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Title: | Thr668磷酸化狀態對APP與其它蛋白質的交互作用與分佈的影響 Effects of Thr668 Phosphorylation on Protein Interaction and Distribution of APP |
Authors: | I-Ting Wang 王怡婷 |
Advisor: | 孔繁璐(Fan-Lu Kung) |
Keyword: | Vivalds,網路座標系統,封包來回時間預測,同儕網路,網際網路拓樸學, amyloid precusor protein,Thr668,phosphorylation, |
Publication Year : | 2007 |
Degree: | 碩士 |
Abstract: | 阿茲海默氏症的其中一種表徵是amyloid-β peptide (Aβ)在細胞外的堆積現象。Aβ是APP經由酵素切割後的產物之一。許多研究指出APP的代謝過程會受到lipid raft或位在lipid raft裡的蛋白質影響,此外它的代謝也會被其位在細胞質內之C端片段之磷酸化情形(特別是在Thr668位置)影響。在阿茲海默氏症的病人裡Thr668的磷酸狀態較正常人明顯的上昇。此外研究也發現當Thr668被磷酸化時,APP的C端(AICD)的構型會因此而有所改變,而可能影響到它跟其他蛋白質間的互相作用,最後導致APP的代謝和分佈有所改變。
本實驗室之前發現flotillin-1跟AICD有交互作用。Flotillin-1是一個lipid raft蛋白質並在AD病人腦中有聚集的現象。為了知道Thr668磷酸化是否會影響AICD與flotillin的交互作用,從而影響APP在細胞中之分佈我們在HEK293T細胞中表現一系列將Thr668這個胺基酸置換成其他胺基酸之AICD mutants,觀察AICD跟flotillin-1的交互作用是否會因有無磷酸化而有所改變。同時我們也探討磷酸化抑制劑與特定免疫抑制劑是否會導致SH-SY5Y細胞裡APP/AICD的分布改變。若可證實Thr668磷酸化是可以影響APP與flotillin之間的相互作用且flotillin是可把APP帶到細胞膜上的重用蛋白之一,那未來或許我們可嘗試著干擾它們的交互作用,讓少一點APP在lipid raft上被BACE processed。這或許是個新的治療AD之 target。 我們的初步實驗結果指出flotillin-1和AICD間有交互作用,而且此交互作用似乎在Thr668磷酸化時較為明顯。目前我們在co-IP的實驗裡看到flotillin-1與AICD的交互作用似乎是跟Thr668磷酸化有關係的。但在sucrose fractionation的實驗裡我們並看不到 p-CTF 與 flotillin-1有co-localize的現象。而且出現的這個大小的fragment在其他的文獻中還尚未報導過。另一方面我們在HEK293T的細胞裡發現FKBP12似乎是影響到APP的磷酸化。在只有over-expressing FKBP12或over-expressing FKBP12與APP的HEK293T我們也同時看到了約34-43和56-72大小的片段出現,並且在over-expressing APP我們只看~34-43的片段增加。 這些磷酸化的片段的切割似乎是與FKBP12的表現有關因為over-expressing APP裡的片段在每個fraction都有出現,但over-expressing FKBP12但似乎磷酸化的片段都會shift到比較輕的fractions裡而且在中間的fractions裡居多。這些磷酸化的片段雖然並沒有聚集在有flotillin較多的fractionations裡,但還是有些co-localization的現象。 Alzheimer’s disease is characterized by deposition of amyloid-β peptide, proteolytic product of amyloid precursor protein (APP). Evidences have suggested that APP processing may be regulated by lipid raft or proteins reside in it, and by cytoplasmic phosphorylation of APP, predominantly on Thr668. Increased levels of Thr668 phosphorylated APP is observed in AD patients. Phosphorylation at Thr668 leads to a conformational change of the intracellular domain of APP (AICD), and may have influences on its interaction with other cytoplasmic protein and eventually lead to the alteration of its processing and localization. We have previously found that AICD interacts with flotillin-1, a lipid raft protein. To further examine the effect of phosphorylation at Thr668 on its interaction with flotillin and its cellular localization, AICD mutants with various single point mutations at the threonine residue are expressed in human embryonic kidney cell (HEK293T) to see whether the distribution of APP and/or AICD are altered. Whether the presence of certain kinase inhibitors affect the localization of APP and/or AICD in neuroblastoma (SH-SY5Y), and which protein or amino acid residues are essential for APP metabolism will be also be discussed. If we can show that Thr668 phosphorylation status modulates the interaction between AICD and flotillin-1 and flotillin-1 is one of the critical protein involve in bring AICD to the plasma membrane, then flotillin-1 may be a new target for treating AD. In our preliminary result, we have show that flotillin-1 does interacts with AICD and their interaction is somewhat enhanced by phosphorylation at Thr668. Consecutively dephosphorylated mutants do not interact with flotillin-1 or their interaction is very weak. We also observe a band ~15-25 kDa whose intensity is significantly increased upon kinase activation. The physiological role of this fragment is still unknown since p-CTF with this molecular weight has not been reported yet. This fragment does not co-localize with flotillin-1 in fractionation and it does not shift to lipid raft fractions as we have proposed so whether flotillin-1 mediates re-distribution of phosphorylated APP is still undetermined. Similarly, we also observe increase in the intensity of the ~15-20 kDa band in either APP and/or FKBP12 transfected HEK293T transfected cells. This observation is consistent with our hypothesis that FKBP12 may affect the phosphorylation of APP. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/29334 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 藥學系 |
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