Binding of longer Aβ to transmembrane domain 1 of presenilin 1 impacts on Aβ42 generation
© Ohki et al.; licensee BioMed Central Ltd. 2014
Received: 8 November 2013
Accepted: 10 January 2014
Published: 13 January 2014
Amyloid-β peptide ending at 42nd residue (Aβ42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aβ through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood.
We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aβ42 production. Aβ42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aβ species including Aβ45 that are the precursor of Aβ42. We further found that the helical surface of TMD1 is involved in the binding of Aβ45/48 and that the binding was altered by GSMs as well as TMD1 mutation.
Binding between PS1 TMD1 and longer Aβ is critical for Aβ42 production.
KeywordsPresenilin Secretases Alzheimer disease Intramembrane proteolysis γ-Secretase modulator
Several lines of evidence suggest that the accumulation of amyloid-β peptide (Aβ), a major component of senile plaques, is a common pathological feature in Alzheimer disease (AD) . Aβ is generated through sequential cleavage by β- and γ-secretases of amyloid-β precursor protein (APP). γ-Secretase primarily cleaves APP to produce a C-terminal stub of APP (APP-CTF). Then, scission of APP-CTF by γ-secretase results in generation of various forms of Aβ with different C-terminal lengths. Especially, Aβ ending at the 42nd residue (Aβ42), the most aggregable species, is initially and predominantly deposited in AD brains . Moreover, familial AD-linked mutations in Psen (Presenilin; PS) 1, Psen2 or APP genes cause an increase in Aβ42 generation. Thus, Aβ42 is considered as the most pathogenic species causative for AD .
γ-Secretase is an intramembrane-cleaving protease complex composed of four membrane spanning proteins: PS, Nicastrin, Aph-1 and Pen-2 [4, 5]. Extensive biochemical studies showed that the γ-secretase-mediated intramembrane cleavage of APP occurs in a processive manner ; APP-CTF is primarily cleaved at the ϵ-site located around the membrane-cytoplasm boundary to produce Aβ49 or Aβ48. Subsequently, these longer Aβ peptides are processed by stepwise cleavages to secrete shorter Aβ in two predominant production lines: Aβ49 is processed to Aβ43/40 via Aβ46 (Aβ40 production line), and Aβ48 is processed to Aβ42/38 via Aβ45 (Aβ42 production line). PS forms a channel-like catalytic pore structure within the membrane, and is endoproteolyzed to generate N- and C-terminal fragments (NTF and CTF, respectively) during the assembly of the protease-active complex [7, 8].
Recently, small compounds that selectively regulate Aβ42 production without affecting ϵ-cleavage emerged, which are termed γ-secretase modulators (GSMs) . We have shown that a potent Aβ42-lowering compound, GSM-1, directly targets the PS1 TMD1 . Moreover, using substituted cysteine accessibility method (SCAM), we identified two different regions within TMD1 of PS1, i.e., a hydrophobic luminal region and a hydrophilic portion facing the catalytic site , that are differently involved in the action of GSM-1 . However, the precise molecular mechanism whereby γ-secretase generates Aβ42, as well as the role of TMD1 in Aβ42 production, remains elusive. In this study, we identified TMD1 of PS1 as a regulatory domain for the processive cleavage of the Aβ42 production line.
Fenofibrate directly targets the N-terminal fragment of presenilin 1
Intermediate longer A was secreted by TMD1 mutant PS1
GSMs and P88L mutation affected the interaction between TMD1 and longer Aβ species
Understanding the molecular mechanism of the processive cleavage by γ-secretase is critical to the development of effective GSMs. We previously reported that phenylpiperidine-type GSMs are bound to TMD1 of PS1 . Here, we further showed that fenofibrate, an Aβ42-raising GSM, also directly targets TMD1, while Fen-B was reported as APP-targeting photoprobe . Recently, some papers reported that large amount of Aβ42 or C99 forms aggregates that cause non-specific binding to GSMs [14, 15]. Therefore, we have used brain microsomes obtained from wild-type mouse for the photo-crosslinking experiment.
