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Alternative Selection of β-Site APP-Cleaving Enzyme 1 (BACE1) Cleavage Sites in Amyloid β-Protein Precursor (APP) Harboring Protective and Pathogenic Mutations within the Aβ Sequence*

  • Ayano Kimura
    Footnotes
    Affiliations
    From the Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-Nishi 6, Kita-ku, Sapporo 060-0812, Japan
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  • Saori Hata
    Footnotes
    Affiliations
    From the Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-Nishi 6, Kita-ku, Sapporo 060-0812, Japan
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  • Toshiharu Suzuki
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-11-706-3250, Fax: 81-11-706-4991;
    Affiliations
    From the Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-Nishi 6, Kita-ku, Sapporo 060-0812, Japan
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  • Author Footnotes
    * This work was supported in part by Grants-in-aid for Scientific Research 262930110 and 16K14690 (to T. S.) and 15K18854 (to S. H.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Bilateral Joint Research Project of the Japan Society for the Promotion of Science (to S. H.). The authors declare that they have no conflict of interest with the contents of this article.
    This article contains supplemental Figs. S1 and S2.
    1 Both authors contributed equally to the results of this work.
Open AccessPublished:September 29, 2016DOI:https://doi.org/10.1074/jbc.M116.744722
      β-Site APP-cleaving enzyme 1 (BACE1) cleaves amyloid β-protein precursor (APP) at the bond between Met671 and Asp672 (β-site) to generate the carboxyl-terminal fragment (CTFβ/C99). BACE1 also cleaves APP at another bond between Thr681 and Gln682 (β′-site), yielding CTFβ′/C89. Cleavage of CTFβ/C99 by γ-secretase generates Aβ(1-XX), whereas cleavage of CTFβ′/C89 generates Aβ(11-XX). Thus, β′-site cleavage by BACE1 is amyloidolytic rather than amyloidogenic. β′ cleavage of mouse APP is more common than the corresponding cleavage of human APP. We found that the H684R substitution within human Aβ, which replaces the histidine in the human protein with the arginine found at the corresponding position in mouse, facilitated β′ cleavage irrespective of the species origin of BACE1, thereby significantly increasing the level of Aβ(11-XX) and decreasing the level of Aβ(1-XX). Thus, amino acid substitutions within the Aβ sequence influenced the selectivity of alternative β- or β′-site cleavage of APP by BACE1. In familial Alzheimer's disease (FAD), the APP gene harbors pathogenic variations such as the Swedish (K670N/M671L), Leuven (E682K), and A673V mutations, all of which decrease Aβ(11–40) generation, whereas the protective Icelandic mutation (A673T) increases generation of Aβ(11–40). Thus, A673T promotes β′ cleavage of APP and protects subjects against AD. In addition, CTFβ/C99 was cleaved by excess BACE1 activity to generate CTFβ′/C89, followed by Aβ(11–40), even if APP harbored pathogenic mutations. The resultant Aβ(11–40) was more metabolically labile in vivo than Aβ(1–40). Our analysis suggests that some FAD mutations in APP are amyloidogenic and/or amyloidolytic via selection of alternative BACE1 cleavage sites.

      Introduction

      β-Site APP-cleaving enzyme 1 (BACE1),
      The abbreviations used are: BACE1, β-site APP-cleaving enzyme 1; AD, Alzheimer's disease; FAD, familial Alzheimer's disease; APP, amyloid β-protein precursor; Aβ, amyloid β-protein; sAPP, soluble large extracellular N-terminal domain of APP truncated at the primary cleavage site; CSF, cerebrospinal fluid; CTF, C-terminal fragment of APP truncated at the primary cleavage site; IP-MS, immunoprecipitation MALDI-TOF/MS; 3mut, triple mutation R676G/Y681F/H684R; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
      a type I transmembrane aspartic protease, was identified as the β-secretase that cleaves amyloid β-protein precursor (APP) to generate neurotoxic amyloid β (Aβ) (
      • Hussain I.
      • Powell D.
      • Howlett D.R.
      • Tew D.G.
      • Meek T.D.
