Paul J. Barrett

The Amyloid Precursor Protein Has a Flexible Transmembrane Domain and Binds Cholesterol

Insights into Amyloidogenesis The amyloid-β (Aβ) peptides associated with Alzheimers disease are generated by cleavage of the transmembrane C-terminal domain (C99) of the amyloid precursor protein by the enzyme γ-secretase. Barrett et al. (p. 1168) used nuclear magnetic resonance (NMR) and electron paramagnetic resonance spectroscopy to show that C99 contains surface-associated N- and C-terminal helices and a flexibly curved transmembrane helix that is well suited to processive cleavage by γ-secretase. Elevated cholesterol levels have been found to increase Aβ generation. NMR titration together with mutagenesis revealed a binding site for cholesterol within C99 that included a motif previously implicated in protein oligomerization. The structure of the amyloid precursor protein transmembrane domain allows processive cleavage and cholesterol binding that may enhance cleavage. C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer’s disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer’s therapeutics.

Direct binding of cholesterol to the amyloid precursor protein: An important interaction in lipid-Alzheimer's disease relationships?

It is generally believed that cholesterol homoeostasis in the brain is both linked to and impacted by Alzheimers disease (AD). For example, elevated levels of cholesterol in neuronal plasma and endosome membranes appear to be a pro-amyloidogenic factor. The recent observation that the C-terminal transmembrane domain (C99, also known as the beta-C-terminal fragment, or beta-CTF) of the amyloid precursor protein (APP) specifically binds cholesterol helps to tie together previously loose ends in the web of our understanding of Alzheimers-cholesterol relationships. In particular, binding of cholesterol to C99 appears to favor the amyloidogenic pathway in cells by promoting localization of C99 in lipid rafts. In turn, the products of this pathway-amyloid-beta and the intracellular domain of the APP (AICD)-may down-regulate ApoE-mediated cholesterol uptake and cholesterol biosynthesis. If confirmed, this negative-feedback loop for membrane cholesterol levels has implications for understanding the function of the APP and for devising anti-amyloidogenic preventive strategies for AD.

The backbone dynamics of the amyloid precursor protein transmembrane helix provides a rationale for the sequential cleavage mechanism of γ-secretase.

The etiology of Alzheimers disease depends on the relative abundance of different amyloid-β (Aβ) peptide species. These peptides are produced by sequential proteolytic cleavage within the transmembrane helix of the 99 residue C-terminal fragment of the amyloid precursor protein (C99) by the intramembrane protease γ-secretase. Intramembrane proteolysis is thought to require local unfolding of the substrate helix, which has been proposed to be cleaved as a homodimer. Here, we investigated the backbone dynamics of the substrate helix. Amide exchange experiments of monomeric recombinant C99 and of synthetic transmembrane domain peptides reveal that the N-terminal Gly-rich homodimerization domain exchanges much faster than the C-terminal cleavage region. MD simulations corroborate the differential backbone dynamics, indicate a bending motion at a diglycine motif connecting dimerization and cleavage regions, and detect significantly different H-bond stabilities at the initial cleavage sites. Our results are consistent with the following hypotheses about cleavage of the substrate: First, the GlyGly hinge may precisely position the substrate within γ-secretase such that its catalytic center must start proteolysis at the known initial cleavage sites. Second, the ratio of cleavage products formed by subsequent sequential proteolysis could be influenced by differential extents of solvation and by the stabilities of H-bonds at alternate initial sites. Third, the flexibility of the Gly-rich domain may facilitate substrate movement within the enzyme during sequential proteolysis. Fourth, dimerization may affect substrate processing by decreasing the dynamics of the dimerization region and by increasing that of the C-terminal part of the cleavage region.

Nonspecificity of Binding of γ-Secretase Modulators to the Amyloid Precursor Protein

Evidence that certain gamma-secretase modulators (GSMs) target the 99-residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of gamma-secretase, but not the protease complex itself has been presented [Kukar, T. L., et al. (2008) Nature 453, 925-929]. Here, NMR results demonstrate a lack of specific binding of these GSMs to monodisperse C99 in LMPG micelles. In addition, results indicate that C99 was likely to have been aggregated in some of the key experiments of the previous work and that binding of GSMs to these C99 aggregates is also of a nonspecific nature.

The Quiet Renaissance of Protein Nuclear Magnetic Resonance

From roughly 1985 through the start of the new millennium, the cutting edge of solution protein nuclear magnetic resonance (NMR) spectroscopy was to a significant extent driven by the aspiration to determine structures. Here we survey recent advances in protein NMR that herald a renaissance in which a number of its most important applications reflect the broad problem-solving capability displayed by this method during its classical era during the 1970s and early 1980s.

