The amyloid precursor protein and its homologues: Structural and functional aspects of native and pathogenic oligomerization

European Journal of Cell Biology 91 (2012) 234–239

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Review

The amyloid precursor protein and its homologues: Structural and functional aspects of native and pathogenic oligomerization Daniela Kaden a , Lisa Marie Munter a , Bernd Reif b,c , Gerd Multhaup a,∗ a

Institut für Chemie und Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany Technische Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany c Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und Umwelt, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany b

a r t i c l e

i n f o

Article history: Received 27 December 2010 Received in revised form 24 January 2011 Accepted 24 January 2011 Keywords: Amyloid precursor protein (APP) APLP1 APLP2 Dimer Homodimerization Heterodimerization Structure Amyloid-beta generation

a b s t r a c t Over the last 25 years, remarkable progress has been made not only in identifying key molecules of Alzheimer’s disease but also in understanding their meaning in the pathogenic state. One hallmark of Alzheimer pathology is the amyloid plaque. A major component of the extracellular deposit is the amyloid-␤ (A␤) peptide which is generated from its larger precursor molecule, i.e., the amyloid precursor protein (APP) by consecutive cleavages. Processing is exerted by two enzymes, i.e., the ␤-secretase and the ␥-secretase. We and others have found that the self-association of the amyloid peptide and the dimerization and oligomerization of these proteins is a key factor under native and pathogenic conditions. In particular, the A␤ homodimer represents a nidus for plaque formation and a well defined therapeutic target. Further, dimerization of the APP was reported to increase generation of toxic A␤ whereas heterodimerization with its homologues amyloid precursor like proteins (APLP1 and APLP2) decreased A␤ formation. This review mainly focuses on structural features of the homophilic and heterophilic interactions among APP family proteins. The proposed contact sites are described and the consequences of protein dimerization on their functions and in the pathogenesis of Alzheimer’s disease are discussed. © 2011 Elsevier GmbH. All rights reserved.

Introduction It became widely accepted that the proteolytic processing of APP is a central event in the onset of Alzheimer’s disease. Following the initial ectodomain shedding of APP by ADAM10 (a disintegrin and metalloprotease, also known as ␣-secretase) or BACE1 (␤-site APP cleaving enzyme, also ␤-secretase), the remaining membranebound C-terminal stubs are degraded by the ␥-secretase complex. This process has been named regulated intramembrane proteolysis (RIP) (reviewed in De Strooper, 2010). The concerted action of BACE1 and ␥-secretase leads to the generation of A␤ peptides. Soluble oligomers of A␤ are regarded as the toxic agent and are most likely responsible for neurodegeneration observed in Alzheimer’s disease (Harmeier et al., 2009; Schmechel et al., 2003; Walsh et al., 2002). APP processing by the ␣-secretase creates the soluble APP ectodomain (sAPP␣), which exerts neuroprotective activities (Furukawa et al., 1996; Mattson, 1997; Small et al., 1994). Insulin and insulin growth factor-1 (IGF-1) have been shown to increase ␣-secretase cleavage of APP as well as the ectodomain shedding of the APP-like proteins APLP1 and APLP2 in human neuroblastoma (SH-SY5Y) cells (Adlerz et al., 2007; Jacobsen et al., 2010). Accu-

∗ Corresponding author. Tel.: +49 30 83855533; fax: +49 30 83856509. E-mail address: [email protected] (G. Multhaup). 0171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2011.01.017

