Peptides based on the presenilin-APP binding ... - The FASEB Journal

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Peptides based on the presenilin-APP binding domain inhibit APP processing and A␤ production through interfering with the APP transmembrane domain Cary Esselens,*,§ Ragna Sannerud,*,§ Rodrigo Gallardo,‡,储 Veerle Baert,*,§ Daniela Kaden,¶ Lutgarde Serneels,†,§ Bart De Strooper,†,§ Frederic Rousseau,‡,储 Gerd Multhaup,¶,# Joost Schymkowitz,‡,储 Johannes P. M. Langedijk,**,1 and Wim Annaert*,§,2 *Laboratory for Membrane Trafficking and †Laboratory for the Research of Neurodegenerative Diseases, Center for Human Genetics, and ‡Department of Molecular Medicine, Katholieke Universiteit Leuven, §Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, and 储VIB Switch Laboratory, Gasthuisberg, Leuven, Belgium; ¶Institut für Chemie und Biochemie, Freie Universität, Berlin, Germany; #Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada; and **Pepscan Therapeutics, Lelystad, The Netherlands Presenilins (PSENs) form the catalytic component of the ␥-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid ␤ (A␤) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and A␤ generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered A␤ production (IC50: 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct ␥-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect Notch⌬E ␥-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of ␥-secretase, most likely through steric hindrance.—Esselens, C., Sannerud, R., ABSTRACT

Abbreviations: A␤, amyloid ␤; APP, amyloid precursor protein; APPFL, full-length amyloid precursor protein; APPSwe, amyloid precursor protein with Swedish mutation; COS, CV-1 origin SV-40; CTF, carboxy-terminal fragment; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; ECL, electrochemiluminescence; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol tetraacetic acid; FAD, familial Alzheimer’s disease; FTIR, Fourier transform infrared; HBTU, 2⬙-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HEK, human embryonic kidney; KLH, keyhole limpet hemocyanin; MEF, mouse embryonic fibroblast; MES, 2-(N-morpholino)ethanesulfonic acid; Notch⌬E, Notch with deleted ectodomain; NTF, aminoterminal fragment; pAb, polyclonal antibody; PSEN, presenilin; sAPP, secreted APP; SDS-PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; WT, wild type 0892-6638/12/0026-3765 © FASEB

Gallardo, R., Baert, V., Kaden, D., Serneels, L., De Strooper, B., Rousseau, F., Multhaup, G., Schymkowitz, J., Langedijk, J. P. M., Annaert, W. Peptides based on the presenilin-APP binding domain inhibit APP processing and A␤ production through interfering with the APP transmembrane domain. FASEB J. 26, 3765–3778 (2012). www.fasebj.org Key Words: Alzheimer’s disease 䡠 amyloid precursor protein 䡠 Notch-sparing 䡠 viral translocation peptide Most mutations associated with familial early onset Alzheimer’s disease are found in the gene encoding presenilin 1 (PSEN1; refs. 1–3). PSEN1 forms the catalytic subunit of a tetrameric ␥-secretase complex and is thereby directly involved in the final proteolytic step liberating amyloid ␤ (A␤) peptides from the amyloid precursor protein (APP; ref. 4). As such, PSENs have been catching a lot of attention since their discovery in 1995 and are still considered a major therapeutic target in A␤-lowering strategies (5). Two aspartate residues (Asp257 and Asp384 in the human sequence) in transmembrane regions 6 and 7 are critical for catalytic activity of PSEN1 (6, 7). Second, ␥-secretase cleavage of type I transmembrane proteins such as APP and Notch occurs within their transmembrane domain and hence in the plane of the lipid bilayer. Because cleavage of a peptide bond requires water molecules, these catalytic residues are facing a 1

Current address: Crucell, Leiden, The Netherlands. Correspondence: Laboratory for Membrane Trafficking, Center for Human Genetics (KULeuven) and VIB Center for the Biology of Disease, O&N4, Rm 7.159, Gasthuisberg, B-3000 Leuven, Belgium. E-mail: [email protected] doi: 10.1096/fj.11-201368 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

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water-accessible cavity in the transmembrane environment of PSENs (8, 9). Furthermore, as substrates are themselves confined to lateral movement within the lipid bilayer, proteolytic cleavage is preceded by docking of the substrate on the ␥-secretase complex. This appears to occur on a site distinct from the catalytic site, as initially suggested through the identification of the distinct PSEN1-binding domains for APP (1). Further evidence came from the experiments demonstrating that the APP substrate could be coeluted with ␥-secretase components on affinity isolation with an immobilized active site inhibitor (10). From a therapeutic point of view, the presence of a spatially distinct docking or binding site for APP on PSEN1 can now be exploited, in particular if the docking characteristics are distinct for the different ␥-secretase substrates. After all, compounds that interfere with the catalytic site of PSEN/␥-secretase have a higher chance to not only affect A␤ production but also to interfere with the cleavage of an increasing number of substrates, such as Notch (11), syndecan-3 (12), N-cadherins (13), ErbB-4 (14), and CD44 (15, 16). For several substrates, it has been demonstrated that this intramembrane proteolysis is required for subsequent downstream signaling. In the case of Notch, interference with its normal proteolysis has significant consequences for physiological processes such as neuronal migration (17), hematopoiesis (18), and later in life tumor suppression (19). In PSEN1, we previously identified 2 binding domains (1), namely, the first transmembrane domain and the C terminus. The corresponding PSEN1 binding domain in APP encompassed the second half of the transmembrane domain downstream of the ␥40 secretase cleavage site, covering many critical residues mutated in familial Alzheimer’s disease (FAD). In this study, these binding domains were used to design peptide-based inhibitors. We provide evidence that peptides mimicking the PSEN1 binding domain in APP selectively interfere with cleavage of APP but not, for instance, with Notch. The combination of biophysical and cell biological data allow us here to provide a working model in which such peptides interfere with APP processing, likely through direct binding to its cognate transmembrane domain in endosomes.

