| Summary: | 碩士 === 國立臺灣大學 === 化學工程學研究所 === 107 === Cataract is one of the main causes of visual impairment in the world. It is the disease associated with protein aggregation in the eye lens. Several risk factors including the exposure to ultraviolet irradiation may induce unfolding of the lens proteins, followed by aggregation of the lens proteins, thus leading to the opacification of the crystalline lens. Crystallin proteins are abundant in the nucleus of the human eye lens. They mainly function to maintain the optical properties of the lens. However, exposure to the sun''s ultraviolet radiation for a prolonged period of time will cause the crystallin proteins to form aggregates, leading to age-related cataract. In addition, evidence suggests that crystallin proteins are able to form amyloid fibrillar species under the acidic conditions. It is considered to be one of the paths of the development of age-related cataract.
This thesis was aimed at investigating the effects of several potential small molecules on the aggregation and/or fibrillogenesis of human γD-crystallin proteins (HγDC). In the first part of the thesis, the small molecules, such as ortho-vanillin, myricetin, and rosmarinic acid were tested to explore the inhibitory effects against UV-C-induced aggregation using several techniques including turbidity, right angle light scattering, SDS-PAGE, TEM, far-ultraviolet circular dichroism spectroscopy, ANS fluorescence spectroscopy and molecular docking simulation. Our results demonstrated that, when the molar ratio of HγDC to small molecule was at 1:10, the percentage reduction of light scattering was found to be ~76.85±2.48%, ~73.53±1.95%, ~12.85±4.60% for ortho-vanillin, myricetin, or rosmarinic acid, respectively. Results from right angle light scattering, TEM, and SDS-PAGE suggested that ortho-vanillin is the most potent UV-C triggered aggregation inhibitor among the three agents examined. Molecular docking studies also revealed that there were various kinds of interacting forces (e.g., hydrogen bonding, electrostatic interactions, and π-π stacking) involved in the binding between these small molecules and native HγDC.
In the second part, we studied how chlorazol Black E, myricetin, and 5-hydroxy-1,4-naphthoquinone affected amyloid fibril formation of HγDC which was induced under the acidic conditions (e.g., pH 2.0) using the techniques including ThT fluorescence spectroscopy, tryptophan fluorescence spectroscopy, ANS fluorescence spectroscopy, SDS-PAGE, TEM, far-ultraviolet circular dichroism spectroscopy, and molecular docking simulation. It was observed that, when the concentration of small molecule was 300 μM, the inhibitory activity followed the order: chlorazol Black E (the percentage reduction of ThT fluorescence = ~82.47±4.03%) > myricetin (the percentage reduction of ThT fluorescence = ~65.45±1.84%) > 5-hydroxy-1,4-naphthoquinone (the percentage reduction of ThT fluorescence = ~57.91±3.61%). We also performed molecular docking studies and learned that these potential small molecules may interfere with amyloid fibrillogenesis of HγDC via binding with the fibril formation hot spots or amyloidogenic regions of HγDC.
Overall, our study successfully demonstrated that amorphous aggregation and amyloid fibril formation of HγDC could be suppressed by certain small molecules. We believe the outcome from this work may, in part, help to design clinical strategies and develop inhibitory compounds against cataracts.
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