The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol.

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.

Alzheimer's disease (AD) is characterized by the deposition of amyloid in the extracellular compartment of the brain in the form of congophilic amyloid angiopathy (CAA) and amyloid plaques (APs). Intracellular neurofibrillary tangles (NFTs) (88) formed from the abnormally phosphorylated cytoskeletal protein tau are also seen (52). The identification of the amyloid β protein (Aβ) in CAA and APs (28 ; 58) led to the cloning of the amyloid protein precursor (APP) (44). The discovery of familial AD (FAD) mutations in the APP gene (10 ; 29 ; 63 ; 64 ; 86) has supported the view that a defect in APP metabolism or function is directly involved in AD pathogenesis. The demonstration that mutations in the tau gene can lead to non-Alzheimer dementias with neurofibrillary pathology, lacking Aβ plaques (reviewed by 81), has reinforced the view that the NFTs are a secondary phenomenon in the pathogenesis of AD. It has long been argued that the deposition of amyloid is an early step in AD pathogenesis (58 ; Hardy and Higgins, 1992 ; 57). The term amyloid refers to insoluble proteinaceous deposits that are congophilic and exhibit red-green birefringence in the presence of plane polarized light (45). Implicit in much of the research on the role of APP and Aβ has been the assumption that deposits of amyloid are toxic to the brain (42) and that these deposits are the underlying cause of AD. The observation that Aβ peptides when "aged" (incubated to form amyloid fibrils) become toxic to neurons in culture (94 ; 22 ; 46 ; 66 ; 38) has further supported this view. The amyloid cascade hypothesis of AD, as formalized by Hardy and Higgins (35), states that Aβ"precipitates to form amyloid and, in turn, causes neurofibrillary tangles and cell death." However, this hypothesis has been challenged (see, e.g., 16 ; 34). It has been argued that the deposition of amyloid does not correlate with dementia (89 ; 2 ; 71 ; 72 ; 5), although the failure to observe a correlation may be related to the method by which AP load is measured (14). Whether amyloid deposits have a pathogenic role remains a controversial issue. Some of the neuropathological changes occurring in the AD brain were first described by Alzheimer (1). Extracellular deposits of amyloid in the form of APs and CAA, as well as intracellular NFTs, are major features of AD pathology (68). The major protein constituent of CAA and APs is a 4-kDa polypeptide termed the amyloid protein or Aβ (28 ; 58). A partial amino acid sequence of Aβ was used to clone a cDNA encoding a protein now referred to as the APP, which has features of an integral type I transmembrane glycoprotein (44). The APP gene contains 18 exons spanning >170 kb (95). The region encoding the Aβ sequence comprises part of exons 16 and 17 and contains between 40 and 43 amino acid residues that extend from the ectodomain into the transmembrane domain of the protein (Fig. 1). Primary structure of APP. Upper panel : APP is expressed initially as a type I transmembrane glycoprotein containing a large N-terminal ectodomain, a short transmembrane (TM) domain of hydrophobic amino acid residues, and a relatively short C-terminal cytoplasmic domain. The Aβ sequence makes up part of the ectodomain and extends partly into the TM domain. The APP gene encodes a protein containing a signal peptide sequence (SP), a cysteine-rich domain, a region rich in acidic residues, a domain with homology to Kunitz-type protease inhibitors (KPI), and a region sharing homology to the OX-2 protein. mRNA splicing can produce forms that lack the KPI or OX-2 domain. Lower panel : Amino acid sequence of the Aβ region of APP. Large arrows show the major sites of cleavage for α-, β, and γ-secretases. Small arrows show known FAD mutations. FIG. 1. It is now known that Aβ is a normal product of APP processing (32 ; 30 ; 21). The major route of APP processing is by α-secretase, an enzyme that cleaves within the Aβ sequence (20). Cleavage by β- and γ-secretases at the N- and C-terminal ends of the Aβ sequence liberates the Aβ polypeptide, which can subsequently be secreted from cells (21 ; 30 ; 31). The major form of Aβ that is secreted contains 40 amino acids (Aβ1-40). However, minor species containing 42 or 43 amino acid residues (Aβ1-42/43) are also produced. These extended forms of Aβ aggregate more readily and may seed amyloid fibril polymerization during the early stages of plaque formation (42). The strongest evidence for a pathogenic role for APP or Aβ comes from genetic studies of early-onset FAD (33). Several FAD mutations have been found in the APP gene. All of these mutations have been found to cluster close to the amyloid sequence in APP. Mutations at codon 716 (Florida), 717 (London), and 723 (Australian) cause an increased proportion of γ-secretase cleavage at position 42 or 43 in the amyloid sequence (Fig. 1 and Table 1). A mutation found at codons 670 and 671 in a Swedish kindred results in increased β-secretase cleavage (8 ; 43), whereas a point mutation at codon 612 (Flemish) inhibits α-secretase cleavage (32). The consequence of the Swedish and Flemish mutations is to increase processing of APP via the β-secretase pathway. All of the FAD mutations in the APP gene result in increased production of Aβ1-42/43 (Table 1). More than 40 FAD mutations in the presenilin 1 and presenilin 2 genes have also been reported (17 ; 75). The common feature of all these mutations is that they also cause an increase in the production of Aβ1-42/43 (73). Thus, presenilins are involved in regulating the proteolytic breakdown of APP by γ-secretase (18). However, the mechanism by which this