bodies immunoprecipitated ␥-secretase activity from detergent- is consistent with other evidence that ␥-secretase is a high solubilized microsomes. Unexpectedly, C83, the major endogenous molecular weight, multiprotein complex (5–7). Identification of amyloid-␤ precursor protein substrate of ␥-secretase, was also these other components, however, has been elusive. Immuno- quantitatively associated with the complex. These results provide affinity purification with PS antibodies led to the identification direct biochemical evidence that nicastrin is a member of the active of nicastrin, a single-pass membrane protein involved in Notch ␥-secretase complex, indicate that ␤-catenin, calsenilin, and pre- signaling in Caenorhabditis elegans and A␤ production in human senilin-associated protein are not required for ␥ activity, and cell lines (28). The role of this protein in ␥-secretase activity is suggest an unprecedented mechanism of substrate–protease not known. Here we report an activity-dependent purification interaction. method for ␥-secretase using an immobilized transition-state analogue inhibitor and show that PS heterodimers, nicastrin, and equential proteolysis of the amyloid-␤ precursor protein an APP ␥-secretase substrate (C83) bind to and elute from this S(APP) by ␤- and ␥-secretases releases the amyloid-␤ peptide matrix under conditions tightly correlating with ␥-secretase (A␤), the major protein component of the cerebral plaques of activity. These results provide compelling biochemical evidence Alzheimer’s disease (1). Mutations in APP and the presenilins that nicastrin is a member of the active ␥-secretase complex and (PSs) PS1 and PS2 cause familial, early-onset Alzheimer’s suggest that substrates for this protease associate with an initial disease, and these mutations increase the production of A␤ in binding site before entry into the active site. ␤ general or the highly fibrillogenic A 42 in particular (2). For these reasons, ␤- and ␥-secretases are considered important Methods therapeutic targets for Alzheimer’s disease (3). ␤-Secretase is a Preparation of Affinity Resins. The compounds WPE-III–31C and membrane-tethered aspartyl protease in the pepsin family (4), WPE-III–112 were synthesized in solution phase by using standard whereas ␥-secretase remains an enigmatic enzyme and has an methods (29). Details are described in Figs. 7 and 8, which are unusual mode of action. This protease processes the transmem- published as supporting information on the PNAS web site, www. brane domain of APP, an otherwise water-excluded region of the pnas.org. The methyl esters were hydrolyzed by using LiOH in substrate, and seems to be a complex of multiple integral aqueous dioxane. The carboxylic acids then were coupled in DMSO membrane proteins (5–7). by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (3 equiv) The polytopic PSs are required for ␥-secretase processing of to the primary amine of a 6-atom hydrophilic linker present on APP (8, 9) and themselves are processed into two pieces, an the agarose resin affi-gel 102 (BioRad). Affi-gel 102 was empir- N-terminal fragment (NTF) and a C-terminal fragment (CTF), ically selected because it showed no background binding to PS1 that remain associated (6, 10). The NTF–CTF complexes are NTF, PS1 CTF, or nicastrin at pH 7.0. The reaction mixtures metabolically stable (11, 12), and their formation is tightly were incubated with continuous gentle inversion for Ͼ12 h. The regulated by unidentified stoichiometric activators (13). Each

subunit possesses one conserved transmembrane aspartate crit- This paper was submitted directly (Track II) to the PNAS ofﬁce. ical to ␥-secretase activity (14–16), and aspartyl protease tran- ␥ Abbreviations: APP, amyloid-␤ precursor protein; A␤, amyloid-␤ peptide; PS, presenilin; sition-state analogue inhibitors of -secretase bind directly and NTF, N-terminal fragment; CTF, C-terminal fragment; PSAP, PS-associated protein. specifically to PS heterodimers (17, 18). Thus, heterodimeric PSs †W.P.E. and W.T.K. contributed equally to this work. apparently comprise an unusual intramembranous active site §To whom reprint requests may be addressed. E-mail: [email protected] or and are among the founding members of a new class of polytopic [email protected]. membrane proteases (19). The publication costs of this article were defrayed in part by page charge payment. This PSs are required also for the transmembrane processing of the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Notch receptor (20), part of a signaling pathway critical for §1734 solely to indicate this fact.