      • Chapman C.
      • Gloger I.S.
      • Murphy K.E.
      • Southan C.D.
      • Ryan D.M.
      • Smith T.S.
      • Simmons D.L.
      • Walsh F.S.
      • Dingwall C.
      • Christie G.
      Identification of a novel aspartic protease (Asp 2) as β-secretase.
      • Sinha S.
      • Anderson J.P.
      • Barbour R.
      • Basi G.S.
      • Caccavello R.
      • Davis D.
      • Doan M.
      • Dovey H.F.
      • Frigon N.
      • Hong J.
      • Jacobson-Croak K.
      • Jewett N.
      • Keim P.
      • Knops J.
      • Lieberburg I.
      • et al.
      Purification and cloning of amyloid precursor protein β-secretase from human brain.
      ,
      • Vassar R.
      • Bennett B.D.
      • Babu-Khan S.
      • Kahn S.
      • Mendiaz E.A.
      • Denis P.
      • Teplow D.B.
      • Ross S.
      • Amarante P.
      • Loeloff R.
      • Luo Y.
      • Fisher S.
      • Fuller J.
      • Edenson S.
      • Lile J.
      • et al.
      Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.
      • Yan R.
      • Bienkowski M.J.
      • Shuck M.E.
      • Miao H.
      • Tory M.C.
      • Pauley A.M.
      • Brashier J.R.
      • Stratman N.C.
      • Mathews W.R.
      • Buhl A.E.
      • Carter D.B.
      • Tomasselli A.G.
      • Parodi L.A.
      • Heinrikson R.L.
      • Gurney M.E.
      Membrane-anchored aspartyl protease with Alzheimer's disease β-secretase activity.
      ). BACE1 cleaves APP at the peptide bond between Met671 and Asp672 (β-site; sequence numbering refers to the APP770 isoform) (
      • Haass C.
      • Schlossmacher M.G.
      • Hung A.Y.
      • Vigo-Pelfrey C.
      • Mellon A.
      • Ostaszewski B.L.
      • Lieberburg I.
      • Koo E.H.
      • Schenk D.
      • Teplow D.B.
      • Selkoe D.J.
      Amyloid β-peptides is produced by cultured cells during normal metabolism.
      ,
      • Roher A.E.
      • Lowenson J.D.
      • Clarke S.
      • Wolkow C.
      • Wang R.
      • Cotter R.J.
      • Reardon I.M.
      • Zürcher-Neely H.A.
      • Heinrikson R.L.
      • Ball M.J.
      • Greenberg B.D.
      Structural alteration in the peptide backbone of β-amyloid core protein may account for its deposition and stability in Alzheimer's disease.
      ). This primary cleavage of APP generates the secreted form of the amino-terminal large fragment (sAPPβ) and the membrane-associated carboxyl-terminal fragment (CTFβ/C99) (reviewed in Ref.
      • Cole S.L.
      • Vassar R.
      The role of amyloid precursor protein processing by BACE1, the β-secretase, in Alzheimer's disease pathophysiology.
      ). Because CTFβ/C99 includes the complete amino acid sequence of the Aβ region, and subsequent cleavage of CTFβ/C99 by γ-secretase generates the Aβ(1-XX) peptides (Aβ1 indicates the position of Asp672), the β-site cleavage is referred to as amyloidogenic processing of APP (reviewed in Ref.
      • Thinakaran G.
      • Koo E.H.
      Amyloid precursor protein trafficking, processing, and function.
      ). Alternatively, APP can also be cleaved by α-secretase (mainly ADAM10/17), at the peptide bond between Lys687 and Leu688, generating sAPPα and CTFα/C83 including the Aβ(17-XX) region; accordingly, this cleavage is referred to as amyloidolytic processing of APP (
      • Buxbaum J.D.
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      ,
      • Lammich S.
      • Kojro E.
      • Postina R.
      • Gilbert S.
      • Pfeiffer R.
      • Jasionowski M.
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      • Allinson T.M.
      • Parkin E.T.
      • Turner A.J.
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      ADAMs family members as amyloid precursor protein α-secretase.