occurs is unknown. TABLE 1. Studies on transgenic mice that overexpress FAD mutant forms of human APP also strongly argue for a central role of APP or Aβ in disease pathogenesis. Games et al. (26) demonstrated Alzheimer-like pathology in a transgenic mouse overexpressing a mutant (V717F) form of APP. Subsequently, Hsiao et al., (40) demonstrated similar pathology in a mouse expressing another mutant form of APP (Swedish mutation). It is interesting that the Hsiao mouse had behavioral changes indicative of a cognitive defect occurring before the appearance of robust plaque pathology. More recently, AD-like pathology has been reported in two other APP transgenic mice carrying FAD mutations (84). Although these mice do not show NFTs, there are behavioral abnormalities, abnormal neuritic processes, neuron and synapse loss, and biochemical abnormalities reminiscent of AD (26 ; 40 ; 56 ; 26 ; 9 ; 23 ; 41 ; 65 ; 79). The validity of these transgenic mice as models of AD pathology is reinforced by the observation that the AD-like phenotype is accelerated in APP transgenic mice that also contain an FAD mutant presenilin 1 transgene (37). Studies on the genetics of AD and on APP transgenic mice provide compelling evidence that a disturbance in APP metabolism or function is the underlying cause of AD. However, these studies do not prove that Aβ is the causative agent. The strongest evidence implicating Aβ in the pathogenesis of AD comes from the observation that Aβ peptides are toxic to neurons in culture (94 ; 22 ; 46 ; 66 ; 38). This toxicity is enhanced if the peptides are "aged" (incubated from hours to days), a procedure that increases amyloid fibril formation (66). Although the process of aging increases the number of amyloid fibrils formed from Aβ, this is not proof per se that fibrils are the major toxic form of Aβ. It is likely that the levels of soluble oligomeric species of Aβ are also increased by the process of aging (see next section). The mechanism of neurotoxicity is unclear. Some studies suggest that Aβ can disrupt calcium homeostasis (Mattson et al., 59, 61,60), perhaps by interfering with L-type voltage-dependent calcium channels (15 ; 90), Aβ may reduce Na+,K+-ATPase activity (55), thereby influencing membrane depolarization. Furukawa and Mattson (25) have reported that cytochalasin D, a compound that inhibits actin polymerization and calcium entry, can reduce Aβ neurotoxicity. Other studies suggest a role for reactive oxygen species in Aβ toxicity (4 ; 6 ; 36). Disturbances in redox potential may lead to disruption of calcium homeostasis, as reactive oxygen species can impair ATPase activities (55). Aβ may cause lipid peroxidation and affect superoxide dismutase, which may contribute to its neurotoxicity in culture (7). The receptor that transduces the effects of Aβ is unknown, although the receptor for advanced glycation end products (RAGE) (91) has been implicated. Studies by Yan et al. (92) suggest that Aβ may also bind an intracellular hydrosteroid dehydrogenase known as ERAB. The neurotoxicity may be mediated by an indirect action of Aβ on a nonneuronal cell. For example, microglial cells are often found in association with neuritic plaques (69), and Aβ has been shown to activate microglia in culture (11). Therefore, the possibility that Aβ stimulates release of an unidentified neurotoxic agent from a nonneuronal compartment must also be considered. Recent work by Geula et al. (27) has shown that when aged Aβ is injected into the brains of old rhesus monkeys, it is neurotoxic. However, injection of the same material into young monkeys has little toxic effect. This suggests that although Aβ may be pathogenic, there must be other age-related susceptibility factors that are also important to generate a toxic reaction in vivo. There is now considerable evidence that the type 4 allele of the apoE gene is a major susceptibility factor for late-onset AD (82). ApoE is a 299-amino acid glycoprotein that is principally involved in lipid transport and related functions (reviewed by 54). The N-terminal domain (residues 1-191) contains a receptor-binding region (residues 136-150) that exists as a four-helix bundle. The C terminus is hydrophobic and contains the lipoprotein-binding determinants. Genetic heterogeneity leads to three common isoforms, designated as apoE2, apoE3, and apoE4, encoded by three alleles called ε2, ε3, and ε4, respectively. The isoforms differ from each other by cysteine-arginine interchanges at positions 112 and 158 (54). The ε4 allele frequency is significantly increased in late-onset AD patients (82). Furthermore, those individuals with one or two copies of the ε4 allele have higher amounts of Aβ immunoreactivity in their brains than those individuals without ε4 (74), suggesting that apoE4 promotes fibrillation of Aβ to form amyloid. There is evidence from several studies to suggest that apoE4 could be involved in the polymerization of Aβ to form amyloid in vivo. For example, apoE4 has been found to promote Aβ fibrillogenesis in vitro more readily than apoE3 (Strittmatter et al., 82,83 ; 53). Also studies in which apoE knockout mice have been crossed with human APP transgenic mice show that the expression of apoE is necessary for amyloid deposition in vivo (3). However, the mechanism by which apoE4 influences the risk of AD is still unknown. Although apoE can bind Aβ (Strittmatter et al., 82,83), most studies showing that apoE4 stimulates Aβ aggregation have used apoprotein, i.e., delipidated, forms of apoE, which do not possess a native conformation. Indeed, studies using native forms of apoE isoforms suggest that apoE4 binds less well to Aβ than the other isoforms (49 ; 96 ; 93). On the basis of this finding it