2720–2725 ͉ PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052436599 Downloaded by guest on September 27, 2021 Fig. 1. A (hydroxyethyl)urea peptidomimetic inhibits ␥-secretase and binds the PS–␥-secretase complex. (a) Structures of WPE-III–31C and WPE-III–112. The transition-state mimicking hydroxyethyl moiety is shown in red. (b) In vitro ␥-secretase assays were performed by using CHAPSO-solubilized HeLa cell membranes and the substrates C100Flag and N100Flag in the presence or absence of indicated concentrations of 31C or 112. Shown are M2 anti-Flag Western blots that detect the Flag-tagged substrate (upper bands) and the C-terminal cleavage product that retains the Flag epitope (arrow). Note that C100Flag is highly proneto aggregation, forming SDS-stable oligomers. Also shown are anti-A␤ Western blots using antibody 6E10. (c) Immobilized 31C and 112 were incubated with CHAPSO-solubilized microsomes. Bound or unbound PS1 NTF, PS1 CTF, and nicastrin were detected by Western blotting. (d) Coimmunoprecipitation (Co-IP) using either anti-PS1 NTF antibody X81 (left lanes) or anti-nicastrin antibody R302 (right lanes) from CHAPSO-solubilized HeLa cell microsomes brings down ␥-secretase activity using C100Flag as substrate. Protease activity is blocked by inhibitor 31C in both cases.

resins were washed extensively with DMSO and exchanged into CHAPSO. To measure ␥-secretase activity in Brij-35 eluates, the aqueous buffer. PS complex was immunoprecipitated as described below.

In Vitro ␥-Secretase Assays. Solubilized ␥-secretase was prepared Western Blotting and Immunoprecipitation. For western analysis of essentially as described by Li et al. (7) except that membranes PS1 NTF, PS1 CTF, and nicastrin, the samples were run on were washed in 0.1 M sodium carbonate, pH 11.3, to remove 8–16% Tris-glycine PAGE gels, transferred to polyvinylidene nonintegral membrane proteins before solubilization in 1% difluoride, and probed with Ab14 (for PS1 NTF, 1:2,000), 13A11 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- (for PS1 CTF, 5 ␮g͞ml), and nicastrin antibodies (1:2,500). In propanesulfonate (CHAPSO). To monitor ␥-secretase activity, addition, polyclonal antisera directed against PS2 NTF (2972), the solubilized preparation (at Ϸ0.2 mg of protein͞ml) was PS-associated protein (PSAP), and calsenilin and monoclonal BIOCHEMISTRY incubated with either a Flag-tagged APP-based substrate antibodies to ␤-catenin (BD Transduction Laboratories, Lex- (C100Flag) or Notch-based substrate (N100Flag) at 37°C for 0 ington, KY) and APP (8E5 and 13G8) were used to probe or 4 h. The substrates and C-terminal cleavage products were selected polyvinylidene difluoride membranes. Samples from detected by Western blot using anti-Flag antibody M2 (Sigma) or the ␥-secretase activity assays were run on 4–20% Tris-glycine anti-A␤ antibody 6E10 (Signet Labs, Dedham, MA). The re- gels and transferred to polyvinylidene difluoride. The Flag combinant substrate C100Flag was prepared as described by Li epitope (in both the substrate and CTF generated by ␥-secretase et al. (7). Plasmid encoding N100Flag in vector pET21a(ϩ) proteolysis) was detected by using anti-Flag M2 or anti-A␤ 6E10 (Novagen) was prepared by cloning the Notch sequence from antibodies. PS1 or nicastrin coimmunoprecipitations were per- Val-1711 to Glu-1809 from mouse Notch1 ⌬E (21) using PCR formed by using anti-sera X81 (raised to the first 81 residues of (simultaneously incorporating an N-terminal methionine and a the PS1 NTF) or R302 (raised to the last 12 residues of nicastrin), C-terminal Flag epitope). The