Scissile bonds for processive cleavage by γ-secretase have hypothetically been mapped on different surfaces in the α-helical model of APP TMD . This raises the possibility that the distinct processive cleavages by γ-secretase, i.e., those leading to production of Aβ49-46-43-40 or Aβ48-45-42-38, are determined by the recognition of one or the other of the specific helical surfaces. However, the domain on γ-secretase that recognizes the helical surface on the substrate is yet to be identified. It has previously been suggested that TMD1 of PS1 is involved in the binding of APP-CTF, a direct substrate of γ-secretase [21, 22]. Here we found that longer Aβ peptides that are generated as intermediate products in the Aβ42 production line (i.e., Aβ45 and Aβ48), which also are direct substrates for the processive cleavage, retain the capacity to interact with TMD1 of PS1. It is highly likely that the “gripping tenacity” of the substrate binding site facing the catalytic pore would determine the processivity of Aβ48 and Aβ45 on the Aβ42 production line, which can be modulated by small compounds. Consistently, Okochi et al. have recently reported that Aβ42 is bound to the γ-secretase complex  and the binding was modulated by GSMs, although they have not identified the binding site of Aβ within the enzyme complex. Thus, we propose that TMD1 of PS1 functions as a binding site of longer Aβ species for γ-secretase during the processive cleavage, which specifically determines the efficiency of the processive cleavage of the Aβ42 production line. Structural analyses suggested that the catalytic cavities of rhomboid protease , another intramembrane-cleaving enzyme, or those of FlaK  and PSH , archaeal GxGD proteases, are unable to accommodate all the amino acid residues of the transmembrane sequence of the substrates. This suggests that a major part of the TMD of substrates remains within the membrane and is gripped by enzymes to incorporate the cleavage site into the intramembrane catalytic site during proteolysis. While the precise structure of human PS1 still remains unclear, our SCAM results on PS1 , as well as the recently reported x-ray crystal structure of PSH , the latter being composed of 9-transmembrane domains similarly to human PS1, altogether suggested that TMD1 locates in proximity to the catalytic aspartate in TMD7. The results of these structural analyses also support our notion that TMD1 functions as a substrate binding domain during the processive cleavage by γ-secretase.
Fenofibrate directly bound to TMD1 of PS1 to induce the conformational changes in the catalytic site of the γ-secretase. P88L mutation in TMD1 caused an aberrant secretion of longer Aβ polypeptides (i.e., Aβ45 or Aβ46), indicating that TMD1 is involved in the regulation of C-terminal length of Aβ. Finally, we found that TMD1 contains a binding site for the longer Aβ species, and GSMs affect Aβ42 production by changing the affinity between TMD1 and longer Aβ. Our results suggest that TMD1 functions as a substrate binding domain during the processive cleavage by γ-secretase.
Compounds, peptides and antibodies
GSM-1, GSM-1-BpB, NS-1017, GSM-1-amide-BpB, Fen-B and DAPT were synthesized as described [10, 12, 36]. L-685,458 and fenofibrate were purchased from Bachem and SIGMA, respectively. L-852,646  was kindly provided from Dr. Y. Li (Sloan-Kettering Cancer Center). Synthetic longer Aβ peptides (i.e., β-amyloid (1-43, #23573), (1-45, #61956-01), (1-46, #62076-01), (1-48, #61965-01), (1-49, #61963-01) were purchased from Anaspec. Aβ (1-40) (#4307-v) and Aβ (1-42) (#4349-v) peptides were purchased from Peptide institute. The rabbit polyclonal antibodies anti-PS1 NTF (G1Nr5), anti-PS1 CTF (G1L3) and anti-Pen-2 (PNT3) were raised as described [38–40]. Anti-PS1 NTF (PS1NT)  and anti-SPP (SPPc)  were kindly gifted from Drs. G. Thinakaran (The University of Chicago) and T. Golde (University of Florida). Anti-nicastrin N1660 (SIGMA), anti-APP CTF (Immuno-Biological Laboratories), anti-Aph-1aL O2C2 (Covance), anti-human Aβ 82E1 (Immuno-Biological Laboratories) and anti-biotin (Bethyl) were purchased from indicated vendors. The monoclonal antibody anti-α-tubulin AA4.3 developed by Dr. C. Walsh was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health, and maintained by The University of Iowa, Department of Biology, Iowa City, IA.
Plasmid construction, cell culture manipulation and cell based assay
cDNAs encoding PS1 and APP carrying Swedish mutation (APPNL) were inserted into pMXs-puro . cDNAs encoding mutant PS1 were generated by long PCR-based QuikChangeTM strategy (Stratagene). To produce recombinant proteins, cDNAs encoding PS1 were cloned into pGEX-6P-1 vector (GE healthcare) . Maintenance of cultured cells, transfection, retroviral infection, two-site enzyme-linked immunosorbent assay (ELISA), or immunoblotting using Urea/SDS-PAGE gel system as described [10, 39, 44, 45].