has been proposed that apoE may be involved in clearance of Aβ (92), although this hypothesis would not explain why apoE knockout inhibits Aβ deposition (3). Molecular genetic studies indicate a central role for Aβ in AD pathogenesis. However, these studies do not indicate the form or site of action of Aβ neurotoxicity. Until the mechanism of Aβ neurotoxicity is understood, it will be difficult to explain the topography of neurodegeneration (77). It has been presumed that deposits of amyloid constitute the entire Aβ load, but recent studies indicate that some of the Aβ in the brain exists in a soluble form. Soluble Aβ is unlikely to be detected by routine fixation and immunostaining. Aβ is probably secreted as a monomer and subsequently aggregates into soluble oligomers or fibrils (67). There is good evidence for the existence of low-molecular-weight Aβ oligomers in the brain. Studies by Kuo et al. (47) have isolated watersoluble Aβ oligomers from normal and AD brains. Perhaps of greatest interest in this study was the finding that not only was the level of soluble Aβ greater in the AD brain compared with controls, but as with some familial mutations, the proportion of soluble Aβ1-42/43 was significantly increased over soluble Aβ1-40 species in AD patients. Similar results have been obtained by Funato et al. (24). In the study by Kuo et al. (47), the watersoluble Aβ species ranged in size from monomers of <10 kDa to oligomers of > 100 kDa. Studies by Roher et al. (70) suggest that the watersoluble dimeric species are neurotoxic, whereas in a recent study Lambert et al. (50) found that small, low-molecular-weight oligomers of Aβ1-42 are several orders of magnitude more potent neurotoxins than high-molecular-weight fibrillar species of Aβ1-40. These studies have implications for our view of amyloid toxicity. If oligomeric soluble forms of Aβ have a pathogenic role, then is it possible that APs are not the major toxic form of Aβ in the brain (Fig. 2) ? Model describing pathways of APP processing. APP can be cleaved by β- and γ-secretases to yield Aβ1-42/43 (γ42/43) or Aβ1-40 (γ40), which can be actively secreted. Alternatively, APP can be cleaved by α-secretase (α) to yield sAPPα, which several studies (reviewed by 78) have shown may have neuroprotective or trophic functions. Aβ1-42/43 can aggregate to form soluble oligomeric species or may seed the polymerization of Aβ1-40 to form insoluble amyloid fibrils, which are deposited in the form of APs. FIG. 2. Recently, Crook et al. (13) described an unusual variant of AD involving a deletion of exon 9 of the presenilin 1 gene from the mRNA. Both NFTs and Aβ-immunopositive plaques were present, but the plaques were of the diffuse nonneuritic (nonfibrillar) type. Like the other FAD mutations in the presenilin 1 gene, the exon 9 deletion also increases Aβ1-42/43 production (62). There are at least two possible explanations for the existence of an AD variant with only diffuse plaques. As previously considered, 42- or 43-residue-long forms of Aβ may be secreted to exert a neurotoxic action (Fig. 2). However, a second possibility is that intraneuronal Aβ plays a role in pathogenesis. Although the molecular genetic studies strongly argue for a direct role of Aβ1-42/43 in AD pathogenesis, they do not provide any indication of whether the Aβ is extracellular or intracellular. Skovronsky et al. (76) have shown that Aβ1-42/43 accumulates preferentially in an insoluble intracellular fraction where it is more abundant than Aβ1-40. Lee et al. (51) have localized intracellular Aβ1-42/43 to the endoplasmic reticulum. Clearly, if Aβ accumulates within intracellular organelles, it could have profound effects on normal cellular protein trafficking and metabolism. If Aβ1-42/43 is the real culprit in AD, then an inverse correlation between the amount of Aβ1-42/43 production and the age of onset of the disease might be predicted. Mutations causing high levels of Aβ1-42/43 in cell culture should cause an early age of onset of clinical symptoms. However, studies with fibroblasts taken from FAD patients (73) show that this is not the case (17). There are several possible explanations for this. It is possible that the level of Aβ1-42/43 production in cell culture does not reflect the level in brain. Another possibility is that FAD mutations influence other cellular events that affect the age of onset. A third possibility is that Aβ1-42/43 is not the only (or even the major) pathogenic form of Aβ. Although Aβ peptides terminating in positions 39-43 are the major forms produced, it is possible that very low levels of previously undetected longer forms of Aβ may also exist (12). If they do exist, they could also be neurotoxic. This would be consistent with observations that C-terminal fragments of APP containing the Aβ sequence are more toxic than Aβ (80). There is very good evidence that Aβ accumulation is the underlying cause of FAD, and there is strong circumstantial evidence to suggest that a similar process underlies the pathogenesis of sporadic (late-onset) AD. Although amyloid deposits (APs and CAA) are markers of the disease, insoluble fibrillar Aβ may not be the main neurotoxic form. Low-molecular-weight diffusible forms of Aβ1-42/43 may also be important. The fact that aged Aβ in vitro contains more amyloid fibrils does not necessarily prove that amyloid is neurotoxic. It is likely that aging also produces increased amounts of soluble oligomeric Aβ species. Therefore, more work is needed to define the precise nature of the toxic form of Aβ and to delineate the mechanism of this toxicity. D.H.S. is supported by grants from the National Health and Medical Research Council of Australia and the Rebecca L. Cooper Foundation.