mulating evidence suggests that APP and its homologous proteins APLP1 and APLP2 are capable of forming homo- and heterodimers in living cells with a direct impact on APP processing and A␤ generation (Kaden et al., 2008, 2009; Munter et al., 2007). The APP family proteins are type I transmembrane proteins with a large, glycosylated extracellular domain and a short conserved cytoplasmic tail. For APP, dimerization likely occurs as early as in the endoplasmic reticulum and follows a zipper-like mechanism starting from the N to the C terminus involving multiple contact sites. Three different interaction sites are described in the literature, two reside in the ectodomain and one in the transmembrane sequence (TMS) (Beher et al., 1996; Kaden et al., 2008; Munter et al., 2007; Rossjohn et al., 1999; Soba et al., 2005; Wang and Ha, 2004). The physiological functions of APP are still not understood in detail, however, a functional role in cell development, cell–cell and/or cell–matrix interaction is likely. Oligomerization of cell surface receptors and activation in response to ligands is a common mechanism to transfer signals across the cell membrane. A proper signal recognition and such a transduction could not be verified for APP yet although it had been postulated when the full-length form of the molecule was first published (Kang et al., 1987). The Notch receptor is a substrate of the same set of proteases and the Notch intracellular domain (NICD) exhibits important signaling functions in neural development (for review see Woo et al., 2009). However, for Notch dimerization it could not be shown to be decisive for processing (Vooijs et al., 2004).

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Structural features of APP and APLP The APP family proteins contain different domains as shown in Fig. 1A. The linker domains are supposed to be unstructured and their main function is to ensure the flexibility of the other individual domains. In this review, we will discuss structural aspects and proposed functions of the E1 and E2 domains, the ectodomain as such, the transmembrane part and the cytoplasmic domain as the structures help to understand the dimerization processes important for A␤ generation.

The E1 and E2 domains The E1 domain of APP (residues 18–207) contains two independent folding units, the growth factor-like domain (GFLD, 28–123) and the copper-binding domain (CuBD, 127–188) (Barnham et al., 2003; Rossjohn et al., 1999; Small et al., 1994). The crystal structure of the APP GFLD revealed a highly charged basic surface that was supposed to interact with glycosaminoglycans and a hydrophobic surface that being important for ligand binding (Rossjohn et al., 1999). Furthermore, the crystal structure showed a highly flexible region consisting of an N-terminal loop formed by a disulfide bridge between cysteines 98 and 105 (Rossjohn et al., 1999). This socalled loop-region was described to possibly mediate dimerization (Rossjohn et al., 1999). Indeed, biochemical data revealed that the E1 domain itself can dimerize in solution and the self-interaction is gradually diminished by adding a small peptide mimicking the loop region (loop peptide) (Kaden et al., 2008; Scheuermann et al., 2001). Interestingly, the loop peptide decreased the generation of sAPP␤ as well as A␤40 and A␤42 when the synthetic peptide was added to APP-expressing neuroblastoma cells (SH-SY5Y). This indicated a direct or indirect influence of dimerization on APP processing (Kaden et al., 2008). We could further show that the loop’s disulfide bond is indispensable for the effects of the loop peptide, as peptide bearing serine residues instead of cysteines neither bound to APP nor diminished dimerization or influenced APP processing (Kaden et al., 2008). The CuBD (amino acids 127–188/124–189) consists of an ␣helix that is tightly packed on a triple-stranded ␤-sheet (Barnham et al., 2003; Kong et al., 2007). At the copper-binding site, Cu(II) can be reduced to Cu(I), leading to the oxidation of the cysteine residues 144/158 and formation of an intramolecular disulfide bond (Multhaup et al., 1996,1998). This was further supported by structural data showing a tetrahedral or square plane coordination for Cu(II) or Cu(I), respectively (Barnham et al., 2003; Kong et al., 2007). Interestingly, Hesse et al. could show that Cu(II) can inhibit the homophilic binding of an APP fragment to rat APP in vitro (Hesse et al., 1994). Later it was described that treatment of Chinese Hamster Ovary (CHO) cells with copper led to a stimulation of ␣-secretase cleavage (Borchardt et al., 1999). Recently, the structure of the whole APP E1 domain was resolved with a resolution of 2.7 A˚ (Dahms et al., 2010) showing that the two subunits of the E1 domain, the GFLD and CuBD form a rigid entity and do not consist of two independent folding units connected by a flexible linker as earlier suggested (Gralle et al., 2006). Interestingly, the residues that form the structural network between the GFLD and CuBD are conserved between APP and APLP2 but are not conserved between APP and APLP1. This led to the assumption that the E1 domain of APLP1 has an individual substructure (Dahms et al., 2010), which is in excellent agreement with our data published on the specific features of APLP1 (Kaden et al., 2009), which are discussed below (see chapter entitled APP protein family). The E2 domain (amino acids 365–570) is the largest subdomain of the APP ectodomain and consists of six ␣-helices. Wang et al.

