探討人類腎上腺腦白質退化症(ALD)有關的ALDP點突變G677D及T693M對於蛋白自身同源性相互作用的影響

過去的文獻指出在酵母菌體內過氧化體膜上的ABC運輸蛋白Pxa1p及Pxa2p是屬於half-ABC transporter，會形成異源二聚體(heterodimer)，且對於長鏈脂肪酸的代謝作用中扮演了一種重要的角色，同時是哺乳類動物過氧化體膜上ALDP、ALDRP、PMP70這三種ABC運輸蛋白的同源性蛋白。另外，本實驗室正在投稿的實驗結果指出，酵母菌的Pxa2p蛋白可經由其C端之CT2區域的幫助使Pxa2p與Pxa1p的NBD之間產生相互作用，因此我們想進一步探討人類的ALDP的CT2區域是否在ALDP同源性相互作用中扮演重要角色。 本篇論文一開始以本實驗室政益學長構築只包含NBD-CT (從第361~745個胺基酸)而不包含穿膜區(從第1~360個胺基酸)的ALDP蛋白的質體為基礎，並根據政益學長的方法進一步將ALDP之C末端區域(CT)分成CT1序列(從第631~676個胺基酸)及CT2序列(從第677~745個胺基酸)。首先，我先構築出用於酵母菌雙雜交分析之CT2序列刪除突變質體pACT2-ALDP-ΔCT2及pEG202-ALDP-ΔCT2，將此質體組合透過酵母菌雙雜交實驗的方法進行分析，結果發現ALDP的CT2區域在ALDP蛋白同源相互作用上扮演重要的角色。經由搜尋X-ALD資料庫網站，發現兩個與疾病相關的點突變位於此CT2區域，分別為G677D、T693M。接著，我透過SOE (Spliced by overlapping extension)的方法構築出帶有G677D、T693M的點突變質體，同樣透過酵母菌雙雜交的方法進行分析，結果發現G677D這一個突變點會影響ALDP蛋白的相互作用，而進一步導致X-ALD疾病的形成。但是T693M這個突變點對於ALDP的相互作用影響比較不明顯。雖然，T693M對於ALDP蛋白之間的相互作用影響比較少，這並不代表對於其蛋白功能並不會產生任何的影響。有可能影響其他特性，如、ATP結合及水解、物質運輸的過程或是被運輸物質與ALDP蛋白相互作用等等…。 以上的結果是遺傳性的方法來分析蛋白之間的相互作用，我們也計畫進一步使用蛋白免疫共沉澱方法來驗證蛋白之間相互作用特性的變化，有關這一部份的實驗結果是失敗的，可能是抗體有問題，無法將蛋白沉澱下來。

In th past studies,it was showed that Pxa1p and Pxa2p proteins were belonged to half ABC transporter on yeast peroxisome and could form a heterodimer,which plays an essential role in long chain fatty acid metabolism.They are homologus to human ALDP,ALDRP,and PMP70.Besides,our submitting results for publication have indicated that the NBD of Pxa2p could interact with the NBD of Pxa1p by the aid of its C-terminal CT2 region.So, we want to study whether the CT2 of ALDP,Homologus to yeast Pxa2p, plays a role in ALDP homodimeric interaction.In the beginning of this study, I used the previously constructed plasmid by our laboratory, containing only the NBD-CT (residue 361 to 745) and not containing the transmembrane region (residue1 to 360) of ALDP protein. Based on the CT sequence homology between ALDP and Pxa2p, I divided the CT region of ALDP into the CT1 sequence (residue 631 to 676 ) and the CT2 sequence (residue 677 to 745 ). First of all, I builded two plasmids with the CT2 deletion in the NBD_CT, pACT2-ALDP-ΔCT2 and pEG202-ALDP-ΔCT2, and used the yeast two hybrid assay to analysize the protein interaction. I found that the CT2 of ALDP played an essential role in ALDP homodimeric interaction. By searching X-ALD database website and I found that two point mutations, G677D and T693M, in the CT2 region of ALDP were associated with ALD disease. Then, I bulided the G677D and T693M mutation in ALDP plasmids by SOE method. From the result on the yeast two hybrid analysis, I found that G677D might affect ALDP homo-interaction and eventually leads to X-ALD. Although the T693M mutation had no obvious effects on protein interaction, it may affect other aspects of protein properties, such as ATP-binding and hydrolysis, the process ofsubstrate transport across the membrane, the interaction between thesubstrate and ALDP, ect. Because these results mentioned above were generated from genetic method, I performed the immunoprecipitation method to verify the effect of point-mutations on the ALDP homodimeric interaction. This part of the experiment failed and was probablely due to the inability of the anti-HA antibody to precipitate Gal4 AD fusion protein.