      ). BACE1 also cleaves APP at another peptide bond between Tyr681 and Gln682 (β′-site), resulting in generation of sAPPβ′ and CTFβ′/C89 (
      • Liu K.
      • Doms R.W.
      • Lee V.M.
      Glu11 site cleavage and N-terminally truncated Aβ production upon BACE overexpression.
      ,
      • Huse J.T.
      • Liu K.
      • Pijak D.S.
      • Carlin D.
      • Lee V.M.
      • Doms R.W.
      β-Secretase processing in the trans-Golgi network preferentially generated truncated amyloid specific that accumulate in Alzheimer's disease brain.
      ). Cleavage of CTFβ′/C89 by γ-secretase yields Aβ(11-XX), which lacks the first 10 amino acids of the Aβ domain. This β′-site cleavage of APP is also thought to be amyloidolytic, because the structural analysis suggests that Aβ(11–40) and Aβ(11–42) are less toxic than Aβ(1–40) and Aβ(1–42) (
      • Meral D.
      • Urbanc B.
      Discrete molecular dynamics study of oligomer formation by N-terminally truncated amyloid β-protein.
      ), and the Aβ(11–42) showed reduced neurotoxicity compared with Aβ(1–42) and Aβ(3–42) in a study with transgenic fly (
      • Jonson M.
      • Pokrzywa M.
      • Starkenberg A.
      • Hammarstrom P.
      • Thor S.
      Systematic Aβ analysis in Drosophila reveals high toxicity for the 1–42, 3–42 and 11–42 peptides, and emphasizes N- and C-terminal residues.
      ), although the neurotoxicity of Aβ(11-XX) in human brain is controversial (
      • Liu K.
      • Solano I.
      • Mann D.
      • Lemere C.
      • Mercken M.
      • Trojanowski J.Q.
      • Lee V.M.
      Characterization of Aβ11–40/42 peptide deposition in Alzheimer's disease and young Down's syndrome brains: implication of N-terminally truncated Aβ species in the pathogenesis of Alzheimer's disease.
      ).
      Aβ is the major protein component of senile plaques observed in the brain of Alzheimer's disease (AD) subjects, and soluble Aβ oligomer(s) are thought to impair synaptic functions prior to Aβ deposition in the brain (
      • Lue L.F.
      • Kuo Y.M.
      • Roher A.E.
      • Brachova L.
      • Shen Y.
      • Sue L.
      • Beach T.
      • Kurth J.H.
      • Rydel R.E.
      • Rogers J.
      Soluble amyloid β peptide concentration as a predictor of synaptic change in Alzheimer's disease.
      ,
      • McLean C.A.
      • Cherny R.A.
      • Fraser F.W.
      • Fuller S.J.
      • Smith M.J.
      • Beyreuther K.
      • Bush A.I.
      • Masters C.L.
      Soluble pool of Aβ amyloid as determinant of severity of neurodegeneration in Alzheimer's disease.
      • Lesné S.E.
      • Sherman M.A.
      • Grant M.
      • Kuskowski M.
      • Schneider J.A.
      • Bennett D.A.
      • Ashe K.H.
      Brain amyloid-β oligomers in aging and Alzheimer's disease.
      ). Therefore, to understand AD pathogenesis and develop effective AD therapies, it is important to elucidate the molecular mechanisms of Aβ generation and degradation. Regulation of BACE1 activity represents a promising therapeutic option for decreasing Aβ production (reviewed in Ref.
      • Yan R.
      • Fan Q.
      • Zhou J.
      • Vassar R.
      Inhibiting BACE1 to reverse synaptic dysfunction in Alzheimer's disease.
      ).
      In familial AD (FAD), several amino acid mutations have been reported in the APP gene, especially in or around the Aβ sequence. Some of these mutations are pathogenic, e.g. the Swedish (K670N/M671L), Leuven (E682K), and A673V mutations increase Aβ generation by promoting APP β-site cleavage by BACE1 (
      • Mullan M.
      • Crawford F.
      • Axelman K.
      • Houlden H.
      • Lilius L.
      • Winblad B.