SummaryThe various membranes of eukaryotic cells differ in composition, but it is at present unclear if this results in differences in physical properties. The sequences of transmembrane domains (TMDs) of integral membrane proteins should reflect the physical properties of the bilayers in which they reside. We used large datasets from both fungi and vertebrates to perform a comprehensive comparison of the TMDs of proteins from different organelles. We find that TMDs are not generic but have organelle-specific properties with a dichotomy in TMD length between the early and late parts of the secretory pathway. In addition, TMDs from post-ER organelles show striking asymmetries in amino acid compositions across the bilayer that is linked to residue size and varies between organelles. The pervasive presence of organelle-specific features among the TMDs of a particular organelle has implications for TMD prediction, regulation of protein activity by location, and sorting of proteins and lipids in the secretory pathway.

More than half of the mutations associated with familiar Alzheimer's disease have been found in the transmembrane domain of amyloid precursor protein (APP). These pathogenic mutations presumably influence the APP transmembrane domain structural and dynamic properties and result in its conformational change or/and lateral dimerization. Despite much data about the pathogenesis of Alzheimer's disease, the initial steps of the pathogenesis remain unclear so far. For the investigation of the molecular basis of Alzheimer's disease, we selected amyloid precursor protein fragment APP671-726 containing the transmembrane and metal-binding domains. This fragment is the substrate of the γ-secretase complex whose abnormal activity leads to the formation of amyloidogenic Aβ42 peptides. This work for the first time describes a highly effective cell-free APP671-726 production method and improved method of bacterial synthesis. Both methods yield milligram quantities of isotope-labeled protein for structural study by high resolution NMR spectroscopy in membrane mimicking milieus.

