鑑別FKBP12與APP交互作用的關鍵胺基酸位點之方法開發

Abstract

FK506-binding protein 12 (FKBP12)為一個能各別與FK506及rapamycin鍵結並形成複合體，且抑制下游phosphatase活性，進而影響T細胞活化、細胞生長甚至蛋白質生合成的蛋白質。而FKBP12本身具有peptidyl-prolyl cis-trans isomerase (PPIase)的酵素活性，可以將蛋白質中位於特定胺基酸proline前的胜肽鍵進行cis/trans轉變，進而改變與其他蛋白質之間的交互作用，而影響此蛋白質走向不同的路徑。本實驗室曾利用酵母菌雙雜交法以及免疫共沉澱法確認FKBP12與APP intracellular domain (AICD)有一定程度的交互作用，且發現當在HEK293T細胞株過量表現FKBP12時，C99/C83比例有上升的趨勢，意即有較多APP以amyloidogenic pathway代謝，而此現象在加入FK506之後會被反轉回來。由先前的FKBP12結構研究中已知FKBP12的Asp37、Arg42、Phe46、Trp59、Tyr82等殘基位於FKBP12與FK506的結合位置，本研究企圖利用一系列在這些位置突變的FKBP12 (FKBP12D37V、FKBP12R42I、FKBP12F46L、FKBP12W59A、FKBP12W59L、FKBP12Y82F)來鑑別對APP-FKBP12交互作用重要的位點，計劃將不同的FKBP12各別與wile-type APP混合，利用免疫共沉澱法觀察其交互作用的差異。 已知免疫共沉澱實驗會受到許多因素影響，例如: detergent、還原劑的種類及濃度、以及溶液的pH值等等，又由於FKBP12與APP可能為酵素與受質之關係，兩者之間的交互作用可能極為短暫，因此在實驗進行過程中，將研究方向轉為克服前述困難之方法開發。此研究結果將可作為未來執行免疫共沉澱實驗鑑別對APP-FKBP12 interaction重要的位點時選擇條件之依據。另外，我們也初步嘗試使用遠西方墨點法，或可作為未來進行APP-FKBP12交互作用研究之輔助方法。

FK506-binding protein 12 (FKBP12) is a protein capable of forming complex with FK506 or rapamycin, leading to the inhibition of downstream phosphatase activity, thus affecting T cell activation, cell growth and protein biosynthesis. FKBP12 has peptidyl-prolyl cis-trans isomerase (PPIase) activity. PPIase can accelerate the cis and trans isomerization of the Xaa-Pro peptide bond, thus changing the interaction with other proteins and may lead these proteins toward different pathways. Our previous study indicated FKBP12 can interact with APP intracellular domain (AICD) by using yeast-two-hybrid and co-immunoprecipitation (co-IP) methods. The C99/C83 ratio has been found to be increased when overexpressing FKBP12 in HEK293T cell line, suggesting a shift in APP processing to the amyloidogenic pathway, but this phenomenon was reverted when adding FK506. It’s known that Asp37, Arg42, Phe46, Trp59 and Tyr82 are at the FK506 binding site of FKBP12 from previous FKBP12 structural study. In this study, we intend to use co-IP method to identify amino acid residues of FKBP12 critical for APP-FKBP12 interaction by using a series of FKBP12 mutants with missense mutations in these amino acid residues (FKBP12D37V, FKBP12R42I, FKBP12F46L, FKBP12W59A, FKBP12W59L and FKBP12Y82F). Co-IP experiment is known to be affected by many factors such as the type and concentrations of detergents and reducing agents present and the pH of the solution, etc. In addition, the interaction between APP and FKBP12 could be too transient to be detected by co-IP as APP may be a substrate of FKBP12 enzyme. The focus of this research, as a result, has shifted to method development during the course of study. Our results could serve to guide future co-IP assays to investigate the interaction between APP and FKBP12. We have also explored the possibilities of using far Western blotting method as a supportive approach for studying APP-FKBP12 interaction in the future.