RESUMEN
In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.
Asunto(s)
Peptidilprolil Isomerasa de Interacción con NIMA/química , Péptidos/química , Agregación Patológica de Proteínas , Dominios WW , Proteínas tau/química , Amiloide/química , Amiloide/metabolismo , Sitios de Unión/genética , Dominio Catalítico , Humanos , Espectroscopía de Resonancia Magnética , Mutación , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Alzheimer's disease involves accumulation of senile plaques in which filamentous aggregates of amyloid beta (Aß) peptides are deposited. Recent studies demonstrate that oligomerization pathways of Aß peptides may be complicated. To understand the mechanisms of Aß(1-42) oligomer formation in more detail, we have established a method to produce (15)N-labeled Aß(1-42) suited for nuclear magnetic resonance (NMR) studies. For physicochemical studies, the starting protein material should be solely monomeric and all Aß aggregates must be removed. Here, we succeeded in fractionating a "precipitation-resistant" fraction of Aß(1-42) from an "aggregation-prone" fraction by high-performance liquid chromatography (HPLC), even from bacterially overexpressed Aß(1-42). However, both Aß(1-42) fractions after 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) treatment formed amyloid fibrils. This indicates that the "aggregation seed" was not completely monomerized during HFIP treatment. In addition, Aß(1-42) dissolved in HFIP was found to display a monomer-dimer equilibrium, as shown by two-dimensional (1)H-(15)N NMR. We demonstrated that the initial concentration of Aß during the HFIP pretreatment altered the kinetic profiles of Aß fibril formation in a thioflavin T fluorescence assay. The findings described here should ensure reproducible results when studying the Aß(1-42) peptide.