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published the X-ray structure of an E2 antiparallel dimer (Wang and Ha, 2004), showing that the N-terminal double stranded coiled coil structure of the first monomer packs against the C-terminal triple stranded coiled coil structure of the second monomer (Wang and Ha, 2004). This structural feature supports earlier data of Hesse et al. (1994), who found that the collagen-binding site in the E2 domain may be involved in APP–APP interactions (Beher et al., 1996). However, in contrast to Wang et al., Dulubova et al. found that the E2 domain does not dimerize in solution (Dulubova et al., 2004). Of note, the fragment analyzed by Dulubova et al. was much shorter (amino acids 460–576) and lacked the N-terminal double stranded helices of the fragment used by Wang et al. (amino acids 365–566), which may explain the contrasting data of the dimerization state. However, somewhat puzzling is the fact that the E2 domain of C. elegans APL-1 was recently also found as a monomer in solution by the same group (Hoopes et al., 2009). This could be due to differences in the human and worm sequences, but could also reflect the fact that dimers are preferentially crystallized over monomers since monomeric proteins can form non-physiological dimers in crystals and then oligomerization may be an artifact of the crystallization conditions (Hoopes et al., 2009). Additional experiments by using more convenient methods need to be performed to clarify the physiological relevance of the oligomeric state and orientation of the E2 domains of APP, its orthologs APPL and APL-1, and its homologous proteins APLP1 and APLP2. The ectodomain There are only few data on the structure of the APP ectodomain as a whole and most are based on small angle X-ray scattering (SAXS) modeling. Conflicting data about dimerization exist for soluble APP generated by ADAM10 cleavage (sAPP␣). While we found the APP ectodomain (sAPP␣) purified from Pichia pastoris dimerizes in solution by cross-linking and size-exclusion chromatography, others only described sAPP monomers, or rather found dimers only in the presence of heparin (Gralle et al., 2002, 2006; Gralle and Ferreira, 2007; Kaden, 2007). This discrepancy could be due to differences in methods and buffers used for purification. Our data further show that the ectodomains of APLP1 and APLP2 were not only dimeric but can also form tetramers in solution, further supporting the hypothesis of self interactions in the APP protein family (Kaden, 2007). The ectodomains of APP and APLPs possess multiple binding sites for metal ions and components of the extracellular matrix substantiating possible functions of the APP family proteins in cell–matrix interactions. These ligands include copper, zinc, collagen and heparan sulfate that all influence each other in their binding strength (Beher et al., 1996; Breen et al., 1991; Bush et al., 1993; Multhaup et al., 1996; Small et al., 1994). Interestingly, APP and APLP2 can bind heparan sulfate in their E1 and E2 domains, whereas APLP1 has only one binding site in the E2 domain, again emphasizing the differences between the E1 domain of APP or APLP2 and APLP1 (Bush et al., 1994; Multhaup et al., 1994, 1995). Furthermore, there might be a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the heparan sulfate degradation in glypican1 as the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts (Cappai et al., 2005). The transmembrane region and the A␤ sequence The A␤ peptide encompasses the N-terminal juxtamembrane region (28 amino acid residues) as well as half of the TMS (Fig. 1B).