BL21(DE3) expression host was respectively, in 1% CHAPSO or 1% Brij-35 and were analyzed transformed with the purified constructs. After a 2-h induction for PS1 NTF and PS1 CTF as described above. Activity measured (1 mM isopropyl ␤-D-thiogalactoside at 37°C), bacteria were after PS1 or nicastrin precipitation was performed in 0.25% lysed in 10 mM Tris, pH 7.0͞200 mM NaCl͞1% Nonidet P-40 by CHAPSO buffer containing 0.05% phosphatidylethanolamine. using a French press. The N100Flag was purified by MonoQ anion exchange chromatography. The protein elutes between Results and Discussion 195 and 240 mM NaCl. Alternatively, the proteins were purified A (Hydroxyethyl)urea Transition-State Analogue Inhibits ␥-Secretase by using an M2 anti-Flag affinity column using 100 mM glycine, Activity and Retains PS1 Heterodimers. Cleavage of the scissile bond pH 2.7, with 1% Nonidet P-40 for elution. by an aspartyl protease proceeds through a transient gem-diol intermediate, or ‘‘transition state,’’ in the substrate. Compound Affinity Chromatography. For batch mode experiments, the solu- WPE-III–31C (Fig. 1a) is a (hydroxyethyl)urea peptidomimetic, bilized ␥-secretase preparation (adjusted to 1% CHAPSO) was a transition-state analogue that mimics this gem-diol intermedi- incubated with compound resin for 2 h at room temperature with ate. This compound is similar structurally to a hydroxyethylene gentle rocking, and the resin was washed twice with several resin transition-state analogue recently reported to inhibit ␥-secretase volumes of buffer (50 mM Pipes or Hepes, pH 7.0͞150 mM activity in vitro and to bind directly to heterodimeric PSs (7, 17). NaCl͞1% CHAPSO). The bound proteins then were released as Compound 31C inhibited A␤ production at the ␥-secretase level described in the text for each given experiment. For preparative in whole cells with an IC50 of 300 nM (data not shown). The scale experiments, 2-ml columns of each resin were prepared and ability of this compound to inhibit cleavage of a Notch-based and run on a Pharmacia AKTA-FPLC chromatography system. an APP-based substrate also was examined in cell-free assays. After sample injection, the columns were washed with 10 column The APP-based substrate, C100Flag, is the endogenous ␥-secre- volumes of binding buffer, and the bound material was eluted in tase substrate C99 plus an N-terminal methionine start site and either 1% Brij-35 or 100 mM glycine, pH 2.7, containing 1% a C-terminal Flag epitope (7). The Notch-based substrate,

Esler et al. PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2721 Downloaded by guest on September 27, 2021 Fig. 2. Disrupting ␥-secretase activity and PS heterodimers prevents binding of the PS complex to the affinity resin. (a) CHAPSO-solubilized ␥-secretase was treated with Triton X-100 (final concentration 2%) and dialyzed into 0.25% CHAPSO buffer to remove Triton. Controls are dialyzed, but lysates are untreated. These preparations were assayed for ␥-secretase activity by using the N100Flag substrate (Left). Li et al. previously showed that Triton disrupts ␥-secretase cleavage of C100Flag (7). PS1 NTF-specific X81 antibody was used to coimmunoprecipitate PS1 CTF from the samples (Center). These samples also were incubated with 31C resin, and the amounts of PS1 NTF, PS1 CTF, and nicastrin bound to the resin were assessed (Right). (b) Solubilized ␥-secretase was heated to 65°C for 0, 10, 30, and 60 min. Samples were assayed for protease activity by using N100Flag (anti-Flag western) or C100Flag (anti-A␤ western) (Left) and for nicastrin binding to the affinity resin (Right).

