人類腦腎上腺白質退化症蛋白ALDP上與遺傳疾病有關的C端突變在酵母雙雜交分析中對蛋白穩定性的影響

過氧化體扮演著代謝解毒、分解細胞內老舊胞器、脂肪酸代謝等重要的角色。過氧化體上之膜蛋白，腦腎上腺白質退化症蛋白(ALDP)，屬於 ATP-binding cassette(ABC)half-transporter protein，會形成同源二聚體(homodimer)並且運送脂肪酸。人類 X 染色體上長臂第二區第八帶的ABCD1 基因發生突變，導致人類過氧化體膜蛋白 ALDP 的功能發生異常，無法形成同源二聚體及 β 氧化作用，最後導致疾病腦腎上腺白質退化症(Adrenoleukodystrophy)。酵母菌的過氧化體上也有類似人類ABC 運輸蛋白 Pxa1p 及 Pxa2p 也是屬於 half-ABC-transporter，會形成異源二聚體(heterodimer)而且和人類的 ALDP 蛋白同功同源(orthologs)。為了研究基因突變對於 ALDP 蛋白同源交互作用的影響，政益學長首先以酵母菌過氧化體膜蛋白的 Pxa1p 及 Pxa2p 做研究，發現 Pxa2p 的CT2 區域對於 Pxa1p 和 Pxa2p 交互作用是相當重要的。刪除 Pxa2p 的CT2 區域會影響 Pxa1p 和 Pxa2p 交互作用。導致酵母菌無法再油酸培養基生長。本實驗先針對先前實驗室所構築之人類 ALDP 刪除 CT2 序列及點突變 G677D、W679R、L684P、T693M 對於其蛋白之同源相互作用再做一次酵母菌雙雜交試驗確認，發現只有點突變 T693M 不會影響蛋白相互作用。接著以西方點墨法分析蛋白的穩定性，發現所有的蛋白都是穩定的。另外，我的實驗在做打破酵母菌細胞的時候，發展出了新的酵母菌破細胞的方法。以往的方法是用玻璃珠震盪打破酵母菌細胞，而我的實驗發展出了以 100℃加熱煮破酵母菌細胞的方法。這個方法比起用玻璃珠震盪的方法，步驟更簡單而且效果更好。所以我的實驗發展的新的以 100℃加熱煮破酵母菌細胞的方法，也提供其他研究者做參考。

Peroxisome plays an important role in metabolic detoxification,degradation of old organelles and metabolism of fatty acid. There is amembrane protein Adrenoleukodystrophy Protein (ALDP) in peroxisomemembrane, which belongs to ATP-binding cassette (ABC) half-transporterprotein, and two of ALDP form a homodimer before transporting fatty acid.The mutation of ABCD1 gene located in human X chromosome q armregion 2 band 8 (Xq2.8) result in human peroxisomal membrane proteinALDP dysfunction, including the failure to become homodimer andinability to perform β-oxidation, finally leading to the diseaseAdrenoleukodystrophy. There are two yeast peroxisomal half-ABCtransporter proteins Pxa1p and Pxa2p, which are similar to human ALDP,and they form a heterodimer. They are orthologs of human ALDP proteins.In order to find out the gene mutations of ALDP defective in homodimericinteraction, a previous study from our lab by Chuang et. al. about the yeastperoxisome membrane proteins Pxa1p and Pxa2p found that the CT2region of Pxa2p is very important for the interaction between Pxa1p andPxa2p. The CT2-deleted Pxa2p is defective in the interaction betweenPxa1p and Pxa2p, resulting in the disability of this yeast to grow on oleicmedium. In this study, first I reconfirmed the previous studies from our labby Lin et.al. and Tzong et.al., by using the plasmids with CT2-deletedmutation and point mutation G677D, W679R, L684P, T693M of ALDP tocheck ALDP homodimeric interaction by yeast two hybrid assay. Only thepoint mutation T693M didn’t affect the ALDP homodimeric interaction.Then I checked these mutated ALDP protein stability by Western Blottingand Commassie blue staining. The results showed that all these mutatedALDP proteins were stable in yeast cell. On the other hand, in my study, Ideveloped a new method for yeast cell disruption. The common method todisrupt yeast cell is using beads vibration. However, during my experiment,I found that the bead vibration method did not work well and only smallamounts of yeast lysate were obtained. So, I developed a method by using100 0C-boiling to directly disrupt yeast cell. This method is much easy andeffective than using beads vibration as demonstrated by Western blottingand Commassie blue staining in my studies. This method is very usefullyand can be utilized by other researchers for their studies.