Transmembrane Domain Of Amyloid Precursor Protein Research Articles

Insights into Amyloidogenesis

The structure of the amyloid precursor protein transmembrane domain allows processive cleavage and cholesterol binding that may enhance cleavage.

Peptides based on the presenilin‐APP binding domain inhibit APP processing and Aβ production through interfering with the APP transmembrane domain

Presenilins (PSENs) form the catalytic component of the γ-secretase complex, responsible for intramembrane proteolysis of amyloid precursor protein (APP) and Notch, among many other membrane proteins. Previously, we identified a PSEN1-binding domain in APP, encompassing half of the transmembrane domain following the amyloid β (Aβ) sequence. Based on this, we designed peptides mimicking this interaction domain with the aim to selectively block APP processing and Aβ generation through interfering with enzyme-substrate binding. We identified a peptide sequence that, when fused to a virally derived translocation peptide, significantly lowered Aβ production (IC(50): 317 nM) in cell-free and cell-based assays using APP-carboxy terminal fragment as a direct γ-secretase substrate. Being derived from the APP sequence, this inhibitory peptide did not affect NotchΔE γ-cleavage, illustrating specificity and potential therapeutic value. In cell-based assays, the peptide strongly suppressed APP shedding, demonstrating that it exerts the inhibitory effect already upstream of γ-secretase, most likely through steric hindrance.

Dimeric structure of transmembrane domain of amyloid precursor protein in micellar environment

Some pathogenic mutations associated with Alzheimer’s disease are thought to affect structural-dynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln686-Lys726 was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy. The APP membrane-spanning α-helix Lys699-Lys724 self-associates in a left-handed parallel dimer through extended heptad repeat motif I702X3M706X2G709X3A713X2I716X3I720X2I723, whereas the juxtamembrane region Gln686-Val695 constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-β peptide. Structured summary of protein interactionsAPPjmtm and APPjmtmbind by comigration in gel electrophoresis (View interaction)APPjmtm and APPjmtmbind by nuclear magnetic resonance (View interaction).

Structure of Transmembrane Domain and Dimerization Mechanism of Amyloid Precursor Protein

Alzheimer's disease (AD) is the most prevalent form of dementia that affects senior people regardless of religious conviction, nationality or color of skin. The central role in understanding the underlying mechanisms of AD plays biosynthesis of amyloid-β peptide (Aβ). Aβ is a product of sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretase. More than half of mutations that cause so called familial forms of AD are situated in membrane domain of APP and in presenilin-1/presenilin-2 that are part of γ-secretase complex and cleavage APP within the membrane. The pathogenic mutations presumably affect the structure and dynamic properties of the APP transmembrane domain by changing its conformational stability, lateral mobility and /or dimerization. In the present study, the structure of right-handed dimer and dynamics properties of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined. The structure was solved in the membrane mimetic environment composed of dodecylphosphocholine micelles using NMR spectroscopy. The conformation of transmembrane region each subunit does not alter from published monomer structure. Residues GLY709 and ALA713 form classical GxxxA motif for helix-helix interaction. Juxtamembrane part of investigated peptide does not form tight secondary structure. In this research for the first time dimerization mechanism of APP in atomic resolution is described.

Mapping the Backbone Dynamics of the Alzheimer's APP Transmembrane Domain

The Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease. Its dimeric transmembrane domain (APP-TMD) is subject to several consecutive cleavage events by the gamma-secretase leading to a certain product pattern of amyloidogenic peptides (Munter et al., 2007). Here, we address the question how backbone dynamics of the alpha-helical APP-TMD is mapped.We have investigated the backbone dynamics of several APP-TMD model peptides with hydrogen-deuterium exchange (HDX) experiments coupled to mass spectroscopy. The underlying principle is the exchange of amide-protons for deuterons which is dependent on the dynamics of the alpha-helix.The investigation demonstrates that the N-terminal part of the APP transmembrane domain exhibits higher backbone dynamics than the C-terminal part. Further results show that exchanging threonines for valines along the APP-TMD sequence changes the helix backbone flexibility. To characterize the dynamics of each cleavage region along the APP-TMD a hybrid peptide set was examined. The hybrid peptides consist of a constant poly-leucine sequence and a varying 8 residue long APP-TMD-region where the last and last but two/ three residues represent cleavage sites.To this end, we could discover local differences in APP-TMD helix dynamics. The findings are discussed in terms of substrate recognition and binding and could explain the A beta product pattern in gamma-secretase proteolysis.Munter, L. M., Voigt, P., et al. (2007). “GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of A beta 42.” EMBO Journal 26(6): 1702-1712.