      • Lannfelt L.
      A pathogenic mutation for probable Alzheimer's disease in the APP gene at N-terminus of β-amyloid.
      ,
      • Zhou L.
      • Brouwers N.
      • Benilova I.
      • Vandersteen A.
      • Mercken M.
      • Van Laere K.
      • Van Damme P.
      • Demedts D.
      • Van Leuven F.
      • Sleegers K.
      • Broersen K.
      • Van Broeckhoven C.
      • Vandenberghe R.
      • De Strooper B.
      Amyloid precursor protein mutation E682K at the alternative β-secretase cleavage β′-site increase Aβ generation.
      • Di Fede G.
      • Catania M.
      • Morbin M.
      • Rossi G.
      • Suardi S.
      • Mazzoleni G.
      • Merlin M.
      • Giovagnoli A.R.
      • Prioni S.
      • Erbetta A.
      • Falcone C.
      • Gobbi M.
      • Colombo L.
      • Bastone A.
      • Beeg M.
      • et al.
      A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis.
      ). On the other hand, at least one protective mutation exists: the Icelandic mutation, A673T, decreases Aβ generation (
      • Peacock M.L.
      • Warren Jr, J.T.
      • Roses A.D.
      • Fink J.K.
      Novel polymorphism in the A4 region of the amyloid precursor protein gene in a patient without Alzheimer's disease.
      ,
      • Jonsson T.
      • Atwal J.K.
      • Steinberg S.
      • Snaedal J.
      • Jonsson P.V.
      • Bjornsson S.
      • Stefansson H.
      • Sulem P.
      • Gudbjartsson D.
      • Maloney J.
      • Hoyte K.
      • Gustafson A.
      • Liu Y.
      • Lu Y.
      • Bhangale T.
      • et al.
      A mutation in APP protects against Alzheimer's disease and age-related cognitive decline.
      ). Although the effect of the Swedish mutation, which is located outside the amino terminus of the Aβ sequence, is obvious, the molecular mechanisms by which amino acid alterations within Aβ affect the level of Aβ in the brain remain controversial. Because BACE1 cleaves APP either at the amyloidogenic β-site or the amyloidolytic β′-site, we investigated the effect of alternative BACE1 cleavage sites in APP on generation and degradation of Aβ.

      Discussion

      It is widely accepted that β-secretase BACE1 is the primary APP-cleaving enzyme responsible for generation of the Aβ(1-XX) species, and that the combined cleavage of APP by BACE1 and the γ-secretase complex generates multiple types of Aβ species with distinct carboxyl termini, e.g. Aβ(1–40) and Aβ(1–42) (reviewed in Refs.
      • Thinakaran G.
      • Koo E.H.
      Amyloid precursor protein trafficking, processing, and function.
      and
      • Steiner H.
      • Fluhrer R.
      • Haass C.
      Intramembrane proteolysis by γ-secretase.
      ). Previous work showed that BACE1 also cleaves APP at the β′-site, but the biological significance of this reaction remained unclear because the β′-cleaved products of hAPP, CTFβ′/C89, and Aβ(11-XX) are scarce relative to CTFβ/C99 and Aβ(1-XX) (
      • Liu K.
      • Doms R.W.
      • Lee V.M.
      Glu11 site cleavage and N-terminally truncated Aβ production upon BACE overexpression.
      ,
      • Moore B.D.
      • Chakrabarty P.
      • Levites Y.
      • Kukar T.L.
      • Baine A.M.
      • Moroni T.
      • Ladd T.B.
      • Das P.
      • Dickson D.W.
      • Golde T.E.
      Overlapping profiles of Aβ peptides in the Alzheimer's disease and pathological aging brains.
      ). Along with previously reported data, our results confirm that mAPP is preferentially cleaved at the β′-site within the Aβ sequence and predominantly generates Aβ(11-XX); by contrast, hAPP mostly generates Aβ(1-XX) in vivo and in vitro (
      • Gouras G.K.
      • Xu H.
      • Jovanovic J.N.
      • Buxbaum J.D.
      • Wang R.
      • Greengard P.