Alzheimer disease (AD) is characterized by senile plaques, which are mainly composed of beta amyloid (Abeta) peptides. Abeta is cleaved off from amyloid precursor protein (APP) with consecutive proteolytic processing: beta-secretase, followed by gamma-secretase. Here, we show that BRI3, a member of the BRI gene family that includes the familial British and Danish dementia gene BRI2, interacts with APP and serves as an endogenous negative regulator of Abeta production. BRI3 colocalizes with APP along neuritis in differentiated N2a cells; endogenous BRI3-APP complexes are readily detectable in mouse brain extract; reducing endogenous BRI3 levels by RNA interference results in increased Abeta secretion. BRI3 resembles BRI2, because BRI3 overexpression reduces both alpha- and beta-APP cleavage. We propose that BRI3 inhibits the various processing of APP by blocking the access of alpha- and beta-secretases to APP. However, unlike BRI2, the binding of BRI3 to the beta-secretase cleaved APP C-terminal fragment is negligible and BRI3 does not cause the massive accumulation of this APP fragment, suggesting that, unlike BRI2, BRI3 is a poor gamma-cleavage inhibitor. Competitive inhibition of APP processing by BRI3 may provide a new approach to AD therapy and prevention.

The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.

E-mail: yakim@hufs.ac.krReceived December 27, 2012, Accepted January 21, 2013The syndecan family consists of four transmembrane heparan sulfate proteoglycans present in most cell typesand each syndecan shares a common structure containing a heparan sulfate modified extracellular domain, asingle transmembrane domain and a C-terminal cytoplasmic domain. To get a better understanding of themechanism and function of syndecan-4 which is one of the syndecan family, it is crucial to investigate its three-dimensional structure. Unfortunately, it is difficult to prepare the peptide because it is membrane-bound proteinthat transverses the lipid bilayer of the cell membrane. Here, we optimize the expression, purification, andcharacterization of transmembrane, cytoplasmic and short extracellular domains of syndecan4 (syndecan-4eTC). Syndecan-4 eTC was successfully obtained with high purity and yield from the M9 medium. Thestructural information of syndecan-4 eTC was investigated by MALDI-TOF mass (MS) spectrometry, circulardichroism (CD) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. It was confirmed thatsyndecan-4 eTC had an α-helical multimeric structure like transmembrane domain of syndecan-4 (syndecan-4 TM) in membrane environments. Key Words : Syndecan, Syndecan-4, Transmembrane, NMR spectroscopy, Membrane proteinIntroductionThe syndecan family consists of heparan sulfate proteo-glycans, which are present on the surface of all cell types inhumans. This family regulates cell-to-cell interaction, celladhesion, cell proliferation and angiogenesis, as well ashelps in healing wounds by activation of growth factors.

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by beta-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an alpha-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.

Bacterial expression, purification, and model membrane reconstitution of the transmembrane and cytoplasmic domains of the human APP binding protein LR11/SorLA for NMR studies

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.

Accumulation of the amyloid beta (Abeta) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Abeta generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Abeta generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced beta-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Abeta generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.

One of the most prominent drug targets for the treatment of Alzheimer disease is gamma-secretase, a multi-protein complex responsible for the generation of the amyloid-beta peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of gamma-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp257 and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-beta peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.

The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

Cholesterol-protein interactions play a critical role in lipid metabolism and maintenance of cell integrity. To elucidate the molecular mechanisms underlying these interactions, a growing number of studies have focused on determining the crystal structures of a variety of proteins complexed with cholesterol. These include structures in which cholesterol binds to transmembrane domains, and structures in which cholesterol interacts with soluble ones. However, it remains unknown whether there are differences in the prerequisites for cholesterol binding to these two types of domains. Thus, to define the molecular determinants that characterize the binding of cholesterol to these two distinct protein domains, we employed the database of crystal structures of proteins complexed with cholesterol. Our analysis suggests that cholesterol may bind more strongly to soluble domains than to transmembrane domains. The interactions between cholesterol and the protein in both cases critically depends on hydrophobic and aromatic residues. In addition, cholesterol binding sites in both types of domains involve polar and/or charged residues. However, the percentage of appearance of the different types of polar/charged residues in cholesterol binding sites differs between soluble and transmembrane domains. No differences were observed in the conformational characteristics of the cholesterol molecules bound to soluble versus transmembrane protein domains suggesting that cholesterol is insensitive to the environment provided by the different protein domains.