N100Flag, contains 99 residues of the Notch1 sequence begin- somes (Fig. 1d, left lanes). CHAPSO solubilization not only ning from the ligand-dependent S2 cleavage site and likewise retains ␥-secretase activity but also keeps PS NTF–CTF com- includes an N-terminal methionine and C-terminal Flag se- plexes together (7). Immunoprecipitation with the anti-nicastrin quence. Bicarbonate-washed HeLa cell membranes were solu- antibody R302 (to the C terminus of nicastrin) under these bilized with the detergent CHAPSO and incubated with conditions likewise brought down ␥-secretase activity, as as- C100Flag (7) or N100Flag in the presence or absence of various sessed by using either C100Flag (Fig. 1d) or N100Flag (data not concentrations of inhibitor. Compound 31C inhibited cleavage shown) as substrate. This precipitated activity was blocked fully of both N100Flag and C100Flag in the low nM range (Fig. 1b), by the inhibitor 31C (Fig. 1d), confirming specific proteolysis by as determined by anti-Flag Western blot (N100Flag and ␥-secretase. Anti-PS1 NTF antibodies precipitated PS1 CTF and C100Flag) or anti-A␤ Western blot (C100Flag). We also exam- nicastrin, and antinicastrin antibodies brought down PS1 NTF ined the inhibitory activity of a control compound, WPE-III–112 and CTF under these same conditions (data not shown). To- (Fig. 1a), which is of similar structure to 31C and has the same gether, these findings implicate nicastrin as a member of the empirical formula but lacks the transition-state mimic. In con- active ␥-secretase complex along with PS NTF and PS1 CTF (see trast to 31C, the control peptide 112 did not inhibit cleavage of also below). N100Flag or C100Flag (Fig. 1b) at all concentrations tested (up to 1 ␮M). Binding of PS1 Heterodimers and Nicastrin to the Inhibitor Matrix Inhibitor 31C was covalently linked to a solid support suitable Requires Active ␥-Secretase. To probe the specificity of the inter- for use in affinity chromatography to enrich for the active action of the affinity resin with PS1 NTF, PS1 CTF, and nicastrin ␥-secretase complex. The compound was covalently linked to the further, we reasoned that pretreatments that disrupt enzymatic free primary amine of a 6-atom hydrophilic spacer on an agarose activity should interfere also with binding to the resin. We first affinity support (Affi-gel 102, BioRad). To control for nonspe- explored treatment with the detergent Triton X-100, which is cific hydrophobic interactions between the immobilized com- known to eliminate ␥-secretase activity (7) and dissociate PS1 pound and the proteins of interest, we also prepared a resin with heterodimers (6). We therefore treated a solubilized ␥-secretase the inactive 112. We then evaluated their ability to deplete PS1 preparation with 2% Triton for 20 min. Because this detergent NTF, PS1 CTF, and nicastrin from a CHAPSO-solubilized also could alter hydrophobic interactions, we removed the Triton HeLa microsome preparation that retains ␥-secretase activity. by exchanging it with CHAPSO through extensive dialysis. As a The ␥-secretase preparation was incubated with the affinity control, a similar sample was treated identically except that no resins in batch format, and the PS1 NTF, PS1 CTF, and nicastrin Triton detergent was added. To document the effects