Substrate Sequence Influences γ-Secretase Modulator Activity, Role of the Transmembrane Domain of the Amyloid Precursor Protein

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aβ42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aβ40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.

Expression, purification, and reconstitution of the transmembrane domain of the human amyloid precursor protein for NMR studies

Alzheimer’s disease (AD) is the most common type of dementia in elderly people. Senile plaques, a pathologic hallmark of AD, are composed of amyloid β peptide (Aβ). Aβ aggregation produces toxic oligomers and fibrils, causing neuronal dysfunction and memory loss. Aβ is generated from two sequential proteolytic cleavages of a membrane protein, amyloid precursor protein (APP), by β- and γ-secretases. The transmembrane (TM) domain of APP, APPTM, is the substrate of γ-secretase for Aβ production. The interaction between APPTM and γ-secretase determines the production of different species of Aβ. Although numerous experimental and theoretical studies of APPTM structure exist, experimental 3D structure of APPTM has not been obtained at atomic resolution. Using the pETM41 vector, we successfully expressed an MBP–APPTM fusion protein. By combining Ni-NTA chromatography, TEV protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically-labeled APPTM for NMR studies. The reconstitution of APPTM into micelles yielded high quality 2D 15N–1H HSQC spectra. This reliable method for APPTM expression and purification lays a good foundation for future structural studies of APPTM using NMR.

Classification of γ-secretase modulators and their effect on pharmacological profiles of amyloid β peptides

γ-Secretase modulators (GSM) decrease the cleavage of amyloid precursor protein (APP) at the site that generates 42-amino acid amyloid ß protein (Aß42) while sparing the normal cleavage of Notch. However, how GSMs modulate γ-secretase is not clear. We have examined the cleavage profile of APP in the presence of various GSMs by immunoprecipitation/MALDI-TOF and quantified key Aß peptides by LC/MS/MS. Structurally distinct classes of GSMs changed Aß1-38 and Aß1-39 in opposite directions, i.e., those Aß38-increasing GSMs decreased Aß39 production, and those Aß38-decreasing GSMs increased Aß39 production, while both classes reduce Aß1-42 production. These two classes of GSMs did not dramatically change the levels of total Aß (Aß1-x) at the concentrations that reduced Aß1-42, presumably because increases in the production of shorter peptides (including Aß1-37) compensate for the loss of longer peptide like Aß1-42. Quantification of individual peptide (Aß37, 38, 39, 40 and 42) by LC/MS/MS revealed a significant concentration of these shorter Aß species, supporting our hypothesis. GSMs may slightly shift γ-secretase along the a-helical domain of APP transmembrane domain without changing total γ-secretase activity by shifting the cleavage from longer to shorter peptides.

Structural and Dynamic Study of the Transmembrane Domain of the Amyloid Precursor Protein

Alzheimer’s disease affects people all over the world, regardless of nationality, gender or social status. An adequate study of the disease requires essential understanding of the molecular fundamentals of the pathogenesis. The amyloid β-peptide, which forms amyloid plaques in the brain of people with Alzheimer’s disease, is the product of sequential cleavage of a single-span membrane amyloid precursor protein (APP). More than half of the APP mutations found to be associated with familial forms of Alzheimer’s disease are located in its transmembrane domain. The pathogenic mutations presumably affect the structural-dynamic properties of the APP transmembrane domain by changing its conformational stability and/or lateral dimerization. In the present study, the structure and dynamics of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined in the membrane mimetic environment composed of detergent micelles using NMR spectroscopy. The structure obtained in dodecylphosphocholine micelles consists of two α-helices: a short surface-associated juxtamembrane helix (Lys687-Asp694) and a long transmembrane helix (Gly700-Leu723), both connected via a mobile loop region. A minor bend of the transmembrane α-helix is observed near the paired residues Gly708-Gly709. A cholesterol-binding hydrophobic cavity is apparently formed under the loop region, where the juxtamembrane α-helix comes into contact with the membrane surface near the N-terminus of the transmembrane α-helix.

Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.

Expression and purification of a recombinant transmembrane domain amyloid precursor protein associated with Alzheimer’s disease

More than half of the mutations of the amyloid precursor protein (APP) discovered in familiar forms of Alzheimer's disease are located in the transmembrane domain. The pathogenic mutations presumably affect the lateral dimerization of the APP transmembrane domain in the membrane and change the dimer conformation and/or stability. Thus, the mutations cause an alternative APP digestion pattern in the membrane and neurotoxic amyloid beta-peptide generation. For the detailed study of the specific protein-protein and protein-lipid interactions of the APP transmembrane domain, an E. coli recombinant expression construct was made. The recombinant protein contains an APP transmembrane domain (APPtm(686-726)) with adjacent extramembrane N and C ends. Here, we report the method of isotope-labeled APPtm expression and purification in quantities necessary for a heteronuclear NMR spectroscopy structure and dynamics study. On the basis of the (1)H-(15)N-HSQC spectra, we developed APPtm(686-726) solubilization conditions in the membrane-emulated milieu detergent micelles and lipid bicelles.

Assessment of the Molecular Determinants Required for Dimerization of the Amyloid Precursor Protein Transmembrane Domain by a Combined Experimental and Computational Approach

Removal of the ectodomain of the amyloid precursor protein (APP) by beta-secretase yields a carboxyl-terminal fragment (betaCTF) that is then degraded by the gamma-secretase to produce the neuropathogenic amyloid beta peptides (Abetas) involved in Alzheimer's disease (AD). Considerable evidence indicates that betaCTF is a transmembrane domain-mediated dimer and that dimer dissociation reduces the Abeta42/40 ratio, thus lowering the risk of AD. Little is known about the structural and thermodynamic features of betaCTF dimerization. Employing both coarse-grained and all-atom models coupled to metadynamics, we studied the free energy of dimerization of betaCTF transmembrane domain in an explicit dipalmitoyl phosphatidyl choline (DPPC) membrane bilayer. We show that the dimeric state consists of several stable configurations, featuring interfaces at different locations in the betaCTF transmembrane helix. The effect that point mutations along the transmembrane helix have on the stability of the dimer is assessed computationally and compared to experimental data. This information sets the stage to identify interface-derived small peptides as lead structures to guide the development of novel peptidomimetics that specifically target betaCTF dimerization and display therapeutic potential in AD.

Dexras1 Interacts with FE65 to Regulate FE65-Amyloid Precursor Protein-dependent Transcription

FE65 is an adaptor protein that binds to and forms a transcriptionally active complex with the gamma-secretase-derived amyloid precursor protein (APP) intracellular domain. The regulatory mechanisms of FE65-APP-mediated transcription are still not clear. In this report, we demonstrate that Dexras1, a Ras family small G protein, binds to FE65 PTB2 domain and potently suppresses the FE65-APP-mediated transcription. The suppression is not via competition for binding of FE65 between Dexras1 and APP because the two proteins can simultaneously bind to the FE65 PTB2 domain. Phosphorylation of FE65 tyrosine 547 within the PTB2 domain has been shown to enhance FE65-APP-mediated transcription but not to influence binding to APP. Here we find that this phosphorylation event reduces the binding between Dexras1 and FE65. We also demonstrate that Dexras1 inhibits the FE65-APP-mediated transcription of glycogen synthase kinase 3beta (GSK3 beta). Moreover, small interfering RNA knockdown of Dexras1 enhances GSK3 beta expression and increases phosphorylation of Tau, a GSK3 beta substrate. Thus, Dexras1 functions as a suppressor of FE65-APP-mediated transcription, and FE65 tyrosine 547 phosphorylation enhances FE65-APP-mediated transcription, at least in part, by modulating the interaction between FE65 and Dexras1. These findings reveal a novel regulatory mechanism for FE65-APP-mediated signaling.