NSAID-Based γ-Secretase Modulators Do Not Bind to the Amyloid-β Polypeptide

γ-Secretase modulators (GSMs) have received much attention as potential therapeutic agents for Alzheimers disease (AD). GSMs increase the ratio between short and long forms of the amyloid-β (Aβ) polypeptides produced by γ-secretase and thereby decrease the amount of the toxic amyloid species. However, the mechanism of action of these agents is still poorly understood. One recent paper [Richter et al. (2010) Proc. Natl. Acad. Sci. U. S. A.107, 14597-14602] presented data that were interpreted to support direct binding of the GSM sulindac sulfide to Aβ(42), supporting the notion that GSM action is linked to direct binding of these compounds to the Aβ domain of its immediate precursor, the 99-residue C-terminal domain of the amyloid precursor protein (C99, also known as the β-CTF). Here, contrasting results are presented that indicate there is no interaction between monomeric sulindac sulfide and monomeric forms of Aβ42. Instead, it was observed that sulindac sulfide is itself prone to form aggregates that can bind nonspecifically to Aβ42 and trigger its aggregation. This observation, combined with data from previous work [Beel et al. (2009) Biochemistry48, 11837-11839], suggests both that the poor behavior of some NSAID-based GSMs in solution may obscure results of binding assays and that NSAID-based GSMs do not function by directly targeting C99. It was also observed that another GSM, flurbiprofen, fails to bind to monomeric Aβ42 or to C99 reconstituted into bilayered lipid vesicles. These results disfavor the hypothesis that these NSAID-based GSMs exert their modulatory effect by directly targeting a site located in the Aβ42 domain of free C99.

Topologically Diverse Human Membrane Proteins Partition to Liquid-Disordered Domains in Phase-Separated Lipid Vesicles.

The integration of membrane proteins into “lipid raft” membrane domains influences many biochemical processes. The intrinsic structural properties of membrane proteins are thought to mediate their partitioning between membrane domains. However, whether membrane topology influences the targeting of proteins to rafts remains unclear. To address this question, we examined the domain preference of three putative raft-associated membrane proteins with widely different topologies: human caveolin-3, C99 (the 99 residue C-terminal domain of the amyloid precursor protein), and peripheral myelin protein 22. We find that each of these proteins are excluded from the ordered domains of giant unilamellar vesicles containing coexisting liquid-ordered and liquid-disordered phases. Thus, the intrinsic structural properties of these three topologically distinct disease-linked proteins are insufficient to confer affinity for synthetic raft-like domains.

Binding of cholesterol to the C99 domain of APP competes with homodimerzation of the protein

AApeptide and also their potential on anti-Alzheimer’s diseases was evaluated.Methods: Herein, one bead one compound AApeptide library was designed for the selection of ligand that targets on Abeta. Selected AApeptides was evaluated for their effect on Aß1-40monomer peptides in their progress of aggregation. Methods are listed as follows: 1. Thioflavin T assay, ß-sheet formation assay for anti-aggregation screening. 2. TEM assay was used to image the morphological change of Aß fibrils with/without presence of AApeptide. 3. Western Blot, in vitro aggregation of HFIP treated Aß1-40 was produced to aggregate under room temperature, theformation of oligomers were detected using 6E10 anti-Aß antibodies. Following LI-COR western was used and quantification was performed for evaluation. Tissue culture, MTT assay on Neuroblastoma cell to test their toxicity. Kit using commercial available methods from Roche. Results: One of screened AApeptides shows great potential of anti-aggregation at concentration of 2.5mM at 1:1 ratio with Abeta. TEM assay also confirmed clearance of Aß fibrils in the presence of AApeptide. Toxicity of neuroblastma cells of AApeptide is not obvious at effective concentration. Conclusions: As a new generation of peptidomimetics, short AApeptide has great potential on Anti-Alzheimer’s diseases as an anti-aggregation drug.

3-D structure of the transmembrane c-terminal domain of the amyloid precursor protein (C99), the immediate precursor of amyloid-β

Background: The soluble As oligomers have been proposed to be the disease causing entity in the pathogenesis of Alzheimer’s disease (AD), but the conformation and time course of the As oligomers are not well established. Genetically modified mouse models recapitulate pathological and biochemical processes involved in the pathogenesis of AD, and are instrumental in studying disease mechanisms and testing therapeutic strategies. Methods: In the present study, we investigated the As assemblies in the cortex of 4-20 months of age APPSwedish transgenic Tg2576 mouse, and compared these with the As assemblies that were detected in autopsy brain tissue from AD patients (58-88 years) (Bao et al. submitted). We serially extracted As species into TBS (soluble), TBST (detergent soluble) and GuCHl (insoluble) fractions. The conformation and level of the As species were determined by ELISA and immunoblotting with Ab-derived diffusible ligands (ADDLs) specific antibodies. Results: As assemblies ranging from dimers to hexamers were detected in all three extraction fractions in the Tg2576 mice, and showed a gradual increased level from 6 months of age. The dodecamers were detected from 6 months of age in Tg2576 mice brain extractions, and the levels remained the same in the older mice. Monomeric As was also detected in the TBST and GuHCl fractions, and interestingly a sharp increased level in the GuHCl fraction of 20 months old Tg2576 mice, which is the age known to show very high plaque load in this transgenic mice. Lower molecular weight As species were generally detected in the cerebral cortex of transgenic mice similar to those detected in AD patients carrying the same APPSwedish mutation. In contrast, no lower molecular weight As assemblies were detected in cortical brain tissue from sporadic AD patients. Conclusions: These results suggest that the soluble As assemblies in the Tg2576 mice brain were not same as the As species presented in the brain tissue from AD patients, and suggest that there may be a different As dynamic processes in this transgenic mice compare to AD patients, and it need to be taken into consideration when using this mouse model to evaluate anti-As therapy.