of Triton, remaining in the supernatant after incubation (‘‘unbound’’) were we first tested the two preparations for ␥-secretase activity and detected via Western blotting. The resins were washed exten- the ability to coimmunoprecipitate PS1 heterodimers. Treat- sively, and bound proteins were released with Laemmli sample ment with Triton irreversibly disrupted ␥-secretase activity, buffer. PS1 NTF, PS1 CTF, and nicastrin released from each whereas the control preparation retained the ability to cleave resin (‘‘bound’’) were detected likewise. At a loading concen- N100Flag (Fig. 2a Left). To confirm that the detergent dissoci- tration of 5 mg of 31C͞ml of resin, PS1 NTF, PS1 CTF, and ated the PS complex, we coimmunoprecipitated PS1 by using an nicastrin were retained quantitatively on the resin (Fig. 1c). In N terminus-specific antibody (X81). Although X81 immunopre- clear contrast, PS1 NTF, PS1 CTF, and nicastrin remained cipitated PS1 NTF under both conditions, PS1 CTF was coim- completely unbound in the presence of 112 resin (Fig. 1c). This munoprecipitated only in the CHAPSO control (Fig. 2a Center). striking difference strongly supports a specific inhibitor-active Therefore, these two preparations are identical except that the site interaction on the 31C matrix that is required for binding of PS1–␥-secretase complex is disrupted in the Triton-treated PS1 NTF, PS1 CTF, and nicastrin. sample. Incubating the two samples with 31C resin demonstrated The finding that nicastrin specifically associated with the that only active, complexed ␥-secretase can bind (Fig. 2a Right); immobilized transition-state analogue prompted us to ask PS1 NTF, PS1 CTF, and nicastrin bound only in the control whether immunoprecipitation with anti-nicastrin antibodies sample. could bring down ␥-secretase activity. As reported previously by We also considered heating as a method to test specificity of Li et al. (7), ␥-secretase activity was precipitated with anti-PS1 binding to the resin. When the solubilized ␥-secretase prepara- NTF antibodies from CHAPSO-solubilized HeLa cell micro- tion was heated at 65°C for 10, 30, and 60 min, a time-dependent

2722 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052436599 Esler et al. Downloaded by guest on September 27, 2021 Fig. 3. Conditions that abolish ␥-secretase activity elute the PS complex from the affinity resin. (a) CHAPSO-solubilized cell membranes were diluted with 50 mM phosphate buffer to alter the pH as indicated and examined for ␥-secretase activity (Upper). Solubilized ␥-secretase at pH 7.0 was incubated with the affinity resin. PS1 NTF, PS1 CTF, and nicastrin eluted at the indicated pHs (Lower). The pH dependence of C100Flag cleavage has been reported (7). Some proteins nonspecifically associate to the resin at pH 2.7 (data not shown); apparently, nicastrin likewise associates nonspecifically at this pH, accounting for its diminished release. (b) In vitro ␥-secretase assays monitored cleavage of N100Flag (or C100Flag, data not shown) in the presence of various detergents (at 1%; Upper). Elution of the PS complex from the affinity resin was examined also (Lower). Solubilized ␥-secretase was incubated with affinity resin, and the ability of detergents (at 1%) to elute PS1 NTF, PS1 CTF, and nicastrin was determined.