MATERIALS AND METHODS Antibodies The following rabbit polyclonal antibodies were made in house. B7/8 was raised against the N terminus of A␤ (20) and was used to immunoprecipiate A␤ from conditioned media or cell-free assays. B63.1 was generated using a synthetic peptide mimicking the 16 most C-terminal amino acids of APP (NGYENPTYKFFEQMQN) coupled to keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL, USA). B19.2 and B32.1 are directed against the murine PSEN1-amino-terminal fragment (NTF; peptide-antigen residues 32– 46, SQERQQQHGRQRLDN) and PSEN1-carboxy-terminal fragment (CTF; 3766

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peptide–antigen residues 310 –330, PKNPKYNTQRAERETQDSGSG), respectively (1, 21, 22). PSEN1-knockout brain tissue was used to confirm the specificity of PSEN1 affinitypurified polyclonal antibodies. Shed or soluble APP was immunoprecipitated using a goat polyclonal antibody (pAb) against the extracellular domain of APP (GB207) and was kindly provided by B. Greenberg (Cephalon, Frazer, PA, USA). A␤ peptides were detected using mab WOII (Millipore, Bedford, MA, USA), directed against residues 4 –10 of human A␤. 2E9 antibody detects syndecan 3 and was provided by J. Schulz and G. David (Katholieke Universiteit Leuven, Leuven, Belgium). The ␥-secretase inhibitor L685,458 was purchased from Calbiochem (San Jose, CA, USA). Monoclonal antibody (mAb) 6E10 to APP was from Signet Laboratories (Dedman, MA, USA); mAb to EEA1 (clone 14) was from BD Bioscience (San Jose, CA, USA). Peptide synthesis Peptides were selected from the APP and PSEN binding domains and synthesized alone or in fusion with the Erns-based translocation peptide (23), palmitoylated or biotinylated. An unrelated control peptide was also synthesized. Peptides were synthesized on an Applied Biosystems 430A synthesizer (Life Technologies Corp., Carlsbad, CA, USA) according to standard procedures or on a Hamilton Microlab 2200 (Hamilton, Reno, NV, USA) using Fmoc/2⬙-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) chemistry (24). After removal of the final Fmoc group, peptides can be biotinylated using 0.45 M activated biotin (15 min in 0.45 M HBTU/1-hydroxybenzotriazole in N,N-dimethylformamide). The reaction was stopped after 1 h by washing 5 times with N-methyl-2-pyrrolidone and 3 times with ethanol. Peptides were purified by reversed phase HPLC. The purity of the peptides was assessed by analytical liquid chromatography/mass spectrometry. Lyophilized peptides were reconstituted in dimethyl sulfoxide (DMSO), portioned into aliquots of 10 mM stocks, and kept at ⫺20°C. Working concentrations were calculated never to exceed 5% DMSO (v/v). Cell-free ␥-secretase assay A bacterial expression plasmid containing the cDNA for APP-C99, including a FLAG-tag, was transformed in Escherichia coli. Induction of this strain results in expression of the recombinant protein. Bound recombinant APP-C99 was eluted from the anti-FLAG column, dialyzed, concentrated, and portioned into aliquots before use in the cell-free assay. Recombinant APP-C99 was mixed with cleared CHAPS extracts from HeLa cells obtained as follows. HeLa cells were harvested and centrifuged. The cell pellet was resuspended in 250 mM sucrose, 5 mM Tris-HCl (pH 7.4), and 1 mM ethylene glycol tetraacetic acid (EGTA) supplemented with protease inhibitors and homogenized using a ball-bearing cell cracker (10 passages, clearance 10 ␮m). After low-speed centrifugation (800 g, 10 min), the postnuclear supernatant was ultracentrifuged (100,000 g, 1 h). The resulting microsomal pellet was washed twice in 0.02% saponin; resuspended in 5 mM Tris and 1 mM ethylene diamine tetraacetic acid (EDTA; pH 7) containing 0.5% CHAPS, and incubated overnight at 37°C with or without peptides. The ␥-secretase inhibitor acted as positive control, and DMSO (solvent) was the negative control. De novo produced A␤ was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA, USA) in 2-(N-morpholino)ethanesulfonic acid (MES) running buffer, followed by Western blotting using WOII (6). Visualiza-

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ESSELENS ET AL.

APP and Notch with deleted ectodomain (Notch⌬E) were cloned into the EcoRV site in front of Gal4VP16 in the pIPAdApt vector. HeLa cells were transfected with 200 ng pFRluc plasmid (UAS-responsive luciferase construct; Stratagene, La Jolla, CA, USA) and 200 ng inducer plasmid using FuGene 6 (Promega, Madison, WI, USA). After 24 h, cells were incubated with or without ␥-secretase inhibitors and after 16 h were lysed and assayed for luciferase activity (Victor2; PerkinElmer, Wellesley, MA, USA). All graphs in this study show means ⫾ se, and differences were analyzed by unpaired, 2-tailed Student’s t test. Values of P ⬍ 0.05 were considered significant, P ⬍ 0.01 highly significant, and P ⬍ 0.001 very highly significant.

from Ni-NTA columns to transfer into eluent buffer and to adjust samples to concentrations of 1 ␮g/ml. C100 aliquots (60 –100 ␮l) were injected at a flow rate of 30 ␮l/min. The integrity of the chip surface charged with C100 (before and after the peptide measurements) was checked by injection of 30 – 60 ␮l of W0-2 antibody solutions (The Genetics Company, Basel, Switzerland) diluted in eluent buffer to 1 ␮g/ml at a flow rate of 20 ␮l/min. The peptides, D7, D7 reverse, and scram, were dissolved in DMSO to a concentration of 1 mM. Before each measurement, DMSO as a control and the peptides were diluted 1:1000 (1 ␮M) in eluent buffer and vortexed immediately. The final concentrations (50 to 125 nM) in eluent buffer were measured immediately to prevent aggregation. Solutions (60 ␮l) were injected at a flow rate of 20 ␮l/min, followed by a 2.5-min dissociation phase in buffer, a regeneration step at 30 ␮l/min using 0.5% SDS for 1 min, and an additional 2 min regeneration in buffer before the next measurement. SDS was used to completely remove the peptides or W0-2 from the C100 surface. Since the C100 is present in SDS micelles from purification (25), the washing step does not negatively influence the integrity of the surface, as tested by W0-2 antibody.

