FKBP12與AICD間交互作用之研究

Abstract

AICD，又名類澱粉前驅蛋白胞內區塊(APP intracellular domain)，是由類澱粉前驅蛋白(amyloid precursor protein，APP)經過不同secretase切割後釋放至細胞質中之片段。目前發現AICD會與一些蛋白質產生交互作用，且發現AICD會進入細胞核中，與一些轉錄因子結合，但目前對於AICD之生理功能仍不清楚。已知類澱粉前驅蛋白被切割之過程與Notch蛋白相似，且Notch最終也會釋放出一段叫作NICD (Notch intracellular domain)之片段。NICD會進入細胞核並對細胞進行生理調控，因此一般推測AICD可能具有類似NICD之角色。本實驗室曾以酵母菌雙雜交法找到一個叫作FKBP12之蛋白質可能與AICD有交互作用。在此，我們初步在酵母菌雙雜交系統中，證明了AICD與FKBP12交互作用之存在，進一步證明實驗室以前觀察到之結果並非是融合蛋白造成之人為現象。此外，以辨識AICD抗體進行免疫沉澱，再以FKBP12抗體進行西方點墨法後，可以看到部分的FKBP12確實有被AICD所抓下來。同時，以免疫螢光染色觀察得到AICD與FKBP12在部分細胞之位置有重疊，而推測兩者可能有colocalization。另外，也觀察到FK506對於MC65細胞株在沒有tetracycline存在時，具有挽救細胞凋亡之保護能力。綜合上述之實驗結果，可得知FKBP12與AICD之間有交互作用之存在，但對於FKBP12與AICD之間彼此之影響仍不了解。希望能更明顯觀察到FKBP12與AICD之交互作用，並觀察FK506是否影響AICD與FKBP12之作用。藉由探討此交互作用對於其他蛋白質表現是否有調控作用，使得我們對於AICD之正常生理功能與FKBP12在阿滋海默症病理發展之分子機轉所扮演之角色有進一步的了解。

AICD, also called APP intracellular domain, is a cytoplasmic fragment released from amyloid precursor protein (APP) processed by different secretases. It is known that AICD interacts with some proteins, and AICD translocates into the nucleus and binds with some transcription factors. But the function of AICD is still not clear. Now it is known that the cleavage of APP is the familiar with Notch. And Notch releases a fragment called NICD (Notch intracellular domain) after been processed. Notch can translocate into nucleus and regulates downstream genes transcription. So it is suggested that AICD may play a role like NICD. We have been found a protein called FKBP12 may interact with AICD by yeast two-hybrid assay. Here our primary data confirmed that FKBP12 interacted with AICD by yeast-two hybrid assay, and further confirmed the results we had were not artifacts. Moreover, we confirmed that FKBP12 could interact with AICD by coimmunoprecipitation. And also we observed AICD overlapped with FKBP12 under microscope by immunostaining, and suggested that FKBP12 might colocalize with AICD. Besides, we observed FK506 exhibited neuroprotective ability by preventing cell apoptosis. In sum, according to the data we had, the interaction between AICD and FKBP12 exists, but we still don’t know the results of the interaction and how they interact. We want to further verify the interaction between AICD and FKBP12 clearly, and to see whether FK506 affects the interaction between FKBP12 and AICD. By investigating whether such an interaction modulates the expression of other proteins, makes us to be able to clarify the physiological and biological function of AICD and the roles of FKBP12 play in the pathogenic pathway of Alzheimer’s disease.