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Aβ and AICD generation

The 37-43 amino acid Abeta peptide is the principal component of beta-amyloid deposits in Alzheimer's disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase. gamma-Secretase also cleaves APP at Val50 in the Abeta numbering (epsilon cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Abeta and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Abeta40 and Abeta42 while increasing Abeta38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Abeta generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N- and C-terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of gamma-secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Abeta and AICD production.

Characterization of a Presenilin-mediated Amyloid Precursor Protein Carboxyl-terminal Fragment γ: EVIDENCE FOR DISTINCT MECHANISMS INVOLVED IN γ-SECRETASE PROCESSING OF THE APP AND Notch1 TRANSMEMBRANE DOMAINS

A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, gamma-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment gamma (CTFgamma) derivatives are dependent on PS expression and inhibited by a highly selective and potent gamma-secretase inhibitor. Unexpectedly, the APP-CTFgamma derivative is generated by processing between Leu-645 and Val-646 (of APP(695)), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid beta protein peptides. Although the relationship of APP-CTFgamma to the production of amyloid beta protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent gamma-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain.

A notable cleavage: winding up with beta-amyloid.

How the β-amyloid peptide (Aβ) is cleaved from its precursor, the amyloid precursor protein (APP), preoccupies a large portion of the community studying Alzheimer’s disease. And for good reason. The Aβ protein, the major component of the amyloid in senile plaques, represents a leading target in the quest for agents to combat Alzheimer’s disease. Its formation involves an unusual proteolytic event within a hydrophobic stretch of amino acids predicted to form an α-helix within the membrane. APP is a type I membrane-spanning protein of 770 residues that secretes its large ectodomain after at least two different types of cleavage. Based on the abundance of the cleavage products, the site most frequently cut is at residue 687, which lies within the Aβ peptide and thus precludes its formation. The activity of the responsible, but elusive, enzyme, dubbed the α-secretase, has the property of recognizing an optimal projection length from the membrane more strongly than any specific sequence determinants (1). The α-secretase cleavage occurs in a post-Golgi compartment, possibly including caveolae (2), and may be a glycosyl-phosphatidylinositol-linked aspartyl protease (3). A small percentage of APP is not cleaved at the α-secretase site, but instead is cleaved at residue 672 of APP (which becomes Asp-1 of Aβ) by an activity dubbed β-secretase, the first step in Alzheimer’s-type amyloidogenesis. Other Aβ species with an elongated or shortened N terminus are known (4). The β-secretase cleavage, once completed, sets the stage for the next cleavage by an activity referred to as γ-secretase. In what is now considered a critical event in the disease pathogenesis, the Aβ peptide is released after cleavage by the γ-secretase either between residues 40 and 41 or between residues 42 and 43. Normally, both forms of the Aβ peptide—Aβ42 and Aβ40—are present, with Aβ40 in great excess of Aβ42. The precise cleavage site of the γ-secretase, and hence the generation of the shorter or longer Aβ peptide, is widely believed to have important pathologic consequences. Compared with Aβ40, Aβ42 deposits earlier in the disease process (5) and assembles more rapidly into filaments in vitro (6). Explanations of the mechanism of Aβ42 vs. Aβ40 cleavage by the γ-secretase are problematic because the two proteolytic sites lie on opposite sides of the predicted α-helical transmembrane domain of APP.