loss in N100Flag cleavage (␥-secretase activity) was observed consistent with an interaction as a complex, and correlated with (Fig. 2b Left). A parallel loss in A␤ production from C100Flag the presence of ␥-secretase activity. also was seen (Fig. 2b Left). We then took the same preparations We found that of the five conditions capable of eluting PS1 and tested for binding to 31C resin. Because extended heating and nicastrin from the inhibitor matrix, only Brij-35 substantially aggregates PSs (W. Xia, unpublished observations), we moni- retained the association of PS1 NTFs and PS1 CTFs as deter- tored only nicastrin binding after this treatment. In agreement mined by coimmunoprecipitation with X81 (Fig. 4a). Because with its effect on ␥-secretase activity, heating reduced the ability 1% Brij-35 is incompatible with our N100Flag or C100Flag of nicastrin to bind the affinity matrix in a time-dependent assays (Fig. 3b), we coimmunoprecipitated ␥-secretase com- manner (Fig. 2b Right). The greater the loss in enzymatic activity, plexes from the Brij-35 eluate off a preparative 31C column and the greater was the reduction in nicastrin binding to the resin. washed the beads with CHAPSO to exchange it into optimal Thus, ␥-secretase activity correlates with binding to 31C resin, detergent. After the addition of either N100Flag or C100Flag, confirming the specificity of this matrix for active ␥-secretase the coimmunoprecipitated material showed clear ␥-secretase complexes. activity (Fig. 4b), demonstrating that the active ␥-secretase

complex could be isolated from the inhibitor matrix. Interest- BIOCHEMISTRY Alterations That Disrupt ␥-Secretase Activity Can Elute the Bound PS ingly, Brij-35 was reported to support ␥-secretase activity in Complex from the Affinity Resin. Because we found a tight corre- microsomes isolated from APP-transfected cells that had been lation between ␥-secretase activity and binding to the immobi- pretreated with a ␥-secretase inhibitor (30). Thus, A␤ produced lized inhibitor, we attempted to identify methods for specific in this assay was generated from cell-derived substrate. Brij-35 elution off the resin. Li et al. (7) have demonstrated that ␥-secretase cleavage of C100Flag has a broad pH range with an optimum of ϷpH 7.0. N100Flag proteolysis displayed a similar pH optimum and no detectable proteolysis at pHs 2.7 and 12.0 (Fig. 3a Upper). To assess the ability of these different pHs to elute proteins off the affinity resin, CHAPSO-solubilized HeLa microsomes were incubated with the immobilized inhibitor at pH 7.0 and then washed extensively. The resin subsequently was exchanged into phosphate buffers adjusted to the various pHs. Only the pHs that eliminated ␥-secretase activity completely, 2.7 and 12.0, were able to elute PS1 NTF, PS1 CTF, and nicastrin (Fig. 3a Lower). We also examined the effect of several detergents on ␥-secretase activity and their ability to elute the PS complex from the affinity matrix. A CHAPSO-solubilized ␥-secretase preparation was spiked with various detergents, and the in vitro cleavage of N100Flag (Fig. 3b Top) and C100Flag (data not shown) were analyzed. The presence of 1% Triton X-100, Nonidet P-40, or Brij-35 abolished ␥-secretase cleavage of both substrates, Fig. 4. The PS complex and ␥-secretase activity can be eluted from the whereas treatment with CHAPS or BIGCHAP [N,N-bis(3-D- afﬁnity resin. (a) Coimmunoprecipitates (Co-IP) were used to determine gluconamidopropyl)cholamide] had little or no effect relative to whether elution conditions disrupt PS heterodimers. CHAPSO-solubilized cell the CHAPSO-treated control. When we examined the ability of membranes were treated as indicated and then immunoprecipitated with X81 these five detergents to elute PS1 NTF, PS1 CTF, and nicastrin (to PS1 NTF). Precipitated PS1 NTF and PS1 CTF were detected by Western blotting. NP40, Nonidet P-40. (b) Solubilized ␥-secretase was passed over a 31C from the affinity resin, only those detergents that disrupted ␥ afﬁnity column, washed in binding buffer, and eluted in 1% Brij-35. The PS -secretase activity (Triton, Nonidet P-40, and Brij-35) eluted complex was coimmunoprecipitated with X81 and exchanged into 0.25% the PS complex (Fig. 3b Bottom). In every case, for both the pH CHAPSO. This material was compared with the starting material in ␥-secretase alterations (Fig. 3a) and the detergent treatments (Fig. 3b), assays using N100Flag and C100Flag in the presence (ϩ) or absence (Ϫ)of release from the affinity matrix was similar for all three proteins, ␥-secretase inhibitor 31C.