Cell-based immunoprecipitation assay

Dynamic light scattering (DLS)

Human embryonic kidney (HEK) 293 cells with stably integrated APP with Swedish mutation (APPSwe; resulting in higher A␤ production) were labeled with 35S-methionine for 3 h in the presence of peptide at 1 and 2 ␮M concentration. Subsequently, full-length APP (APPFL) and APP-CTFs were immunoprecipitated from the cell lysate using pAb B63.1. A␤ peptides and soluble APP (originating from both ␣- and ␤-cleavages) were immunoprecipitated from the conditioned media using pAb B7/8 and Greenberg antibody, respectively. Bound metabolically labeled proteins were eluted from beads and loaded on SDS-PAGE. Dried gels were analyzed using phosphorimaging (Typhoon; Molecular Dynamics, Sunnyvale, CA, USA), and radioactive bands were quantified using ImageQuant software (GE Healthcare, Little Chalfont, UK).

DLS measurements were made at room temperature with a DynaPro DLS plate reader instrument (Wyatt, Santa Barbara, CA, USA) equipped with a 830-nm laser source. Samples (100 ␮l, PBS buffer, 2 mM peptide) were placed into a flat-bottom 96-well microclear plate (Greiner, Frickenhausen, Germany). The autocorrelation of scattered light intensity at a 90° angle was recorded for 10 s and averaged over 40 recordings to obtain a single data point. The Wyatt Dynamics software was used to calculate the hydrodynamic radius by assuming a spherical particle shape.

tion of the signals was done via Western lightning electrochemiluminescence (ECL; Amersham, Piscataway, NJ, USA) or alternatively, for the dose-response curves, by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, ME, USA), using an infrared detectable secondary antibody. Cell-based ␥-secretase luciferase assay

Pulldown assay with biotinylated D7 Mouse embryonic fibroblast (MEF) wild-type (WT), MEF PSEN1/2⫺/⫺, or syndecan-3-expressing MEF cells were scraped and resuspended in sodium-Tris-EDTA buffer, pH 7.4 (cells were cracked with 10 passages through a cell cracker, clearance: 10 ␮m; EMBL, Heidelberg, Germany). After nuclei were spun down (800 g, 10 min), the postnuclear supernatant was subjected to ultracentrifugation (100,000 g, 1 h). The resulting membrane pellet was extracted with 125 mM NaCl and 1% TX-100. Extracts (1 ␮g/␮l) were incubated overnight with different concentrations (2– 0.5 ␮M) of biotinylated D7, followed by incubation with streptavidin Sepharose beads and analysis of the bound fraction by Western blotting. Surface plasmon resonance (SPR) SPR experiments were performed using a BIAcore 3000 instrument and NTA sensor chip (NIHC 200 m; Xantec Bioanalytics, Duesseldorf, Germany) at a constant temperature of 25°C. The sensor chip was charged with 20 ␮l of 500 ␮M NiCl2 in eluent buffer (0.01 M HEPES, 0.15 M NaCl, 1 ␮M EDTA, and 0.005% surfactant P20, pH 7.4). The dispenser buffer consisted of eluent buffer with 3 mM EDTA. C100 was purified as described previously (25). Amicon microcentrifugal tubes (10 kDa cutoff; Millipore) were used as buffer-exchange devices for purified C100 protein eluted

Fourier transform infrared (FTIR) spectroscopy Peptides were dissolved to 1 mM in PBS⫺/⫺, and solutions were applied to the FTIR spectroscopy sample holder. Infrared spectra were recorded on a Bruker Tensor 27 infrared spectrophotometer (Bruker Optik, Ettlingen, Germany) equipped with a Bio-ATR II accessory. Spectra were recorded at a spectral resolution of 4 cm⫺1, and 120 accumulations were performed per measurement. FTIR spectra were recorded every 5 min in situ at a wavelength range from 900 to 3500 cm⫺1. The obtained spectra were baseline subtracted and rescaled in the amide I area, which spans from B1600 to B1700 cm⫺1. Cell surface biotinylation and internalization assay Cell surface biotinylation and internalization assays were performed as described by Sannerud et al. (26). Briefly, HEK cells were plated on poly-l-lysine dishes and starved. HEK cells were biotinylated with sulfo-NHS-SS-biotin, and for internalization the remaining biotin at the cell surface was cleaved off with 2-Na-2-mercaptoethanesulfonate. Finally, cells were extracted in lysis buffer. Total protein was measured, and biotinylated proteins were pulled down from equal amounts of extracts using streptavidin Sepharose beads, loaded on SDS-PAGE, and processed for Western blotting and immunodetection. Confocal microscopy HeLa cells were plated on glass coverslips and cotransfected with pCDNA Cherry-APP-YFP (26) and pCDNA Rab5Q79L

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(expressing the GTP-locked dominant active Rab5) using FuGene 6 according to the manufacturer’s protocol. Cells were further incubated with control DMSO or 1 ␮M D7-biotin for the indicated times. After fixation with 4% paraformaldehyde (10 min) and permeabilization with 0,1% TX-100 in PBS (10 min), D7-biotin was detected using streptavidin-Alexa 647 (Invitrogen). Coverslips were mounted in Mowiol (Sigma, St. Louis, MO, USA), sealed with nail polish, and analyzed on a Radiance 2100 (Carl Zeiss Microimaging, Oberkochen, Germany) connected to an Eclipse E800 Nikon upright microscope (Nikon, Tokyo, Japan) and using an oil-immersion plan Apo ⫻60 A/1.40 NA objective lens. For super-resolution PiMP imaging (27), a stack of 100 images was taken in 3 colors and processed in ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA) using the PiMP macro with a set bleach rate of 5%. Sequential images were corrected for X-Y drift using the StackReg plug-in (28). Cell-based dimerization assay After a 6 h preincubation with peptide, CV-1 origin SV-40 (COS) cells were transfected with pSG5-APP695 using FuGene 6 according to the manufacturer’s protocol. Cells were lysed in PBS with 2% CHAPSO and Complete protease inhibitor (Roche, Indianapolis, IN, USA). Cell extracts were loaded on SDS-PAGE under strict nonreducing conditions and after separation transferred to nitrocellulose. APP mono- and dimers were detected with B63 antibody and quantified with AIDA software (Raytest, Straubenhardt, Germany).

