果蠅中IBMPFD之蛋白質體學分析

Proteomic Analysis of IBMPFD, a Hereditary Disease, in Drosophila melanogaster

詹欣慈(Chan, Chelsea)

指導教授 ： 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2010年)

https://doi.org/10.6843/NTHU.2010.00233

摘要

Inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia, also known as IBMPFD, is a hereditary, autosomal dominant and ultimately lethal multi-system degenerative disorder due to single missense mutations in VCP (Valosin-containing Protein). TER94/VCP is an evolutionary highly conserved AAA-ATPase (ATPases Associated with diverse cellular Activities), which is involved in a great variety of cellular mechanisms such as protein degradation, ERAD (Endoplasmic Reticulum (ER)-associated Protein Degradation), membrane fusion, cell cycle control, transcriptional control, and DNA repair. The mechanisms of how mutations of VCP lead to IBMPFD remain mysterious. Here we utilize a powerful technique, two-dimensional difference gel electrophoresis (2D-DIGE), that when combined with mass spectrometry, can be applied to study IBMPFD disorder at the protein level. With this set up, we are able to employ comparative proteomics to analyze IBMPFD disease using Drosophila melanogaster as our disease model organism. Head proteome of transgenic D. melanogaster expressing TER94/VCP-WT is respectively compared with the head proteome of transgenic D. melanogaster expressing TER94/VCP mutants that correspond to human IBMPFD disease alleles (TER94R152H, TER94R188Q, TER94A229E). Of all the proteins identified, a significant fraction of proteins altered in TER94A229E and TER94R188Q mutants belong to the same functional categories, i.e. eye formation, apoptosis and metabolism. Transferrin, a blood plasma protein for iron ion delivery, is observed to be significantly up-regulated in mutant flies expressing TER94A229E. Knock-down experiment proves transferrin to be a potential key player in IBMPFD disease. The molecular analysis of IBMPFD disease greatly benefit from a proteomics approach that combines the advantages of high throughput analysis and the focus on protein levels.

關鍵字

多重器官退化性疾病

並列摘要

IBMPFD 是一種顯性遺傳，因為VCP的點突變所導致的多重器官退化性疾病。TER94/VCP是一種在演化上具有高度保留的AAA-ATPase。它參與了許多細胞機制，例如：蛋白質降解、內質網相關蛋白質的降解(ERAD) 、膜融合、調控細胞週期、轉錄調控以及DNA修補。然而VCP的突變是如何導致IBMPFD的機轉目前仍是神秘未知的。因此我們利用了2D-DIGE及質譜儀的技術去比較IBMPFD這個疾病在蛋白質體表現上的差異。本研究我們利用了果蠅當作這個疾病的模式生物去比較分析IBMPFD這個疾病的蛋白質表現。將正常表現 ter94基因的果蠅與其他三種ter94突變的果蠅 (TER94R152H, TER94R188Q, TER94A229E) 比較，結果顯示他們共同有眼睛形成蛋白、apoptosis 和代謝相關蛋白表現量的差異。其中，運鐵蛋白 (Transferrin) 在突變果蠅TER94A229E中明顯具有高度表現。因此我們利用了 Knock-down 果蠅運鐵蛋白的實驗中發現它在IBMPFD這個疾病中扮演一個重要的角色。IBMPFD這個疾病經過2D-DIGE及質譜儀的技術分析後對於它蛋白質的表現以及修飾得到非常大的瞭解及助益。

並列關鍵字

IBMPFD ； MALDI-TOF ； 2D-DIGE ； TER94

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