Esler et al. PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2723 Downloaded by guest on September 27, 2021 Fig. 5. Preparative-scale affinity isolation and characterization of the components of the ␥-secretase complex. (a) Solubilized ␥-secretase was passed over a 31C affinity column and eluted with glycine, pH 2.7 (or Brij-35, data not shown). In the UV chromatogram, the first arrow is the injection point, the first (larger) peak is the flow-through, the second arrow is the start of the elution, and the second (smaller) peak corresponds to the proteins eluted from the column. Western blotting demonstrates that essentially all the PS1 NTF, PS1 CTF, and nicastrin were bound to the column and eluted in the fractions corresponding to theUV elution peak. The area under the UV elution peak corresponds to 5% of the total area. (b) The flow-through and elution fractions from the 31C column or a similarly run 112 column were collected and probed for the presence of the indicated proteins using Western blotting.

apparently interferes with the initial interaction of a recombi- tates inefficiently with PS1 in endoplasmic reticulum-enriched nant substrate with the enzyme but does not affect a preas- vesicles (33). However, essentially all the C83 APP CTF (the sole sembled enzyme-substrate complex. Nevertheless, in our hands APP ␥-secretase substrate detectable in HeLa cells) was re- the active ␥-secretase complex clearly is maintained in Brij-35, tained on the affinity resin and coeluted with PS1 and nicastrin. because column elution and immunoprecipitation out of this This result agrees with prior work that showed that C83 and C99 detergent isolated the protease activity (Fig. 4b). coimmunoprecipitated with PS1, in particular in subcellular fractions that exhibit in vitro ␥-secretase activity (33). In this Partial Characterization of the Enriched ␥-Secretase Complex. Several earlier study, our coimmunoprecipitations were not quantitative; proteins have been proposed to be PS-interacting proteins. To however, they were not carried out in the presence of a ␥-secre- ␥ test whether these proteins are involved in the PS– -secretase tase inhibitor and were performed before the discovery of complex, we performed a preparative-scale affinity separation conditions ideal for isolation of the active ␥-secretase complex. by using 31C or 112 resins and probed for the presence or Thus, differences in isolation conditions likely account for the absence of these other PS binding partners. Using a column current observation that C83 is recovered quantitatively from format, the majority of all protein (as monitored by UV280 the immobilized 31C column. ␥ absorbance) from a CHAPSO-solubilized -secretase prepara- Why would a substrate and enzyme copurify after inhibitor tion passed through the 31C column without binding. After affinity chromatography? Because the affinity resin binds to the thorough washing, the bound material was eluted by using acidic active site of ␥-secretase (17, 18), we interpret the invariant glycine (Fig. 5a) or 1% Brij 35 (data not shown). In both cases, presence of C83 as evidence for a two-step mechanism for a single UV elution peak was observed. To determine the elution substrate–protease interaction. Specifically, we propose the ex- profiles of PS1 NTF, PS1 CTF, and nicastrin, the starting istence of an initial binding site for the substrate on the surface material and all the fractions were Western-blotted (Fig. 5a). of the ␥-secretase complex before its entry into the active site. Very little PS1 NTF, PS1 CTF, and nicastrin were detected in the This hypothesis makes particular sense in the two-dimensional flow-through peak (fractions 1–5), indicating that these proteins context of a lipid bilayer, in which the water-containing active were almost quantitatively bound to the column. These three site of the protease should be sequestered from the hydrophobic proteins eluted from the column in parallel and were found environment. The substrate first must contact the outer surface primarily in fractions 10–11, corresponding to the elution peak of the complex before its entry into and cleavage at the active site detected by UV. Consistent with the results in Fig. 1c, when the 112 column was run under identical conditions, all the PS1 NTF, PS1 CTF, and nicastrin were found in the flow-through fractions (Fig. 5b). To identify other candidates in the PS–␥-secretase complex, fractions corresponding to the flow-through and elution peaks were probed for the presence or absence of proteins proposed to be PS-interacting partners (Fig. 5b). Although there are many candidates that interact with PS1, we focused on several lead candidates including APP holoprotein, C83, ␤-catenin, PSAP, and calsenilin. ␤-Catenin, PSAP, and calsenilin were not retained by the immobilized inhibitor. Indistinguishable results were obtained by using glycine or Brij-35 for elution. These results suggest that these candidates are not required for activity, ␥ although they may have other indirect or modulatory roles. PS2 Fig. 6. A model for the interaction of -secretase with its substrate. The ␥-secretase complex apparently includes PS NTF (blue), PS1 CTF (green), and NTF, however, was quantitatively bound, which is consistent nicastrin (orange). The water-containing active site in PS is sequestered from with the fact that PS2 is a functional homolog of PS1 and is the hydrophobic membrane lipids, whereas substrate C83 or C99 (red) can associated with proteolytic activity (16, 31, 32). Only a very small move only in two dimensions within the conﬁnes of the bilayer (step 1). The amount of full-length APP associated with the complex, which substrate ﬁrst interacts on the outer surface of the protease (step 2) before is in agreement with previous results that it coimmunoprecipi- accessing the active site (step 3).