RESULTS Screening for peptide inhibitors using a cell-free assay Peptides mimicking PSEN1 and APP binding domains were synthesized and tested for A␤ inhibition in a cell-free assay. Several peptides, derived from PSEN1 as well as APP binding domains, exerted a moderate inhibitory effect, but only at the highest doses tested (100 and 250 ␮M, Fig. 1B, C), which renders them inappropriate for eventual therapeutic use. Most likely, the ␥-cleavage within the plane of the lipid bilayer lowers the accessibility of the peptides to the catalytic and/or docking site environments of the target proteins. To achieve a more efficient delivery to the transmembrane target domains, we linked our binding domain peptides to a so-called translocation peptide. The prototype translocation peptide (GRQLRIAGRRLRGR) we used is based on the carboxy terminus of Erns, an envelope protein of the pestivirus (Fig. 1A). Peptides corresponding to this region are able to translocate across eukaryotic cell membranes (23, 29). Furthermore, other labeled proteins and even active enzymes can gain access to cells when fused to this Erns-derived peptide. Translocation, moreover, is very fast (⬍1 min), requires no energy or any type of receptor, and displays no cell type or species specificity. As shown in Fig. 1B, C, adding this translocation sequence enhanced the inhibitory effect of both PSEN1- and APP-derived peptides from 250 to ⬍10 ␮M. Alternatively, we added a palmitoyl group, which targets peptides to membranes as well, but this turned out 3768

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to be ineffective. As opposed to a translocation peptide, a palmitoyl moiety does not translocate peptides between membranes, and may be less cell permeable. In addition, a palmitoyl moiety likely targets peptides mainly to the inner leaflet of the lipid bilayer contributing furthermore to their lower effectiveness. Overall, fusion peptides including the APP binding domain displayed far stronger inhibitory effects on A␤ production, as compared with those mimicking PSEN1 binding domains, and were therefore selected for further studies (peptides D7 and D9 in Fig. 1C). Particularly peptide D7, which differs from D9 in only 2 additional amino-terminal amino acids, appeared to be slightly more potent, as revealed by a higher degree of inhibition at 1␮M compared with D9 (Fig. 1D). This indicates that short changes in length can have a profound effect on their inhibitory activity. The specificity of the APP binding domain sequence is further underscored by the fact that a scrambled peptide (scram) fused to the translocation peptide was inactive. Nevertheless, the inhibitory potency is still significantly lower than that of small-compound ␥-secretase inhibitors, such as L685,458 (Fig. 1D). This became more obvious when data were quantitatively analyzed, allowing us to generate dose-response curves on inhibition of A␤ production (Fig. 1E). This resulted in an IC50 of 317 nM (n⫽3) for our D7 peptide, as compared with an IC50 of 16 nM (n⫽4) for L685,458. Surprisingly, merely fusing the Erns translocation peptide to the N terminus of the APP binding domain VVIVIATVIVITLVMLK dramatically increased the IC50 (D7 reverse, IC50: 2953 nM; n⫽4) and thus strongly diminished the inhibitory potency. This indicated to us that the orientation in which the fused peptide is presented to the membrane is critical or that the physicochemical properties of the peptides with the N-terminal translocation motif are considerably different compared with D7. To address the aggregation behavior of the different peptides, they were subjected to DLS. This showed that both the D7 and the reversed and scrambled peptides display similar size distributions, accumulating in particles with an average hydrodynamic radius of 30 –70 nm (Supplemental Fig. S1A). In contrast, the naked translocation sequence clearly aggregated and formed particles with radii ⬎ 1 ␮m (Supplemental Fig. S1A). These data show the essential role of the translocation peptide in solubilizing the interaction domain, explaining the inactivity of the naked peptide. However, the data also reveal no significant differences in aggregation potential between the longer peptides, prompting us to compare their secondary structure by FTIR. Overall, the spectra of the different peptides looked very similar and were dominated by the contribution from the arginine residues (maximal absorbances at 1633 and 1673 cm⫺1), which precluded detailed secondary structure analysis (Supplemental Fig. S1B). Overall, the data show that the structure of the D7 peptide as well as the reversed and the scrambled is similar although not identical, supporting the idea that the interaction is mediated by a specific sequence presented to the membrane in an interaction-competent orientation.


ESSELENS ET AL.

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Translocation peptide ENARQGAARVTSWLGRQLRIAGKRLEGRSKTWFGAYA

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Figure 1. Peptides mimicking the binding domains in APP and PSEN1 inhibit ␥-secretase activity in a cell-free assay: A) Envelope glycoprotein Erns is a 20 disulfide-linked homodimer of ⬃90 kDa, and approximately half of the molecular mass is contributed by carbohydrates. It is found on the pestivirus 0 0.1 10 100 1 1000 10,000 100,000 envelope and the surface of pestivirus-infected cells and has low homology Concentration (nM) with high-Mr cycling RNAses. The carboxy-terminal domain is nonglycosylated, highly positively charged, and amphipathic, which are known properties of translocating peptides. Part of this carboxy terminus (GRQLRIAGKRLEGR) maintains the membrane-passing properties, was optimized to GRQLRIAGRRLRGR (5603; ref. 23), and was used as a translocation peptide in fusion with inhibitory peptides. B) L685,458 inhibitor (10 ␮M) completely abrogates A␤ production, while solvent alone (DMSO) displays the amount of A␤ produced without inhibition. Different potencies can be observed for different peptides (250 to 10 ␮M). Fusion with the virally derived translocation peptide clearly increases the inhibitory potential (PALM, palmitoyl group). Peptides based on the APP binding domain show the most efficient A␤ inhibition. C) Truncation of peptides from B to assess optimal peptide length. Peptides D7 and D9 give complete inhibition at 10␮M and are tested in subsequent assays. D) Top panel: D7 and D9 inhibit ␥-secretase cleavage in a dose-dependent manner. Bottom panel: reaction specificity of D7 peptide sequence. Scrambling the peptide sequence or moving the translocation peptide to the N terminus results in loss of inhibitory activity. E) Dose-response curve of various peptides compared with L685,458. D7 has a calculated IC50 of 317 nM, a 20 times lower efficacy than obtained for L685,458 (IC50: 16 nM). D7 Austrian (a FAD mutation) displays only minor if any effect within the tested concentration range. D7 reverse (IC50: 2953 nM) again illustrates the importance of the orientation in which the fused peptide is presented to the membrane. 40

