- 學位論文
瑞舒伐他汀藥物調節血管內皮細胞蛋白質轉譯後修飾質體學與發炎體之研究
Rosuvastatin modulates post translational modified proteomic and inflammasome in vessel endothelial cells
摘要
史他汀類 (statins) 是目前被常用的降膽固醇藥物。其中，瑞舒伐他汀 (Rosuvastatin) 的使用比例相當高。除了降血脂外，史達汀藥物的更具有多效性作用 (Pleiotropic) 。在內皮細胞的訊息傳遞中，轉譯後修飾 (Posttranslational modification, PTM) 扮演相當重要的角色。本實驗以血管細胞株 (EA.hy 926) 以及人類臍帶靜脈內皮細胞 (HUVEC) 為材料，針對蛋白質胺基酸的磷酸化、乙醯基化和硝基化等來探討史他汀藥物可能經由何種蛋白質分子的修飾進而啟動下游生理訊號傳遞。本研究，利用固定化金屬離子親和性層析法 (Immobilized metal affinity chromatography, IMAC) 進行磷酸化胜肽純化後，以串聯質譜儀 (MS/MS) 進行分析並藉由MASCOT軟體與資料庫進行比對，共鑑定到530個可被瑞舒伐他汀誘導進行磷酸化的蛋白質。在硝基化方面，利用生物素分子 (Biotin) 進行置換(Biotin-switch)，將原本結合在半胱胺酸 (Cysteine, Cys) 的一氧化氮 (Nitric oxide, NO) 置換成生物素。之後利用二維電泳並結合西方墨點法，共鑑定到17個硝基化的蛋白質。進一步分析更發現會被硝基化的胺基酸位置多屬於蛋白質的疏水性區域。至於乙醯基化的研究，發現瑞舒伐他汀可以增加內皮細胞的長壽蛋白 (Sirtuin) 表現。進一步利用二維電泳與西方墨點法，亦鑑定到18個乙醯基化的蛋白質。此外其磷酸化、硝基化以及乙醯基化的胺基酸位置皆可被精準鑑定。除了轉譯後修飾蛋白質的鑑定，在實驗中亦針對AMP-activated protein kinase (AMPK) 及發炎因子的表現進行研究。實驗結果顯示瑞舒伐他汀可以抑制發炎因子NALP3和Caspase1的表現。而處理AMPK抑制劑(Compound C) ，發現COX-2 (Cyclooxygenase 2) 所增加的表現又可以被瑞舒伐他汀抑制；顯示瑞舒伐他汀可透過AMPK途徑降低發炎反應。綜合本研究，藉由蛋白質體資訊，我們可以進一步了解血管內皮細胞在環境刺激下的動態修飾變化，提供未來更專一性藥物設計的方向。
並列摘要
Statin, HMG-CoA reductase inhibitors are widely prescribed as cholesterol-lowering drugs in clinical therapy. Rosuvastatin exhibits the lowest inhibition coefficient and is mostly applied clinically. Rosuvastatin shows the cholesterol-independent pleiotropic effects. Post-translational modifications (PTMs) can profoundly affect the enzymatic activity in endothelial signaling cascade. The endothelial cells such as cell line EA.hy 926 cell line and the primary cells as human umbilical vein endothelia cell (HUVEC) were applied to determine rosuvastatin-mediated protein phosphorylation, acetylation and S-nitrosylation. In the present study, Immobilized Metal Affinity Chromatography (IMAC) was use to purify phosphorylate peptide. 530 phosphorylated proteins were identified with the couple of nLC-MS/MS and MASCOT software. For the determination of S-nitrosylation, the biotin switch was used to replace nitric oxide (NO) and subjected to 2-DE and western blot. There are 17 S-nitrosylation proteins identidied and the S-nitrosylated cysteine also predicted on the hydrophobic region. SIRT1, a deacetylase can be upregulated by rosuvastatin 18 acetylated-protein were identified. Due to the advances of mass spectrometric technology, the amino acid with phosphorylation, S-nitrosylation and acetylation can be predicted directly. Moreover, we also investigate the role of AMPK in modulating inflammasome. The endothelial cells treated with compound C an inhibitor of AMPK, level of NALP3, Caspase1 and COX-2 were increased. Interesting, this increased level can be attenuated by the co-treatment of rosuvastatin. This indicated that rousuvastatin can decreas the expression of inflammasom through AMPK pathway. Taken together, we concluded that rosuvastatin could increase the dynamic posttranslational modifications and reduced the expression of inflammasome which is beneficial to endothelial homeostasis and provide the new direction to drug design.