γ-Secretase Cleavage Is Distinct from Endoplasmic Reticulum Degradation of the Transmembrane Domain of the Amyloid Precursor Protein

One of the critical cleavage events that generates Alzheimer's amyloid Abeta peptide occurs within the transmembrane domain (TMD) of the amyloid precursor protein (APP) and is carried out by a poorly understood enzyme activity known as gamma-secretase. To investigate this processing, a probe molecule, H26-57C, was constructed containing the TMD of APP flanked immediately on each side by unique epitope tags. H26-57C-transfected cells secrete a approximately 2.9-kDa fragment, indicating that the lumenal and cytosolic domains of APP are not required for gamma-secretase processing. Pulse-chase experiments indicate that the probe turns over with a half-life of 8 min. No degradation intermediates are detected during the chase period, indicating that TMD turnover is a highly processive mechanism. The protease inhibitors, ALLN and MG132, cause a dramatic (50-fold) increase in the steady-state amount of the probe. All of the inhibitors that prevent degradation of the probe in the rough endoplasmic reticulum increase the amount of the approximately 2.9-kDa fragment that is secreted into the media and also causes a similar increase the secretion of 4 kDa Abeta from APP-transfected cells. These results indicate that the system responsible for the degradation of the probe in the rough endoplasmic reticulum and the intramembrane cleavage by gamma-secretase that produces soluble, secreted Abeta are distinct and opposing processes.

Mutations in the Transmembrane Domain of APP Altering γ-Secretase Specificity

Alzheimer's disease (AD) beta-amyloid peptide (Abeta and betaA4) is derived from the amyloid precursor protein (APP) by the subsequent action of the so-far unidentified beta- and gamma-secretases. gamma-secretase, which generates the C-terminus of Abeta, cleaves within the transmembrane domain of APP, preferentially after Abeta-residue 40 (Abeta 40) but also after residue 42 (Abeta 42). This Abeta 42 represents the major subunit of the plaques in AD. Since the position of gamma-secretase cleavage is crucial for understanding the pathogenic pathway, we investigated the effect of different point mutations at Thr43 on gamma-secretase specificity in SPA4CT (SPC99)-expressing COS7 cells. These constructs only require gamma-cleavage for Abeta release. We observed that all Thr43 mutations altered the specificity of gamma-secretase. Small hydrophobic residues favored the generation of Abeta 42, leading to an increase in the 42/40 ratio of Abeta (1.6-2.8-fold). The increase was even stronger (5.6-5.8-fold) when combined with the familial mutation Val46Phe. Thus, these constructs might be highly valuable for the generation of animal models for AD. Processing of full-length APP or SPA4CT yielded the same 42/40 ratio of Abeta (4. 7%). Both constructs, bearing the familial AD mutation Val46Phe, led to a similar increase in the 42/40 ratio (3.3- versus 3.6-fold). The p3 fragment, produced by alpha- and gamma-secretase, showed 42/40 ratios similar to Abeta when derived from wild-type and mutant proteins. These results suggest that the different Abeta- and p3-species are generated by gamma-cleavage activities with a similar enzymatic mechanism.

Exchanging the Extracellular Domain of Amyloid Precursor Protein for Horseradish Peroxidase Does Not Interfere with α-Secretase Cleavage of the β-Amyloid Region, but Randomizes Secretion in Madin-Darby Canine Kidney Cells

Secretory processing and polarized sorting of horseradish peroxidase fused to the amyloid precursor protein transmembrane domain were compared with those of wild-type amyloid precursor protein in COS and polarized Madin-Darby canine kidney (MDCK) cells. The cellular and secreted forms of the chimeric protein were enzymatically active in colorimetric and cytochemical assays after reconstitution with hemin and Ca2+. The peroxidase enzyme was secreted by a proteolytic process, similar to the parent amyloid precursor protein. In polarized MDCK cells, amyloid precursor protein was secreted exclusively in the basolateral compartment, while the peroxidase chimeric protein was secreted in both compartments. The basolateral sorting determinant for secretion must therefore be located in the extracellular domain of amyloid precursor protein. On the other hand, cell surface-associated peroxidase chimeric protein was similar to cell surface-associated wild-type amyloid precursor protein, mainly expressed at the basolateral side. The basolateral cell-surface expression, in contrast to the basolateral secretion, is therefore controlled by determinants in the cytoplasmic domain. Methylamine inhibited and bafilomycin slightly increased the basolateral secretion of both proteins, but both drugs strongly increased apical secretion. The default secretory pathway of COS cells and the basolateral (but not the apical) secretory pathway of MDCK cells are therefore comparably sensitive to methylamine and not to bafilomycin.