L685,458 D7 D7 reverse D7 Austrian

This is supported by the fact that D7 encompasses several FAD mutations in the APP gene, and when we introduced for instance the Austrian mutation T714I (30) in D7 there was barely an inhibitory effect on A␤ production in the concentration range tested. This indicates that certain FAD mutations in APP may affect the binding to ␥-secretase; however, it is not clear in which way APP cleavage is altered. Peptide inhibitors are active in cell-based assays So far, the cell-free assay provided us with a fast readout to test a set of synthetic peptides; however, it monitors A␤ production in a simplified system, i.e., outside the cellular context and without taking pro-

tein orientation, microdomain organization, and complex localization into account. It can be assumed that although active in a cell-free environment, these inhibitory peptides are bound to fail in a cellular situation. To address this, we applied two cell-based assays. First, we employed a cellular luciferase reporter assay (31–33) designed to detect ␥-cleavage of APP-GAL4-VP16 (see Materials and Methods and Fig. 2A). The D7 peptide inhibits luciferase activity, demonstrating the functionality of our peptide in a cellular context (Fig. 2B). Remarkably, no effect of the D7 peptide was observed when using Notch⌬EGAL4-VP16 (Fig. 2C), implying that our peptide selectively targets the ␥-secretase processing of APP


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Figure 2. Luciferase reporter assay. A) A GAL4-VP16 cassette is fused to the C terminus of APP or Notch⌬E, which becomes translocated to the nucleus on ␥-secretase cleavage. There it activates the luciferase gene, resulting in the emission of light, a measure for ␥-secretase activity. B) Dose-dependent inhibition of APP processing by ␥-secretase using D7 (n⫽3). C) D7 inhibits APP cleavage but leaves Notch cleavage unaffected (D7 and scrambled values were normalized to APP GAL4-VP16, and to Notch⌬E GAL4-VP16 for APP and Notch cleavage analysis respectively; n⫽9).

and not Notch. This is in a way unexpected for a peptide based on the binding domain of APP and suggests that either there might be distinct docking sites for different substrates or the peptide’s direct target is not PSEN1 or ␥-secretase. It should be noted that a dose of 1.5 ␮M was needed to acquire a 50% inhibition, as compared with 317 nM for the cell-free assay, something that is not unexpected given the higher accessibility of the peptide to its target in the latter assay. To explore this in more detail and to better understand how D7 interferes with APP processing, we applied a second cell-based assay (31), using HEK293 cells stably transfected with APPSwe (34). Following metabolic labeling in the presence of peptide inhibitors, cell lysates and conditioned media were examined through immunoprecipitation and quantitative phosphorimaging analysis of the different proteolytic APP fragments. Again, the D7 peptide inhibited A␤ secretion in a dose-dependent manner and in line with the results obtained using the luciferase assay (Fig. 3C), while APPFL remained 3770

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largely unaffected; the translocation peptide alone had no effect. However, again to our surprise, D7 dramatically decreased the levels of the secreted APP (sAPP) ectodomain (Fig. 3D), clearly underscoring that this peptide is active upstream of ␥-secretase activity. Although we did not observe strong effects on cellular levels of APP ␣- or ␤-CTF (Fig. 3E, F), the decrease in total sAPP favors the conclusion that overall shedding is affected. This was as well confirmed independently by direct Western blot analysis of the conditioned medium of HEK293 cells stably expressing APPswe (Supplemental Fig. S2). In summary, these data indicate that the inhibitory effects of D7 are not solely attributed to the inhibition of ␥-secretase processing directly (as observed in cellfree and cell-based reporter assays using APP-C99 as a substrate) but, when tested in a cellular context with APPFL, are also affecting APP proteolysis in the ectodomain, i.e., at a step before ␥-secretase cleavage. This suggests that the inhibitory mechanism is not operating on ␥-secretase itself but is acting specifically on the substrate.


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Figure 3. Effect of D7 on APPSwe processing. A) HEK293-APPSwe cells were metabolically labeled for 3 h using 35S, followed by immunoprecipitation of the conditioned medium and cell lysates and detection of APP species by phosphorimaging. Trans, translocation peptide alone. B–F) Quantification of APPFL (B), A␤ (C), sAPP (D), and C-terminal fragments ␤-CTFs (E) and ␣-CTFs (F). Values are normalized to DMSO.

D7 peptide binds to APP As the D7 peptide is based on the APP binding domain, we hypothesized that it would inhibit ␥-secretase cleavage of APP through binding to PSEN1 and interfere with the APP-PSEN1 binding or substrate docking. The strong inhibitory effect on APP ectodomain shedding suggests, however, a different mechanism. To examine this in more detail, we generated a biotinylated variant of D7 that would enable us to perform pulldown assays to study the interaction. MEFs were incubated with different concentrations of biotinylated D7, followed by detergent extraction, incubation with streptavidin beads, and Western blot analysis. As shown in Fig. 4A, the biotinylated peptide pulled down APP, PSEN1-NTF, and PSEN1-CTF in a dose-dependent manner. Since this result does not discriminate between the possibilities that D7 binds either PSEN1 or APP, we performed exactly the same experiment in PSEN1- and PSEN2knockout MEF cells. In the absence of PSEN expression, the biotinylated D7 peptide was still capable of pulling down APPFL from cell extracts, indicating that it binds directly to APP (Fig. 4A, right panel). Furthermore, the specificity of this interaction was verified in MEFs stably transfected with syndecan-3, a known substrate for ␥-secretase (12). As shown in Fig. 4B, no syndecan-3 immunoreactivity was detected in the bound fraction, underscoring that full-length APP is indeed the direct binding target of D7. Independent analysis using surface plasmon resonance confirmed the specific and dose-dependent binding of our D7

