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標題: | 探討FKBP12對APP代謝之影響 Elucidation of the Effects of FKBP12 on APP Processing |
作者: | Tiffany Hsin-Yu Huang 黃歆予 |
指導教授: | 孔繁璐(Fan-Lu Kung) |
關鍵字: | 阿茲海默症,類澱粉前驅蛋白,FKBP12,PPIase,FK506,calcineurin, Alzheimer's disease,Amyloid Precursor Protein,FKBP12,PPIase,FK506,calcineurin, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 阿茲海默症 (Alzheimer’s disease,AD) 為一常見的神經退化性疾病,其中一項病理特徵為在細胞間隙堆積的老年斑塊 (senile plaques),由類澱粉前驅蛋白 (amyloid precursor protein,APP) 經酵素循序切割水解後產生的 amyloid-β (Aβ) 所組成。過去研究發現APP上的Thr668經磷酸化後會提高Thr668-Pro669間胜肽鍵cis構型的比例,並與Aβ的產生呈正相關。
實驗室過去發現一具有PPIase 活性的免疫親合素FK506-Binding-Protein12 (FKBP12) 與類澱粉前驅蛋白胞內區塊 (APP intracellular domain,AICD) 有交互作用,並使得APP的代謝途徑走向amyloidogenic pathway。透過FKBP12的突變株,證實FKBP12對APP代謝的影響與其PPIase活性有關,推測FKBP12可能透過催化APP上Thr668-Pro669胜肽鍵的構形由trans到cis form,進而影響APP的水解途徑。為確認此假說,利用以Ala取代APP上Pro669而產生一無法被FKBP12進行胜肽鍵構形催化的突變株,以及FKBP12 PPIase活性缺陷突變株,可釐清FKBP12影響APP代謝背後的分子機轉。同時,各FKBP12的活性則以chymotrypsin-free assay確認。前人的研究發現,在細胞中給予FK506可以反轉過度表現FKBP12的效果,其機轉可能是干擾FKBP12與AICD的交互作用,同時抑制FKBP12 PPIase 活性。然過去的研究指出FK506的神經保護作用與其對calcineurin的抑制有關,為了解calcineurin是否參與所觀察到FKBP12-mediated的APP水解調控,FKBP12突變株FKBP12R42I與FK506衍生物FK1706將用於實驗中來釐清此問題。 在本實驗當中,以快速蛋白質液相層析系統 (fast protein liquid chromatography,FPLC)純化出七個具不同 PPIase activity的FKBP12蛋白。然而在以chymotrypsin-free assay測定FKBP12的PPIase activity實驗當中,並未測得因受質胜肽鍵構形改變而產生在330 nm波長下的吸收變化,其原因可能來自FKBP12在純化過程中活性的降低,亦或是儀器靈敏度不足。在探討APP代謝是否受FKBP12 PPIase活性影響的部分,APP兩代謝途徑中產物C99和C83的比例,可指出APP代謝途徑受FKBP12作用的改變。然而 C99和C83分子量的差距極小,實驗中未能以西方墨點法成功分離兩者,因此無法確認本實驗的假說。 針對上述實驗的瓶頸,可改以NMR 和chymotrypsin assay分析FKBP12 之PPIase 活性。在APP代謝的部分,則在本論文中針對西方墨點法的步驟以及可能的替代方法進行討論,以用於未來的改善。 Alzheimer’s disease (AD) is a common neurodegenerative disease with the pathological symptom of extracellular senile plaques composed of amyloid-β (Aβ), a serial processed product of amyloid precursor protein (APP). Accumulating evidences have shown that phosphorylation at Thr668 causes an increase of cis form Thr668-Pro669 peptide bond in APP and is correlated with Aβ generation. FKBP12, an immunophilin with PPIase isomerase activity, was found to interact with APP intracellular domain (AICD) and shift APP metabolism to amyloidogenic pathway. A subsequent study using FKBP12 mutants revealed that the effect of FKBP12 on the regulation of APP processing corresponded to its PPIase activity, indicating that FKBP12 might catalyze Thr668-Pro669 peptide bond conformation from trans to cis form. To confirm this hypothesis, APPP669A, a mutant which cannot be catalyzed by FKBP12 due to the substitution of Pro669 with Ala, and PPIase activity-deficient FKBP12 were used to elucidate the molecular mechanism of the effect of FKBP12 on APP processing. Additionally, the PPIase activity was attempted to be evaluated by chymotrypsin-free assay. A Previous study also showed that the effect of FKBP12 can be reversed by the presence of FK506, which may cause the disruption of AICD-FKBP12 interaction and, at the same time, the inhibition of PPIase activity. However, the neuroprotective effect of FK506 has been linked to the inhibition of calcineurin. To elucidate whether or not calcineurin is involved in the FKBP12-mediated APP processing, FKBP12 mutant, FKBP12R42I, and FK1706, a FK506 derivate, are used to address this question. Seven FKBP12 with different PPIase activity were purified using fast protein liquid chromatography (FPLC). While the PPIase activity of purified FKBP12 cannot be validated by detecting the 330 nm absorbance differences between cis and trans form substrates in chymotrypsin-free assay. The failure can be due to the loss of protein activities during purification process and/or the sensitivity limitation of a UV/VIS spectrum. The ratio of the two products, C99 and C83, generated by processed APP in two different pathways were used to verify the shifting of APP metabolism under the effect of FKBP12. However, C99 and C83 signals on western blot cannot be successfully distinguished due to the small size difference between each two. Therefore, the hypothesis of the role of FKBP12 in APP processing cannot be confirmed by the conducted experiment. To improve this study, NMR and chymotrypsin assay can be applied to verified the PPIase activity of FKBP12. Also, the improvement of western blot procedures and alternative methods to observe APP processing are discussed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59408 |
DOI: | 10.6342/NTU201701054 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥學系 |
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