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Fig. 1. Schematic representation of dimers in the APP protein family and the A␤ sequence. (A) E1 domain (blue) with the subdomains GFLD (growth factor like domain) in turquoise and CuBD (copper-binding domain) in green for APP and APLP2. Referring to the structural differences of APLP1 no subdomains of the APLP1 E1 domain are described. The E2 domain is shown in yellow, the transmembrane sequence (TMS) in light grey. Only APP contains functional GxxxG motifs. AcD, acidic domain; ICD, intracellular domain of the individual protein; A␤, amyloid-␤ peptide region; ␣, ␤ and ␥ indicate the approximate position of secretase cleavages (see details in B). The E1 domain mediates homo- and heterophilic interactions of APP and APLP2 mainly through the GFLD. By contrast, the E1 domain is dispensable for dimerization of APLP1. The E2 domain of APLP1 substitutes the function of the E1 domain in initiating the interaction. However, for APP and APLP2 dimerization of the E1 domain is required to initiate the contact, which is then stabilized by the E2 domain. The brown line indicates heparin/heparan sulfate that might have a stabilizing function on the APP and APLP2 dimers. (B) Sequence of the amyloid ␤ (A␤) peptide. Positions of cleavages by the respective proteases are marked by arrows. Red rectangle indicates A␤, the grey rectangle the TMS. Glycines of the three consecutive GxxxG motifs are numbered and shown in bold letters. The juxtamembrane and TMS region of APP is unique as the APLPs do not contain the A␤ sequence. Although they share a high overall percentage of amino-acid identity (Walsh et al., 2007), the percentage of identity in this region is quite low, i.e. 20% identity between APP and APLP1 (overall 56%), 37% for APP and APLP2 (overall 68%) and only 15% for APLP1 and APLP2 (overall 59%).

Amyloid plaques purified from brain tissue contain mostly aggregated A␤ peptides. In the past few years, it turned out that oligomeric intermediate states, not the fibrillar end-products of the aggregation process are responsible for the neurotoxic effects that A␤ exerts in vivo (Hartley et al., 1999). Recently, several attempts to characterize the structure of these oligomers have been undertaken (Ahmed et al., 2010; Chimon et al., 2007; Yu et al., 2009). Although the results differ somehow, all groups consistently demonstrated that the structured regions in A␤ are rich in ␤-sheet secondary structure. Recently, we found that conformational changes induced in the A␤ sequence by substitutions at position G33 unlink toxicity and oligomerization of A␤ and revealed that G33 is the key amino acid in mediating toxic activity (Harmeier et al., 2009). Using solution-state NMR, the oligomeric intermediate state can be monitored via chemical exchange to the random coil monomeric state. This way, the regions of the peptide which are involved in A␤–A␤ interactions could be identified (Narayanan and Reif, 2005). It was postulated, that fibrillization involves an ␣-helical intermediate state that precedes the conversion into ␤-sheets (Kirkitadze et al., 2001). An ␣-helical intermediate state is in fact observed in bacterial inclusion bodies which are formed by the amyloid peptides A␤40 and A␤42 (Dasari et al., 2011). H/D exchange experiments yield a periodic i,i+4 exchange protection pattern at the C-termini of the peptides, which is expected for coiled-coil helices. This is reminiscent of the dimeric glycine zipper type conformation that has been recently suggested for the transmembrane region of APP (Munter et al., 2007). This region encompasses three consecutive GxxxG motifs (G25 SNKGAIIGLMVG38 ) with the central motif representing the third APP–APP contact site. These motifs are known to mediate the dimerization of TMSs as the small glycine residues form a

groove on an ␣-helical surface and permit the close proximity of two helices (MacKenzie et al., 1997). The interaction within the hydrophobic environment of the membrane is then stabilized by backbone hydrogen bonding (C␣ –H. . .O) and interactions of the neighboring amino-acid side chains (Munter et al., 2007). Using a bacterial reporter gene assay (ToxR assay) developed by Langosch et al. (1996), we could show that the APP–TMS strongly dimerizes similar to the well-investigated TMS of Glycophorin A containing a single GxxxG motif (Munter et al., 2007). By a mutational approach glycine residues 29 and 33 were shown to be the key residues in the APP–TMS contact. A substitution to alanine (G33A) already attenuated the dimer stability significantly. Amino acid exchanges of other positions of the TMS had negligible effects on the dimerization strength (Munter et al., 2007). Importantly, a strengthened APP–TMS dimerization correlated with a high level of A␤42 generation whereas a decreased dimer stability facilitated A␤38 production (Munter et al., 2007). We also found that most FAD mutations in APP do not affect APP–TMS dimer stability and molecular mechanisms leading to an enhanced A␤42/A␤40 ratio are located downstream in the processing cascade (Munter et al., 2010). NMR analysis of two independent groups corroborated that G29 and G33 of the GxxxG interface represent key positions in the contact site although alternative interfaces might also be possible (Beel et al., 2008; Sato et al., 2009). Recently, we could show that APP–TMS dimerization is a perfect target side for alternative therapeutic approaches since it can indeed be destabilized by compounds like sulindac sulfide and indomethacin that bind to the GxxxG interface of the APP–TMS and thereby reduced A␤42 production while A␤38 was found to be increased which is a wanted effect of anti-amyloidogenic approaches (Richter et al., 2010).