peptide to bacterial APPC100 (25), corresponding to the APP␤ CTF and therefore the direct substrate for ␥-secretase (Fig. 4C, D). D7 peptide accumulates in APP-positive endosomal organelles We next explored the subcellular localization of the D7 inhibitory peptide in HeLa cells by confocal microscopy, employing streptavidin-Alexa647 to visualize the biotinylated variant of D7 used in previous experiments. After 4 h of incubation at 37°C, the D7-biotin did not colocalize to major subcellular compartments, including the endoplasmic reticulum (visualized by anti-BIP) and the Golgi apparatus (anti-GM130; Fig. 5A). Partial colocalization was detected with EEA1-positive organelles, indicating an early endosomal localization. The finding was surprising, since the translocation peptide on its own or coupled to an enzyme was shown previously to become very rapidly translocated in an energy-independent manner throughout the cell (29). Our findings suggest that the D7-biotin deviates from this and enters the cell in a more restricted manner. To test this, we added D7-biotin for 1, 2, and 4 h to HeLa cells that were cotransfected with cDNA encoding the GTP-locked Rab5Q79L together with APP-YFP. The small GTPase Rab5 controls the fusogenic properties of early endosomes through GTP-dependent recruitment and activation of effector proteins. The dominant active Rab5Q79L mutant is known to cause formation of enlarged endosomes (35, 36), allowing us to follow the


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Figure 4. Specific binding of biotinylated D7 to APP. A) APPFL and PSEN1 can be precipitated using biotinylated D7. Even in the absence of PSEN1 and PSEN2 [MEF PSEN1/2 double-knockout (PSEN DKO) cell line], the peptide is able to precipitate APPFL, illustrating its direct interaction (right panel). Total, total cell extract after lysis; bound, fraction bound on beads; unbound, fraction after incubation with beads; DMSO, precipitation carried out with peptide solvent alone. B) Precipitation of a MEF cell line extract stably expressing syndecan-3, a known ␥-secretase substrate, with biotinylated D7. No significant amount of syndecan-3 was detected, indicating that syndecan-3 did not interact with the peptide. C) SPR analysis of the peptides binding to C100. C100 was coupled to a Ni2⫹-charged NTA surface [500 response units (RU)]. Sensorgrams are representative of 6 measurements each. DMSO and the peptides D7, D7 reverse, and scrambled were diluted to 100 nM and injected at a flow rate of 20 ␮l/min for 3 min, followed by a dissociation phase of 2.5 min. The D7 peptide strongly binds to C100 with almost no dissociation, whereas the D7 reverse and scrambled peptides show a much weaker interaction with C100. The first sharp peak (⬃60 s) might be due to the buffer exchange or an artifact with the injection. We did not subtract the DMSO curve, since the dissociation of the peptides is lower than for DMSO alone. D7 reverse and scrambled show low binding to C100, but the signal is much weaker compared with the D7 peptide. D) D7 peptide binds to C100 in a concentration-dependent manner. DMSO was used as control. Concentration of ⱖ75 nM D7 peptide is required to detect a signal above background (DMSO control).

accumulation of internalized cargo into Rab5 positive organelles. APP-YFP has been previously shown to accumulate in Rab5Q79L-enlarged endosomes following internalization from the cell surface (26, 37). While after 1 h hardly any D7-biotin was detected intracellularly, it clearly and increasingly accumulated in Rab5Q79L-enlarged endosomes from 2 to 4 h (Fig. 5B). The time frame in which this occurred shows that D7 is not merely translocated over membranes but likely internalizes and sorts to early endosomes. Interestingly, there was a marked colocalization of D7-biotin in those Rab5Q79L endosomes that were also accumulating APP-YFP. Indeed, D7-biotin localized to ⬃72% of all endosomes, as identified by the general early endosomal marker EEA1, while this percentage increased to ⬎90% when only considering the APP-YFP/EEA1-positive pool (Fig. 5C). Thus, D7 is likely not just randomly internalized (as in that case it should accumulate in all 3772

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endosomes) but more likely binds APP at the cell surface, allowing it to become cosorted to the endocytic pathway. D7 inhibits APP shedding in endosomes and affects APPFL dimerization We next investigated whether the accumulation of D7-biotin in Rab5Q79L-enlarged endosomes also affected the processing of APP accumulating in these organelles. HeLa cells were cotransfected with Rab5Q79L and Cherry-APP-YFP (26) and incubated with D7-biotin for 4 h. APP shedding can be readily observed through the accumulation of only Cherrylabeled APP ectodomain fragments in the lumen of Rab5Q79L-enlarged endosomes (Fig. 6A, top panels, and ref. 26). YFP remains mainly located in the limiting membrane, as it is fused to the carboxy terminus of APP


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Figure 5. D7 peptide is internalized and sequestered to APP containing late endosomes in a timeAPPYFP D7 biot 4h Vesicle overlap 100% 92% dependent manner. A) Hela cells were incubated with biotinylated D7 for 4 h and subsequently fixed 80% 72% 72% and mounted for confocal analysis. Antibody staining with specific 60% subcellular markers shows that the biotinylated peptide, detected EEA1 merge 40% with streptavidin-Alexa 647, does not colocalize with BIP (ER) or GM130 (Golgi). However, partial 20% colocalization (arrowheads) was observed for D7 and EEA1 (early 0% endosomes), indicating an early APP vesicles D7 vesicles D7 vesicles endosomal localization. B) HeLa containing containing containing EEA1 EEA1 APP cells expressing APPYFP and Rab5QL were incubated with 1 ␮M D7 for the indicated time points. From 2 h onward, D7 is found in enlarged vesicles containing APP. C) Count of vesicles containing APP, D7, or EEA1: 92% of vesicles containing D7 also contain APP, while only 72% of APP- or D7-containing vesicles have EEA1 (n⫽207). Scale bars ⫽ 10 ␮m.