      • Relkin N.R.
      • Gandy S.
      Generation and regulation of β-amyloid peptide variants by neurons.
      ,
      • Cai H.
      • Wang Y.
      • McCaethy D.
      • Wen H.
      • Borchelt D.R.
      • Price D.L.
      • Wong P.C.
      BACE1 is the major β-secretase for generation of Aβ peptides by neurons.
      • Vanderstichele H.
      • De Meyer G.
      • Andreasen N.
      • Kostanjevecki V.
      • Wallin A.
      • Olsson A.
      • Blennow K.
      • Vanmechelen E.
      Amino-truncated β-amyloid42 peptide in cerebrospinal fluid and prediction of progression of mild cognitive impairment.
      ).
      Over the course of this study, we made several discoveries regarding the role of BACE1 in APP metabolism. First, we showed that alternative selection of cleavage sites by BACE1 in both human and mouse is determined by the amino acid at position 684 (His in human, Arg in mouse). This suggests that some FAD-associated pathogenic and/or protective mutations within the Aβ region of the APP gene influence the selection of cleavage sites by BACE1. Second, we showed that the pathogenic mutation A673V decreased β′-site cleavage of hAPP, whereas the protective Icelandic mutation A673T increased β′-site cleavage. This observation strongly indicates that amino acid substitutions at position 673 influenced the alternative selectivity of the cleavage site by BACE1. In other words, A673V induces the pathogenic β-site cleavage of APP by BACE1, whereas A673T raises the protective β′-site cleavage of APP. This is the primary cause of FAD harboring APP A673V, and this is the primary effect to protect AD in subjects harboring APP A673T. Third, we demonstrated that elevation of BACE1 activity due to overexpression of the enzyme results in secondary cleavage of CTFβ/C99 at the β′-site, dramatically increasing Aβ(11-XX) production. This is true, at least in the cell study, even for APP harboring the Swedish mutation, which predominantly produces Aβ(1-XX), although a pilot study in a BACE-overexpressing transgenic mouse suggested that reduction in Aβ deposition is mediated by another mechanism (
      • Lee E.B.
      • Zhang B.
      • Liu K.
      • Greenbaum E.A.
      • Doms R.W.
      • Trojanowski J.Q.
      • Lee V.M.
      BACE overexpression alters the subcellular processing of APP and inhibits Aβ deposition in vivo.
      ). Finally, we showed that Aβ(11-XX) may be more metabolically labile than Aβ(1-XX) in vivo and in vitro.
      Thus, contrary to the popular view, our findings may suggest that activation of BACE1 in AD subjects (including FAD patients), using procedures other than overexpression of BACE1, represents a promising target for AD therapies aimed at decreasing the level of Aβ. Development of inhibitors of BACE1 and/or γ-secretase activities has been pursued since these enzymes were identified (reviewed in Ref.
      • Yan R.
      • Vassar R.
      Targeting the β secretase BACE1 for Alzheimer's disease therapy.
      ). However, we believe that more attention should have been devoted to γ-secretase inhibitors. It is reasonable to predict that attenuation of γ-secretase activity by an inhibitor would decrease generation of neurotoxic Aβ generation. In contrast to this early idea, recent progress in understanding the molecular mechanism by which γ-secretase cleaves APP suggested to us that attenuating γ-secretase activity would result in reduced production of Aβ(1–38) and elevated production of Aβ(1–42), which is a precursor of Aβ(1–38) and is more neurotoxic than Aβ(1–38) and Aβ(1–40) (
      • Takami M.
      • Nagashima Y.
      • Sano Y.
      • Ishihara S.
      • Morishima-Kawashima M.
      • Funamoto S.
      • Ihara Y.
      γ-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of β-carboxyl terminal fragment.
      ) (reviewed in Ref.
      • Takami M.
      • Funamoto S.
      γ-Secretase-dependent proteolysis of transmembrane domain of amyloid precursor protein: successive tri- and tetrapeptide release in amyloid β-protein production.
      ).