Photoaffinity labeling and SCAM experiments
Preparation of samples for photoaffinity labeling experiments  was performed as follows. Brains of C57J/B6 mouse (3-5 month age) or cultured cells were homogenized with homogenize buffer (20 mM HEPES (pH 7.0), 140 mM KCl, 250 mM sucrose, 0.5 mM diisopropyl fluorophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml tosyllysine chloromethyl ketone, 1 μg/ml antipain, 1 μg/ml leupeptin, 10 μg/ml phosphoramidon, 5 mM EDTA, 1 mM EGTA) using Potter-Elvehjem Tissue Grinder (Wheaton), and membrane fractions were collected by ultracentrifugation at 100,000 × g (Beckman) . PAL experiments utilizing avidin-biotin catch principle  and thrombin digestion experiments after PAL were performed as previously described . Briefly, after resuspension of the microsome in the homogenize buffer by 25G needle with syringe, protein content was measured by BCA assay (Thermo Fisher Scientific). 1 mL of microsome-containing solution (1 mg/ml protein) was preincubated with compounds for 30 min on ice. Then photoprobes were added and incubated for 10 min on ice under the dark condition. UV irradiation (352 nm) was performed on ice for 1 hr with a UV lamp (Model XX-15BLB, UVP). The approximate distance from UV lamp to the samples was 10 cm. The biotinylated proteins were precipitated by streptavidin sepharose (GE healthcare) in 1% SDS containing homogenization buffer. For SCAM, all methanethiosulfonate reagents (Toronto Research Chemicals) were dissolved in dimethyl sulfoxide at 200 mM prior to use or stored at 80 degree until use. The methods for SCAM and competition experiments using biotinylaminoethyl methanethiosulfonate have been described in detail before [10, 11]. Briefly, stable DKO cells expressing cysteine mutant PS1 were grown on two 15-cm dishes per single analysis. Cells were scraped in PBS and resuspended in 2 ml of SCAM homogenization buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, Complete protease inhibitor cocktail (Roche Biochemicals)). Cells were disrupted by a Polytron homogenizer (Hitachi), and nuclei and large cell debris were pelleted by centrifugation at 1,500 × g for 10 min. The postnuclear supernatants were centrifuged, and the resultant microsomal pellets were resuspended in 0.2 ml of PBS in a syringe, and 0.1 mM biotinylaminoethyl methanethiosulfonate was added to this fraction. After 30 min incubation at 4 degree, microsomes were centrifuged twice to wash out. The resultant pellets resuspended in 1% SDS/PBS were incubated with the streptavidin sepharose overnight and analyzed as in the intact cell biotinylation experiment. In PAL or SCAM experiments, we loaded 1.5 and 20% of samples as “input” and “bound”, respectively, in all immunoblot analyses.
Protein purification and binding assay
GST-fusion recombinant proteins were expressed in E. Coli (BL21 DE3) (Novagen) and purified by two step procedures using glutathione sepharose and mono Q columns (GE Healthcare) as manufacturer’s instruction. All recombinant proteins were finally diluted with recombinant protein preparation buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.25% CHAPSO). C99-FLAG was purified from Sf9 cells infected with recombinant baculovirus encoding C99-FLAG and diluted at 1 μg/ml in recombinant protein preparation buffer. To perform binding assay, 0.5 μg of GST-fusion recombinant proteins were mixed at 1 μg of C99-FLAG, 1 (for Aβ43, Aβ45, Aβ46, Aβ48 and Aβ49) or 10 (for Aβ40 and Aβ42) nM of synthetic Aβ in 1 ml of recombinant protein preparation buffer, and incubated at 4 degree overnight. After addition of glutathione sepharose, samples were then washed with the buffer and precipitates were eluted by boiling in sample buffer. For binding assay using native PS1 protein, PS1 or P88L mutant PS1 was expressed in DKO cells and solubilized in 10 mM HEPES buffer containing 1% CHAPSO. After addition of 2 ng of Aβ48 peptide, the solubilized fraction was incubated with anti-PS1 antibody G1Nr5 at 4 degree overnight. PS1-Aβ48 complex was then immunoprecipitated using Protein G sepharose 4 Fast Flow (GE Healthcare). Subsequently eluates (i.e., proteins bound to GST-fusion recombinant proteins or native PS1 proteins) were analyzed by immunoblotting. We loaded 0.75 and 20% of samples as “input” and “bound”, respectively, in immunoblot of all pull down assay unless the amount of loaded proteins was otherwise indicated.
Psen1/Psen2 double knockout mouse immortalized fibroblasts
Substituted cysteine accessibility method
The authors are grateful for G. Thinakaran (The University of Chicago), T. Golde (University of Florida), B. De Strooper (VIB Leuven), Y. Li (Sloan-Kettering Cancer Center), and T. Kitamura (The University of Tokyo), S. Funamoto and Y. Ihara (Doshisha University), and Takeda pharmaceutical company for Aβ ELISA for valuable reagents, and our current and previous laboratory members for helpful discussions and technical assistance. This work was supported in part by grants-in-aid for Young Scientists (S) from the Japan Society for the Promotion of Science (JSPS) (T.T.), Scientific Research on Innovative Areas for Foundation of Synapses and Neurocircuit Pathology (for T.I.) and Brain Environment (for T.T.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by the Ministry of Health, Labor and Welfare of Japan (Comprehensive Research on Aging and Health) (for T.T.), by Targeted Proteins Research Program of the Japan Science and Technology Corporation (JST) (for T.T., T.I.), by Core Research for Evolutional Science and Technology of JST (for T.T., T.I.) and by a donation from Mr. Chuichi Imai (for T.T.). Y.O. and T.H. are research fellows of JSPS.
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