探討單點突變與分子對接模擬對人類γD型水晶體蛋白不穩定區域之影響

Abstract

白內障是一種與蛋白質聚集相關的眼疾，也是導致失明的主要原因，而引發白內障的主要原因包括受日光的照射、不正常的飲食、藥物使用或是糖尿病等，其機制主要是來自於水晶體蛋白的聚集，因此為了能有效避免這種眼睛相關疾病，了解水晶體蛋白的結構穩定性以避免聚集的形成是非常重要的。人類γD型水晶體蛋白(Human γD-crystallin, HγDC)是一種由173個殘基組成的蛋白質，其在人類水晶體核中的含量極為豐富，也是與老化型白內障有高度相關的水晶體蛋白之一。 過去研究指出，HγDC在殘基115-118的位置為一不穩定的區域，在本研究中，吾人利用多種生物資訊工具，就分子層面來預測HγDC與其不同突變種的結構穩定性與聚集傾向，並利用一致性排序的方式挑選出每個殘基上結構最穩定與最不穩定的突變種，接著使用分子動力學模擬來印證。根據RMSD、原態接觸分析以及聚集傾向的預測，吾人發現所有的突變種中，R116I和F117A分別擁有最高和最低的聚集傾向，然而其結構穩定性都與原態沒有太大的差異。接著，吾人更進一步發現聚集傾向與其不穩定區域115-118的擾動情形與疏水性有相當程度的關聯。 除此之外，吾人利用分子對接的方式嘗試找出最有可能避免HγDC聚集形成的抑制劑。在分析HγDC與多種小分子間的交互作用力後，因為迷迭香酸(rosmarinic acid)與殘基115-118能產生最多的作用力，吾人推測迷迭香酸應為本研究中最具有治療白內障的潛力。吾人相信此研究結果可以幫助研究者更了解HγDC的結構完整性，並發展未來能治療白內障的可能途徑。

Cataract, an eye disease related to protein aggregation, is the leading cause of blindness world-wide. Factors that cause cataract include exposure of daylight, abnormal diet, drug intake, and diabe-tes. The underlying mechanism that leads to cataract is related to the aggregation of crystallin proteins. Therefore, understanding the structural stability of crystallin proteins to avoid aggregation is of fore-most important for the prevention of this sight-threatening disease. Human γD-crystallin (HγDC), a 173-residue protein, is abundant in the nucleus of human eye lens and is one of the major crystallin proteins involved in the age-related nuclear cataract. Previous studies showed the unstable regions of HγDC are residues 115-118. In the first part of this thesis, the structural stabilities and aggregation propensity of various HγDC mutants from the un-stable regions were investigated at the molecular level by multiple bioinformatics tools. The most sta-ble and unstable mutants were chosen through a consensus ranking method for further verification by molecular dynamics simulations (MDS). By combining the results of RMSD, native contact fraction, and prediction of aggregation propensity, we found that R116I and F117A have the highest and low-est aggregation propensity among the mutants studied, respectively; however, their structural stabilities were almost the same as the wildtype. Moreover, the degree of aggregation propensity was related to the fluctuation and hydrophobicity in the residues 115-118. In the second part of this thesis, attenpts were made to seek the possible inhibitors to prevent the HγDC from forming aggregates by molecular docking. According to the interaction analysis between HγDC and small molecules, we speculated that rosmarinic acid was the best inhibitory molecule in our study because of its binding to the residues 115-118 of HγDC. We believe the outcome from this research may help to achieve a better understanding of the structural integrity of HγDC and pave way for future work in the development of therapeutic strategies against cataract.