2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38501Peroxisomes are the important organelles of a cell for the 芻-oxidation of the fatty acid. At present, it has being known that four ABC (ATP-binding cassette) transporters are located on peroxisome』s membrane in human and one of them, ALDP, is specifically involved in the 芻-oxidation of the VLCFA (very long chain fatty acid). The marked genetic disorder ALD (adrenoleukodystrophy) was due to the mutation of the aldp gene. However, to date, it is unclear what functions ALDP play in the 芻-oxidation. ALDP and the other three peroxisomal ABC transporters (ALDRP, PMP70 and PMP69) were all belong to half-ABC transporter. It has been shown that ALDP can interact with ALDP itself and with other two peroxisomal ABC transporters (ALDRP, PMP70), suggesting that they can form a homo-dimer or hetero-dimer. In yeast, there are two ALDP and PMP70 orthologs with the name of PXA1 and PXA2, respectively. The results from biochemical and genetic studies have suggest that the hetero-dimers of PXA1/PXA2 are involved in 芻-oxidation of the LCFA. Nevertheless, most (if not all) evidences for the hetero-dimers formation of PXA1 and PXA2 came from co-immunoprecipitation, genetic interaction and protein stability dependence, and these results can』t fully prove that these proteins form a dimer, due to inability to precisely determine the molecular weights of the protein complex, and rule out the possibility that PXA1 and PXA2 may form a homo-dimers. So, we want to study the relationships between the structure and function of PXA1 and PXA2. The subtitle of our researches will include:(1) demonstrate whether PXA1 or PXA2 can form the homo-complex and determine the number of monomer in protein complex, (2) demonstrate whether the complex assembly are regulated by ATP binding and hydrolysis, (3) construct PXA1 mutants, corresponding to human ALDP mutants, and determine the effects of mutation on protein structure and function, (4) establish in vitro long chain fatty acid import assay by using purified peroxisome from living cell, (5) reconstitute the functional peroxisome ABC transporters in liposome and characterize the factors involved in transport function. From our research results, we will unequivocally determine the function and structure of PXA1 and PXA2, and the new knowledge from the mutant characterizations can be applied to human ALD study by other scientists.過氧化體是真核細胞中脂肪酸進行芻氧化作用的重要胞器。目前已知人 類過氧化體膜上有四個ABC 運輸蛋白(ATP-binding cassette transporter)，其 中的ALDP 參與極長鏈脂肪酸之芻氧化作用，當此蛋白基因發生變異時，會 導致腎上腺腦白質退化症(簡稱為ALD)。然而，到目前為止仍不清楚ALDP 的功能為何。目前的證據指出，ALDP 與其他兩個過氧化體ABC 蛋白 (ALDRP 及PMP70)會形成同源及異源二聚體。酵母菌中也有ALDP 及 PMP70 的同源性蛋白，分別為PXA1 及PXA2。生化及遺傳的證據指出， PXA1 及PXA2 是以異源二聚體參與長鏈脂肪酸的芻氧化作用。然而，這些 二聚體的證據皆來自於免疫共沈澱、遺傳學上的分析及蛋白質穩定的相關 性，這些方法無法測定分子量而不能完全證明是否為二聚體，也無法排除同 源複合體存在的可能性。因此，我想要研究酵母菌PXA1 及PXA2 蛋白複合 體的構造及功能之間的關係。我們的研究目標如下：(1)證明PXA1 及PXA2 是否會形成同源複合體、是二聚體還是寡聚體(2)複合體聚合過程是否受核 甘酸調控(3)在PXA1 上構築相對於ALD 致病的突變，分析突變對蛋白功能 及複合體結構的影響(4)以純化的過氧化體建立in vitro 脂肪酸運輸分析系統 (5)在微脂粒中重組成有功能的運輸蛋白，並分析影響蛋白功能的因素。這 些方法將使我們清楚知道複合體的功能及其組成，並賦於ALD 的突變新的 意涵。過氧化體ABC 運輸蛋白蛋白複合體脂肪酸運輸ALDperoxisomal ABC transporterprotein complexfatty acid transportALDProject