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and thus represents the membrane-tethered full-length APP or APP-CTF. In contrast, in the presence of D7-biotin, the lumen of Rab5Q79L-enlarged endosomes is largely empty, no filling is observed (Fig. 6A, bottom panel), and both Cherry and YFP fluorescent proteins remain colocalized in the limiting membrane indicating the presence of full-length APP. Quantitative analysis of the Cherry vs. YFP intensities in the limiting membrane and lumen clearly demonstrates that APP shedding is strongly inhibited (Fig. 6B), confirming our previous biochemical data. Subdiffraction imaging using PiMP (27), moreover, allowed us to determine the orientation of D7-biotin within the membrane of Rab5Q79L-enlarged endosomes (Fig. 6C). The greatly increased resolution allowed us to determine that D7biotin is prominently concentrated at the outer rim of vesicles, i.e., outside the colocalized Cherry fluorescent

protein and YFP fused to APP. This strongly suggests that the biotin tag points outward and locates to the cytosolic side of these endosomes. This is in agreement with an amino acid configuration of the APP binding domain parallel to the cognate APP sequence in its transmembrane domain, as schematically illustrated in Fig. 6D and as would also be the case when 2 APP molecules are dimerizing. Since APP processing is affected by dimerization, we further explored this lead for a possible working mechanism of our D7 peptide. APP can form dimers via at least 3 dimerization sites, i.e., two in the ectodomain and a third in the APP transmembrane domain containing a GXXXG motif (38). Interestingly, a slightly reduced dimerization can have a significant effect on A␤42 generation. Mutation of G29A and G33A (numbering according to A␤ sequence) in the GXXXG dimerization domain of APP


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D7 D7 scram scram DMSO Figure 6. APP shedding is inhibited in vesicles containing D7, and 2µM 1µM 2µM 1µM D7 inhibits APPFL dimerization: A) HeLa cells were transfected APP with Cherry-APP-YFP and Rab5Q79L and incubated with biotinylDimer ated D7 for 4 h. Without D7, APP shedding causes the vesicles to fill up with red, i.e., Cherry-APPNTF is released from the limiting APP monomer membrane and fills the lumen of the vesicle, while APPCTF-YFP remains membrane-bound, as expected. When D7 is present, APP is not cleaved, and both Cherry-APPNTF and YFP APPCTF-YFP stay on the limiting membrane. B) Quantification of panels in A clearly shows the shift of localization for Cherry-APPNTF from the lumen to the limiting membrane on D7 inhibition, while leaving APPCTF-YFP unaffected. C) Photobleaching microscopy with nonlinear processing (PiMP) analysis of vesicles (continued on next page)

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caused a corresponding 7 and 23% dimer reduction and lowered A␤42 levels nearly 60% (38). To test the effects of D7 on APP dimerization, we expressed APP695 in COS-7 cells in the presence of D7 or a scrambled peptide at the same concentrations used for cell-based assays previously, and monitored the effects on APP dimerization via quantitative Western blotting. As shown in Fig. 6E, D7 but not the scrambled peptide (nor DMSO) shows a specific and dose-dependent lowering of APPFL dimer. In general, these experiments allow us to conclude that D7 exerts its inhibitory function through direct binding to APP and as such might interfere with its dimerization. This finding further supports a role for the D7 peptide in dimerization and regulated processing of APP.

DISCUSSION In this study, we developed novel peptide inhibitors with sequences based on the PSEN1-APP interaction domains identified previously by our group (1). We demonstrated that a peptide mimicking the PSEN1binding domain in APP, i.e., the carboxy-terminal 11 aa of the APP transmembrane domain, is capable of selectively inhibiting A␤ production in cell-free and cell-based assays without affecting Notch intracellular domain (NICD) production. Notably, this region harbors all FAD-causing missense mutations in APP that promote ␥-secretase cleavage at the ␥42 site (39). Mutating individual residues or altering its length affects ␥-secretase cleavage, underscoring the importance of this region in presenting APP to ␥-secretase (present study and refs. 40, 41). By binding to APP, our peptide inhibitor can compete with the docking of APP on PSEN, with a chaperone or even with APP itself. It has to be noted that the use of peptides as a therapeutic tool faces some major hurdles. To circumvent problems of bioavailability, inhibitors often need to be extensively modified (42). Small proteins or peptides in particular may trigger an immune response, and the activity, stability, and potential side effects complicate a therapeutic approach. Among potent and specific ␥-secretase inhibitors are also a number of small molecules of different structural classes, such as sulfonamides (43), benzodiazepines (44, 45), L685,458 (IC50: 13 nM; ref. 46), and DAPT (IC50: 115 nM; ref. 47), which mainly target the catalytic activity of the ␥-secretase. When the VVIATVIVITLVMLK peptide is fused to a translocation peptide adapted from the pestiviral envelope protein Erns (23, 29), the inhibitory

action is prominently enhanced and now falls in the nanomolar range (IC50: 317 nM; Fig. 1E). Still, there is room for further improvement, since peptides can be further stabilized by chemical alterations, in this way creating so-called peptidomimetics. For example, a peptide analog constructed out of d- instead of l-amino acids can effectively block ␥-secretase in a cell-free system at an IC50 of 30 nM (48), and a helical peptide was shown to inhibit ␥-secretase in a cell-free enzyme assay at subnanomolar concentrations (IC50: 140 pM; ref. 49). It can be foreseen that minimizing the necessary dose equally lowers the risk for potential side effects. In our case, blocking APP cleavage using the D7 peptide, this is addressed in two ways: first, it is not necessary to completely block APP cleavage, since this process occurs also in healthy individuals and likely has a physiological function. A lowering of APP cleavage to nearnormal levels could stop plaque growth and possibly even reverse plaque formation by allowing protein degradation and clearance to handle accumulated A␤. Second, the optimization of our peptide permits its use in nanomolar doses, reducing the possibility of toxic effects. Our data illustrate that D7 does not interfere with Notch ␥-cleavage. In addition, binding studies using a biotinylated peptide or SPR showed direct and specific binding to APP, while it did not interact with syndecan-3, another ␥-secretase substrate. The advantage of using the D7 peptide is therefore not immediately its effective concentration but lies in the fact that it targets APP and not ␥-secretase, thereby avoiding off-target effects, troubling the majority of ␥-secretase inhibitors in a clinical setting (50 –52). Another hurdle that has to be taken is testing the APP-derived peptide in animal models. Certainly, the targeting of D7 toward the brain regions where A␤ formation takes place could prove to be difficult. Moreover, cerebral drug delivery is faced with many additional obstacles due to the characteristics of the blood-brain barrier. It is known that some small peptides can readily enter and exit the brain via this barrier in a passive manner and display effects on the central nervous system (53). The use of antennapedia-mediated transduction of heterologous proteins into cells, discovered in 1992 (54), and other “Trojan horse” peptides, such as Tat, has drawn increased attention as an approach for drug delivery. The demonstration that proteins fused to the Tat protein transduction domain are capable of crossing the intact blood-brain barrier (55, 56) may transform molecular research and neuro-