      Our results suggest that β-secretase inhibitors may face some of the same problems as γ-secretase inhibitors. Suppression of APP cleavage using a BACE1 inhibitor is a potential therapeutic strategy for decreasing Aβ generation. However, many other substrates of BACE1 have been reported to date, suggesting that BACE1 inhibition could have considerable side effects. Nevertheless, suppression of BACE1 activity specifically in the brain may be a practical means for treating AD patients (reviewed in Ref.
      • Yan R.
      • Vassar R.
      Targeting the β secretase BACE1 for Alzheimer's disease therapy.
      ). Our results show that cleavage of APP by excess BACE1 activity can degrade Aβ by cleaving APP at the β′-site. Moreover, if CTFβ/C99 is abundant in FAD subjects, sufficient levels of BACE1 activity could cleave CTFβ/C99 again at the β′-site to generate Aβ(11-XX), which is more metabolically labile. Consequently, the total amount of Aβ in the brain would decrease, as demonstrated in BACE1 transgenic mice (
      • Lee E.B.
      • Zhang B.
      • Liu K.
      • Greenbaum E.A.
      • Doms R.W.
      • Trojanowski J.Q.
      • Lee V.M.
      BACE overexpression alters the subcellular processing of APP and inhibits Aβ deposition in vivo.
      ). Because Aβ(1–34) is degraded by neprilysin, an Aβ-degrading enzyme, faster than Aβ(1–40) (
      • Caillava C.
      • Ranaldi S.
      • Lauritzen I.
      • Bauer C.
      • Fareh J.
      • Abraham J.-D.
      • Checler F.
      Study on Aβ34 biology and detection in transgenic mice brains.
      ), Aβ(11-XX) may also be degraded more easily by such enzymes than Aβ(1-XX) (
      • Iwata N.
      • Tsubuki S.
      • Takaki Y.
      • Shirotani K.
      • Lu B.
      • Gerard N.P.
      • Gerard C.
      • Hama E.
      • Lee H.J.
      • Saido T.C.
      Metabolic regulation of brain Aβ by neprilysin.
      ). At least we showed that Aβ(11–40) was degraded quickly rather than Aβ(1–40) in a study in vitro. Therefore, to treat AD patients (e.g. FAD subjects harboring the Swedish mutation), activation of BACE1 might be a more effective therapy than administration of a BACE1 inhibitor. Moreover, in contrast to development of γ-secretase modulators that enhance the peptidase-like activity of γ-secretase to reduce Aβ(1–42) generation while promoting formation of Aβ(1–38), it may be difficult to develop a drug that modifies the selectivity of BACE1 because selection of a cleavage site (i.e. the β- or β′-site) depends on the sequence of the substrate Aβ domain. However, we cannot rule out other possibilities that the intracellular environment of BACE1 can influence in the selectivity of cleavage site of APP by BACE1.
      In general, it is possible that both BACE1 and γ-secretase activities may decrease with age, although at least one report showed that the BACE1 level is elevated in AD (
      • Fukumoto H.
      • Cheung B.S.
      • Hyman B.T.
      • Irizarry M.C.
      β-Secretase protein and activity are increased in the neocortex in Alzheimer disease.
      ). Reduction of BACE1 activity may attenuate the amyloidolytic β′-site cleavage of APP relative to amyloidogenic β-site cleavage, resulting in the generation of Aβ(1-XX). Attenuation of γ-secretase activity, especially its carboxypeptidase-like activity, promotes production of Aβ(XX-42) at the expense of Aβ(XX-38). Therefore, the combination of altering and/or weakening the activities of both BACE1 and γ-secretase increases production of the most neurotoxic species, Aβ(1–42), whereas decreasing production of Aβ(11-XX) and Aβ(XX-38) species. Based on our understanding of the mechanisms of APP cleavage by BACE1 and γ-secretase, administration of compounds to regulate BACE1 and γ-secretase activities to AD subjects should proceed with scrupulous caution.

      Author Contributions

      A. K., S. H., and T. S. participated in the design of study, and A. K. and S. H. carried out all studies. T. S. conceived the study, and T. S. and S. H. wrote the paper. All authors read and approved the final manuscript.

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