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C-terminal turn stability determines assembly differences between A?40 and A?42.

ABSTRACT: Oligomerization of the amyloid ?-protein (A?) is a seminal event in Alzheimer's disease. A?42, which is only two amino acids longer than A?40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in A?42 that is not present in A?40. The dihedral angles of residues 36 and 37 in an Ile31-Ala42 peptide were consistent with ?-turns, and a ?-hairpin-like structure was indeed observed that was stabilized by hydrogen bonds and by hydrophobic interactions between residues 31-35 and residues 38-42. In contrast, A?(31-40) mainly existed as a statistical coil. To study the system experimentally, we chemically synthesized A? peptides containing amino acid substitutions designed to stabilize or destabilize the hairpin. The triple substitution Gly33Val-Val36Pro-Gly38Val ("VPV") facilitated A?42 hexamer and nonamer formation, while inhibiting formation of classical amyloid-type fibrils. These assemblies were as toxic as were assemblies from wild-type A?42. When substituted into A?40, the VPV substitution caused the peptide to oligomerize similarly to A?42. The modified A?40 was significantly more toxic than A?40. The double substitution d-Pro36-l-Pro37 abolished hexamer and dodecamer formation by A?42 and produced an oligomer size distribution similar to that of A?40. Our data suggest that the Val36-Gly37 turn could be the sine qua non of A?42. If true, this structure would be an exceptionally important therapeutic target.

SUBMITTER: Roychaudhuri R

PROVIDER: S-EPMC3540122 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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C-terminal turn stability determines assembly differences between Aβ40 and Aβ42.

Roychaudhuri Robin R Yang Mingfeng M Deshpande Atul A Cole Gregory M GM Frautschy Sally S Lomakin Aleksey A Benedek George B GB Teplow David B DB

Journal of molecular biology 20121112 2

Oligomerization of the amyloid β-protein (Aβ) is a seminal event in Alzheimer's disease. Aβ42, which is only two amino acids longer than Aβ40, is particularly pathogenic. Why this is so has not been elucidated fully. We report here results of computational and experimental studies revealing a C-terminal turn at Val36-Gly37 in Aβ42 that is not present in Aβ40. The dihedral angles of residues 36 and 37 in an Ile31-Ala42 peptide were consistent with β-turns, and a β-hairpin-like structure was indee ...[more]

PMID: 23154165

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Project description:BackgroundCathepsin D (CatD) is a lysosomal protease that degrades both the amyloid β-protein (Aβ) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aβ proteostasis in vivo and to more completely characterize the degradation of Aβ42 and Aβ40 by CatD.MethodsWe quantified Aβ degradation rates and levels of endogenous Aβ42 and Aβ40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aβ42 vs. Aβ40, and the degradation of aggregated Aβ42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aβ42 and Aβ40, as well as corresponding p3 fragments.ResultsGenetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aβ42 and Aβ40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aβ42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aβ42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aβ42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aβ42 vis-à-vis Aβ40. Specifically, Aβ42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aβ42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aβ42 vs. Aβ40 also extend to p3 fragments ending at positions 42 vs. 40.ConclusionsOur findings identify CatD as the principal intracellular Aβ-degrading protease identified to date, one that regulates Aβ42/40 ratios via differential degradation of Aβ42 vs. Aβ40. The finding that Aβ42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aβ42 and downstream pathological sequelae in AD.

Project description:The relative abundance of two main Abeta-peptide types with different lengths, Aβ40 and Aβ42, determines the severity of the Alzheimer's disease progression. However, the factors responsible for different behavior patterns of these peptides in the amyloidogenesis process remain unknown. In this comprehensive study, new evidence on Aβ40 and Aβ42 amyloid polymorphism was obtained using a wide range of experimental approaches, including custom-designed approaches. We have for the first time determined the number of modes of thioflavin T (ThT) binding to Aβ40 and Aβ42 fibrils and their binding parameters using a specially developed approach based on the use of equilibrium microdialysis, which makes it possible to distinguish between the concentration of the injected dye and the concentration of dye bound to fibrils. The binding sites of one of these modes located at the junction of adjacent fibrillar filaments were predicted by molecular modeling techniques. We assumed that the sites of the additional mode of ThT-Aβ42 amyloid binding observed experimentally (which are not found in the case of Aβ40 fibrils) are localized in amyloid clots, and the number of these sites could be used for estimation of the level of fiber clustering. We have shown the high tendency of Aβ42 fibers to form large clots compared to Aβ40 fibrils. It is probable that this largely determines the high resistance of Aβ42 amyloids to destabilizing effects (denaturants, ionic detergents, ultrasonication) and their explicit cytotoxic effect, which we have shown. Remarkably, cross-seeding of Aβ40 fibrillogenesis using the preformed Aβ42 fibrils changes the morphology and increases the stability and cytotoxicity of Aβ40 fibrils. The differences in the tendency to cluster and resistance to external factors of Aβ40 and Aβ42 fibrils revealed here may be related to the distinct role they play in the deposition of amyloids and, therefore, differences in pathogenicity in Alzheimer's disease.

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C-terminal turn stability determines assembly differences between A?40 and A?42.

Publications

C-terminal turn stability determines assembly differences between Aβ40 and Aβ42.

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