containing D7-biotin again shows the absence of APP cleavage in D7-positive vesicles and hints at the orientation of the D7 peptide in the limiting membrane. A larger-diameter ring is consistently seen in the D7 channel (white), suggesting that the biotinylated moiety is sticking outward; see also the bottom right panel delineating the outer limits of each color. Scale bars ⫽ 10 ␮m. D) Schematic representation of the orientation of Cherry-APP-YFP and D7-biotin in the limiting membrane. E) COS-7 cells were transfected with APP695 and incubated with DMSO, D7, or scrambled peptide. Western blot under stringent nonreducing conditions allows for the detection of APP dimers, and the proportion of the monomers can be quantified. D7 has a dose-dependent effect on APP dimerization, while a scrambled peptide has no effect. APP PROCESSING INHIBITION THROUGH PEPTIDES MIMICKING THE APP-PSEN INTERACTION DOMAIN

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biological therapy. However, it was only recently that protein transduction domain-mediated delivery of proteins with therapeutic potential was achieved in models of neural degeneration in nerve trauma (57) and ischemia (58 – 60). Several groups have published the first positive results using protein transduction domains for the delivery of therapeutic proteins in relevant animal models of human neurological disorders (61– 63). This is why we opted to follow a similar approach by fusing our active peptide to the translocation peptide adapted from pestivirus envelope glycoprotein Erns translocation domain, while this also ensures presence in the cell membrane where APP ␥-cleavage occurs. To monitor the correct localization of our peptide, we used a biotinylated D7 variant as a tool. This allowed us in the first place to demonstrate that unlike the translocation peptide itself (29), the D7 peptide is not translocated rapidly throughout the cell but follows an internalization itinerary to endosomal compartments likely in association with APP (Fig. 5A). Thus, the primary role of the viral translocation peptide here is not to translocate but rather to increase the solubility of the inhibitory peptide, as was demonstrated by physicochemical experiments (Supplemental Fig. S1), and to allow the hydrophobic part to become embedded in the lipid bilayer, where it likely directly binds the APP transmembrane domain at the cell surface. From here, the hydrophobic peptide remains embedded in the membrane and cointernalizes with APP to become sorted to endosomes, where it prevents its cleavage. This could be convincingly shown in Fig. 6A, B, where D7-positive vesicles display a blockade in APP shedding. PiMP analysis of these vesicles positioned the D7-biotin at the outer circumference of the vesicle, which allows us to speculate that D7 is directionally inserted into the membrane and in a parallel fashion with the APP sequence (Fig. 6D). These data also highlight the importance of the C-terminal location of the translocation peptide, which likely only in this configuration allows the parallel orientation of the inhibitory domain to the APP transmembrane domain. In addition, we found that D7 interferes with APP dimerization, although the physiological relevance of this observation could not yet be clearly deciphered. Dimerization of surface proteins is nevertheless a wellestablished mechanism of triggering signal transduction through promoting receptor internalization in eukaryotic cells (64). Moreover, several other ␥-secretase substrates are known to dimerize as part of their physiological function, e.g., Notch (65), Her4 (66), or E-cadherin (67, 68). This strongly implies that APP processing and A␤ production can indeed be positively regulated by dimerization. Alternatively, dimerization of APP could promote a more efficient internalization and sorting to early endosomes, where most of the ␤-site APP cleaving enzyme 1 (BACE1)-mediated shedding appears to occur (26, 37). Despite its attractiveness, D7-biotin readily accumulated with APP in Rab5Q79L-enlarged endosomes, indicating that internalization is not hampered and does not require APP 3776

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dimerization per se. Alternatively, the binding of D7 could interfere with the binding of APP sheddases, such as BACE1, providing an explanation for the inhibitory effect on APP ectodomain processing. Indeed, we and others have shown that APP shedding by BACE1 mainly occurs in endosomes, i.e., the compartments where D7-biotin coaccumulates with APP (Fig. 5). In summary, by exploring the binding domains of APP and PSEN1, we identified a sequence encompassing the second half of the APP transmembrane domain that bears therapeutic potential for a selective interference in APP processing and thus A␤ production without potentially affecting other substrates. Furthermore, it shows that the overall knowledge of APP-PSEN1 binding properties and the mechanisms involved contributes to the functional understanding of the ␥-secretase and its substrates and offers a solid starting ground for developing more effective inhibitors. The work presented here was supported by the Vlaams Instituut voor Biotechnologie (VIB); Katholieke Universiteit Leuven (KU Leuven-Methusalem to B.D.S.); Fonds voor Wetenschappelijk Onderzoek (FWO G.0663.09 and G.0.754.10.N); Stichting Alzheimer Onderzoek (SAO-FRMA/ cycle 2010); federal government IAP P7 (2012–2017); and Alzheimer Association (IIRG-08-91535). C.E. is holder of a return fellowship from the federal government (Belspo). The authors thank S. Munck for help with PiMP analysis.

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Received for publication November 30, 2011. Accepted for publication May 8, 2012.

ESSELENS ET AL.