RESUMO
Prion disease is a neurodegenerative disorder with progressive neurologic symptoms and accelerated cognitive decline. The causative protein of prion disease is the prion protein (PrP), and structural transition of PrP from the normal helix rich form (PrPC) to the abnormal ß-sheet rich form (PrPSc) occurs in prion disease. While so far numerous therapeutic agents for prion diseases have been developed, none of them are still useful. A fluorinated alcohol, hexafluoro isopropanol (HFIP), is a precursor to the inhalational anesthetic sevoflurane and its metabolites. HFIP is also known as a robust α-helix inducer and is widely used as a solvent for highly aggregated peptides. Here we show that the α-helix-inducing activity of HFIP caused the conformational transformation of the fibrous structure of PrP into amorphous aggregates in vitro. HFIP added to the ScN2a cell medium, which continuously expresses PrPSc, reduced PrPSc protease resistance after 24-h incubation. It was also clarified that ScN2a cells are more susceptible to HFIP than any of the cells being compared. Based on these findings, HFIP is expected to develop as a therapeutic agent for prion disease.
Assuntos
Proteínas Priônicas/metabolismo , Propanóis/farmacologia , Multimerização Proteica/efeitos dos fármacos , Animais , Células COS , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Camundongos , Propanóis/toxicidadeRESUMO
Post-translational modifications are often detected in age-related diseases associated with protein misfolding such as cataracts from aged lenses. One of the major post-translational modifications is the isomerization of aspartate residues (L-isoAsp), which could be non-enzymatically and spontaneously occurring in proteins, resulting in various effects on the structure and function of proteins including short peptides. We have reported that the structure and function of an αA66-80 peptide, corresponding to the 66-80 (66SDRDKFVIFLDVKHF80) fragment of human lens αA-crystallin, was dramatically altered by the isomerization of aspartate residue (Asp) at position 76. In the current study, we observed amyloid-like fibrils of L-isoAsp containing αA66-80 using electron microscopy. The contribution of each amino acid for the peptide structure was further evaluated by circular dichroism (CD), bis-ANS, and thioflavin T fluorescence using 14 alanine substituents of αA66-80, including L-isoAsp at position 76. CD of 14 alanine substituents demonstrated random coiled structures except for the substituents of positively charged residues. Bis-ANS fluorescence of peptide with substitution of hydrophobic residue with alanine revealed decreased hydrophobicity of the peptide. Thioflavin T fluorescence also showed that the hydrophobicity around Asp76 of the peptide is important for the formation of amyloid-like fibrils. One of the substitutes, H79A (SDRDKFVIFL(L-isoD)VKAF) demonstrated an exact ß-sheet structure in CD and highly increased Thioflavin T fluorescence. This phenomenon was inhibited by the addition of protein-L-isoaspartate O-methyltransferase (PIMT), which is an enzyme that changes L-isoAsp into Asp. These interactions were observed even after the formation of amyloid-like fibrils. Thus, isomerization of Asp in peptide is key to form fibrils of αA-crystallin-derived peptide, and L-isoAsp on fibrils can be a candidate for disassembling amyloid-like fibrils of αA-crystallin-derived peptides.
Assuntos
Amiloide/química , Ácido Aspártico/metabolismo , Ácido Isoaspártico/metabolismo , Processamento de Proteína Pós-Traducional , Cadeia A de alfa-Cristalina/metabolismo , Envelhecimento/genética , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Ácido Aspártico/química , Benzotiazóis/química , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ácido Isoaspártico/química , Isomerismo , Cristalino/metabolismo , Cristalino/patologia , Microscopia Eletrônica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/química , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Eletricidade Estática , Cadeia A de alfa-Cristalina/genéticaRESUMO
Achieving high mobility in SnO2, which is a typical wide gap oxide semiconductor, has been pursued extensively for device applications such as field effect transistors, gas sensors, and transparent electrodes. In this study, we investigated the transport properties of lightly Ta-doped SnO2 (Sn1-xTaxO2, TTO) thin films epitaxially grown on TiO2 (001) substrates by pulsed laser deposition. The carrier density (ne) of the TTO films was systematically controlled by x. Optimized TTO (x = 3 × 10-3) films with ne ~ 1 × 1020 cm-3 exhibited a very high Hall mobility (µH) of 130 cm2V-1s-1 at room temperature, which is the highest among SnO2 films thus far reported. The µH value coincided well with the intrinsic limit of µH calculated on the assumption that only phonon and ionized impurities contribute to the carrier scattering. The suppressed grain-boundary scattering might be explained by the reduced density of the {101} crystallographic shear planes.