2724 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.052436599 Esler et al. Downloaded by guest on September 27, 2021 (Fig. 6). The presence of the immobilized inhibitor in the active conclude that the ␥-secretase complex apparently contains PS1 site therefore would stabilize the C83 substrate at the initial NTF and PS1 CTF, nicastrin, and one or more additional binding site. This idea is corroborated by evidence that C83 and proteins. It is likely that there is at least one other critical C99 coimmunoprecipitated with wild-type PS1 using lysates member of the complex, because overexpression of nicastrin and from cells pretreated with transition-state analogue inhibitors PS1 neither generates increased PS1 NTF and PS1 CTF nor (33). In these coimmunoprecipitation experiments, however, augments ␥-secretase activity (28). The unknown stoichiometric C83 and C99 levels were increased pharmacologically by treating activator(s) of PS endoproteolysis are expected to remain a part the cells with inhibitors. We show here that endogenous levels of of the PS complex, because these factors otherwise would C83 copurify with PS heterodimers and nicastrin, suggesting that continue to allow PS heterodimer formation (i.e., overexpressed this is a biologically relevant interaction and not simply a result PS would lead to an increased level of NTF–CTF complexes, of elevated protein levels. In contrast, full-length APP appar- which is not observed). The identification of these unknown ently does not interact with the ␥-secretase complex efficiently partners must await complete purification of the ␥-secretase under these conditions, perhaps because the large APP ectodo- complex. Further work toward the purification of this enzyme main sterically interferes with binding. Identifying compounds will yield additional insights into its function and should reveal that block the initial substrate binding site on the ␥-secretase new targets for the treatment and prevention of Alzheimer’s complex might be an alternative means of inhibiting this impor- disease. tant therapeutic target. The present study also confirms the importance of nicastrin, We thank J. Vandrovec for preparation of the N100Flag expression host, ⌬ providing direct biochemical evidence that nicastrin is associated R. Kopan for mouse Notch1 E, S. Gandy for Ab14, P. St. George- with the active ␥-secretase complex in wild-type (nontrans- Hyslop and D. Miller for antibodies to nicastrin, C. Haass for 2972, X. Xu for PSAP antibody, W. Wasco for calsenilin antibodies, and P. fected) human cells. The presence of protease activity correlated Seubert and D. Schenk for 8E5 and 13G8. We also thank D. Walsh, M. tightly with the binding of PS1 and nicastrin to the immobilized LaVoie, and W. Xia for helpful discussions. This work was supported by inhibitor. Conversely, conditions that abolished activity released National Institutes of Health Grants AG 17574, NS 41355 (to M.S.W.), bound PS1 heterodimers and nicastrin from the resin. Moreover, and AG 12749 (to D.J.S.) and a Pioneer Award (to D.J.S.) and a New anti-nicastrin antibodies precipitated ␥-secretase activity. We Investigator Award (to W.P.E.) from the Alzheimer’s Association.

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