探討小分子物質對於人類γD型水晶體蛋白聚集之影響

Exploring the Effects of Small Molecules on the Aggregation of Human γD-Crystallin Protein

呂建宏(Jian-Hong Lu)

指導教授 ： 王勝仕

國立臺灣大學/工學院/化學工程學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201901426

摘要

白內障是全世界視力受損的主要原因之一，為一種眼睛水晶體內之蛋白質聚集相關的疾病，一些風險因素包含紫外光照射會誘導水晶體蛋白去折疊以及隨後的水晶體蛋白聚集，而造成水晶體混濁。人類水晶體核心中富含許多水晶體蛋白，其主要功能為協助維持水晶體的光學性質，然而長時間曝曬於太陽光底下，其紫外光照射會致使水晶體蛋白產生聚集體，導致引發老年性白內障。此外有證據顯示於酸性條件下有類澱粉纖維物種的形成，而被認為是老年型白內障發展過程的路徑之一。
本論文旨在探討幾種潛在的小分子物質對於人類γD型水晶體蛋白(HγDC)聚集與其類澱粉纖維形成之影響，於論文的第一部分，ortho-vanillin、myricetin與rosmarinic acid等小分子物質用於測試並探討由UV-C誘導聚集的抑制效果，使用turbidity、right angle light scattering、SDS-PAGE、TEM、遠紫外光圓二色光譜、ANS螢光光譜以及分子對接模擬等分析，研究結果發現HγDC與小分子物質莫耳比1:10情況下，ortho-vanillin、myricetin與rosmarinic acid於right angle light scattering結果顯示其光散射下降百分比分別為~76.85±2.48%, ~73.53±1.95%, ~12.85±4.60%，並與TEM和SDS-PAGE結果指出ortho-vanillin是三種檢測小分子物質中，對抗UV-C觸發之聚集為最有效的抑制劑，而分子對接模擬揭示出這些小分子物質和原態HγDC之間的結合中存在著氫鍵、靜電作用力和π-π共軛堆積等相互作用力。
第二部分研究chlorazol Black E、myricetin與5-hydroxy-1,4-naphthoquinone如何於酸性條件(pH=2)下影響HγDC之類澱粉纖維形成，使用ThT螢光光譜、tryptophan螢光光譜、ANS螢光光譜、SDS-PAGE、TEM、遠紫外光圓二色光譜與分子對接模擬等分析，發現於小分子物質300μM情況下，其抑制效果依序為: chlorazol Black E (ThT螢光下降百分比= ~82.47±4.03%) > myricetin (ThT螢光下降百分比= ~65.45±1.84%) > 5-hydroxy-1,4-naphthoquinone (ThT螢光下降百分比= ~57.91±3.61%)，使用分子對接模擬並了解到這些潛在的小分子物質，可能透過與HγDC之類澱粉纖維聚集熱點的結合，而干擾其類澱粉纖維之形成。
本研究成功地展示出某些小分子物質可用於抑制HγDC的無定型體聚集以及類澱粉纖維形成，相信這項研究結果有助於設計臨床策略和開發抗白內障的抑制化合物。

關鍵字

白內障 ； 人類γD型水晶體蛋白 ； 小分子物質 ； 蛋白質聚集 ； 無定型體聚集體 ； 類澱粉纖維

並列摘要

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.

並列關鍵字

cataract ； human γD-crystallin ； small molecule ； protein aggregation ； amorphous aggregate ； amyloid fibril

參考文獻

1. Mitchell, P., et al., Prevalence of cataract in Australia: the Blue Mountains eye study. Ophthalmology, 1997. 104(4): p. 581-588.

2. Chua, J., et al., Ancestry, socioeconomic status, and age-related cataract in Asians: the Singapore Epidemiology of Eye Diseases Study. Ophthalmology, 2015. 122(11): p. 2169-2178.

3. Wu, J.W.-R., et al., Human γD-crystallin aggregation induced by ultraviolet C irradiation is suppressed by resveratrol. Biochemical Engineering Journal, 2013. 78: p. 189-197.

4. Asbell, P.A., et al., Age-related cataract. The Lancet, 2005. 365(9459): p. 599-609.

5. Erie, J.C., et al., Risk of retinal detachment after cataract extraction, 1980–2004: a population-based study. Ophthalmology, 2006. 113(11): p. 2026-2032.