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The cytoplasmic domains and their role in signaling The cytoplasmatic tail of APP comprises 47 amino acid residues containing a YENPTY sequence with a proposed dual function. On the one hand, the NPxY motif is a common signal for endocytosis and is necessary for the internalization of APP (Lai et al., 1995). On the other hand, the sequence mediates binding to various interacting partners, like Fe65 and X11 (summarized in Jacobsen and Iverfeldt, 2009). In aqueous solutions, the cytoplasmatic domain shows only transiently structured regions (Ramelot et al., 2000). Binding of the phosphotyrosine-binding domain (PTB) of Fe65 stabilizes two helices that are capped by the TPEE or YENPTY sequence motifs (Kroenke et al., 1997; Radzimanowski et al., 2008). Both motifs contain threonine and/or tyrosine phosphorylation sites that might function as a molecular switch for binding partners (Ramelot and Nicholson, 2001; Suzuki and Nakaya, 2008). The APP intracellular domain (AICD) that is generated after regulated intramembrane proteolysis by the ␥-secretase is thought to modulate gene transcription in concerted action with various adapter proteins (Cao and Sudhof, 2001). Similar to APP, the intracellular domains of APLP1 and APLP2 denoted as AICD-like fragments ALID1 and ALID2, respectively, contain the YENPTY motif but only APLP2 has a TPEE motif. ALID1 and 2 are also likely stabilized by Fe65 and are suggested to regulate gene transcription (Scheinfeld et al., 2002). However, dimerization of the APP C-terminal fragment was only shown for fragments containing the TMS in addition (Beel et al., 2008; Munter et al., 2007) but has so far never been described for AICD, ALID1 or ALID2 as such.

The APP protein family Studies with APP and APLP1 knockout mice have revealed partially redundant functions of the APP family proteins. Whereas all single knockouts are viable and fertile, the double knockouts of APP/APLP2 and APLP1/APLP2 are perinatally lethal (Heber et al., 2000; von Koch et al., 1997; Zheng et al., 1995) suggesting that APLP2 is functionally important and unique within the APP protein family. The APP/APLP1 double knockout is also viable and fertile and no grossly changed phenotype was observed (Heber et al., 2000). However, whereas APP and APLP2 are ubiquitously expressed, APLP1 is exclusively localized to the brain (Bayer et al., 1997) and behaves differentially in terms of subcellular localization and dimerization properties (Kaden et al., 2009). As for many other transmembrane proteins, there are no structures of the full-length APP or APLPs available to date. Meanwhile, independent analyses from several laboratories showed homodimerization of membrane-bound APP by two different FRET (Fluorescence or Förster Resonance Energy Transfer) approaches and a bimolecular fluorescence complementation assay (Chen et al., 2006; Gralle et al., 2009; Munter et al., 2007). FRET measurements revealed that only about 35–40% of APP exist as monomers, when wild-type dimers were compared to covalently disulfide-bonded dimers formed by engineered mutants (Gralle et al., 2009; Munter et al., 2007). Singlemolecule tracking using membrane impermeable quantum dots of APP dimers displayed that an even higher proportion of wild-type APP was claimed to be present as a dimer at the cell surface (Gralle et al., 2009). By characterizing the localization of the individual APP family members we found that APLP1 is mainly localized to the plasma membrane, whereas APP and APLP2 are mostly found in intracellular compartments and only a minor amount of the latter were present on the cell surface (Kaden et al., 2009). Interestingly, coexpression of APLP1 with one of the other family proteins led to a partial retention of APLP1 in intracellular compartments, indicating heterophilic interactions occur. We could clearly demonstrate

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homo- as well as heterodimerization of the APP family proteins in living cells by FRET analysis (Kaden et al., 2009). Furthermore, by using N-terminal deletion mutants we figured out that the mode of interaction of APLP1 noticeably differs from APP and APLP2 (Kaden et al., 2009). The model shown in Fig. 1A illustrates that for the interactions of APP and APLP2 the E1 domain and in particular the GFLD is indispensible, whereas for APLP1 the E1 and E2 domain have a similar impact on dimerization. When deleting the E1 domain of either APP or APLP2, the homodimerization as measured by FRET efficiency is diminished completely, but is only reduced by 50% when the APLP1 E1 domain is absent (Kaden et al., 2009). Further support is obtained by in vivo results from different studies in the brain describing APP and APLP interactions (Scheuermann et al., 2001; Soba et al., 2005). Bai et al. corroborated the homo- and heterophilic interactions of the APP family in an in vivo brain interactome study and could further show that APP, APLP1 and APLP2 exhibit obviously different protein interaction networks, supporting the hypothesis that next to the high abundancy there are also clearly distinct functions in this conserved protein family (Bai et al., 2008). Conclusion Oligomerization is a key factor in the regulation of proteins in general and especially in Alzheimer’s disease as non-native oligomers of A␤ peptides are associated with the pathogenic states. Key molecules in the development of Alzheimer’s disease natively oligomerize, i.e. substrates (APP and APLPs) and the enzymes BACE1 and ␥-secretase (not discussed in this review). Although there is an ongoing discussion if the APP ectodomain and the single domains dimerize in solution, it is quite clear that the full-length membrane bound APP and APLPs interact with each other not only in vitro and in cellular models but also in vivo. Furthermore APP and APLP2 are more redundant in terms of localization and mechanism of dimerization than the brain protein APLP1. Native oligomers of APP, APLP1 and APLP2 are probably needed to carry out their physiological functions. However, dimerization is also a pathogenic event leading to the complex amyloidogenic cascade as we found that dimerization plays a major role in the generation of A␤ peptides. Howsoever we analyzed APP dimerization it always turned out that dimers promote the formation of the causative agent A␤42 whereas monomers or APP–APLP heterodimers would be protective against it. Although we did not discuss the oligomerization of BACE1 and the ␥-secretase, we think it is important to mention that we and others already described the occurrence of BACE1 dimers (Schmechel et al., 2004; Westmeyer et al., 2004) and that several groups also reported multimerization of presenilin-1 subunits in the ␥-secretase complex (Cervantes et al., 2004; Schroeter et al., 2003). Therefore it might be interesting to elucidate how the enzyme’s self interactions are connected to APP or APLP2 dimerization as it could be possible that a dimeric substrate requires also a set of twin enzymes for getting processed. From our point of view, these data lead to the assumption that the protein’s dimerization may constitute a functional mechanism which is intimately linked to the pathogenesis of Alzheimer’s disease. References Adlerz, L., Holback, S., Multhaup, G., Iverfeldt, K., 2007. IGF-1-induced processing of the amyloid precursor protein family is mediated by different signaling pathways. J. Biol. Chem. 282, 10203–10209. Ahmed, M., Davis, J., Aucoin, D., Sato, T., Ahuja, S., Aimoto, S., Elliott, J.I., Van Nostrand, W.E., Smith, S.O., 2010. Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils. Nat. Struct. Mol. Biol. 17, 561–567. Bai, Y., Markham, K., Chen, F., Weerasekera, R., Watts, J., Horne, P., Wakutani, Y., Bagshaw, R., Mathews, P.M., Fraser, P.E., et al., 2008. The in vivo brain interactome of the amyloid precursor protein. Mol. Cell. Proteom. 7, 15–34.

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