Molecular mechanisms and development of enzyme therapy for mitochondrial trifunctional protein deficiency

長鏈脂肪酸的氧化是由長鏈脂酰輔酶A脫氫酶啟動反應開始，接著粒線體三功能蛋白酶 (mitochondrial trifunctional protein, MTP)負責接下來的三個步驟， 粒線體三功能蛋白酶是由四個α次單元(Hadha)與四個β次單元(Hadhb)組成的多功能酵素複合體。與粒線體三功能蛋白酶基因的突變相關的遺傳疾病有長鏈酰基輔酶A脫氫酶缺乏症和粒線體三功能蛋白酶缺乏症，其中粒線體三功能蛋白酶缺乏症伴隨著粒線體三功能蛋白酶的三個酵素活性都顯著下降。在先前的研究當中，我們發現在老鼠β次單元的外顯子14區上發生由脫氧胸腺嘧啶變成脫氧腺嘌呤，因而造成此蛋白第404個胺基酸由甲硫胺酸變成離胺酸(M404K)。西方墨點法與酵素活性實驗顯示粒線體三功能蛋白酶的α和β次單元表現量與活性都減少，意味著這個突變可能會影響此複合體的穩定度。因此，我們推測如果將正常的β次單元送回突變老鼠體內，應該可以回復粒線體三功能蛋白酶的功能。我們利用轉染方式送入正常β次單元基因到含有M404K突變的纖維母細胞，同時用藥物篩選可穩定表現正常β次單元蛋白的突變纖維母細胞，初步的實驗結果顯示其α和β次單元蛋白表現都有增加。另一方面，我們也嘗試用細胞穿膜肽(cell penetrating peptides, CPP)來攜帶正常的β次單元進入細胞裡，我們的假設是與細胞穿膜肽結合的β次單元可以穿越細胞膜、到達並進入到粒線體內取代突變的β次單元，進而回復粒線體三功能蛋白酶的功能。利用轉染的實驗得知表現的細胞穿膜肽融合的α及β次單元可運送到粒線體。然而目前用大腸桿菌表現系統表達重組的α及β次單元都不具酵素活性，推測可能是α及β次單元本身為大分子而且內含許多雙硫鍵，造成無法正確摺疊出有活性的α和β次單元，我們嘗試找出適合的條件能有利於表達有活性α和β次單元。此外，我們也希望找出能增加細胞穿越肽融合α和β次單元蛋白濃度的純化方法，來增加細胞穿越肽融合α和β次單元蛋白進入細胞的效率。我們實驗的結果顯示替換掉突變的β次單元可以增加粒線體三功能蛋白酶的穩定，未來的酵素活性實驗及分析肉鹼脂肪酸結合物含量可進一步證實是否粒線體三功能蛋白酶功能的回復，同時更多的研究可釐清細胞穿越肽融合β次單元蛋白應用在酵素替代治療方法在粒線體三功能蛋白酶缺乏症的可行性。β-oxidation of the long-chain fatty acids is initiated by a catalytic reaction mediated by a long-chain acyl-CoA dehydrogenase, followed by the mitochondrial trifunctional protein (MTP), which is a multienzyme complex composed of four α-subunits (Hadha) and four β-subunits (Hadhb) that catalyses the next three steps of β-oxidation.. Genetic defects in MTP cause the deficiency of isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) or complete MTP deficiency with markedly reduced activities of all three enzymes. In our previous study, we identified mice with a nucleotide T-to-A transversion in exon 14 of Hadhb gene which results in a missense mutation from methionine to lysine at codon 404 of β-subunit. Western blot analysis and enzyme activities showed a significant reduction of both α- and β-subunits, which imply that this mutation may affect MTP complex stability. Therefore, we speculated that delivering normal β-subunits could restore the MTP function in the mutant mice. Stable transfectants carrying normal Hadhb cDNA were generated from Hadhb-/- mutant fibroblasts. Our preliminary data showed that both α- and β-subunit expression were increased in the stable tansfectants. In addition, we attempted to deliver exogenous β-subunit protein into cells by conjugating β-subunit with cell penetrating peptides (CPP). We hypothesized that CPP-β-subunit fusion protein could enter the cell, target to the mitochondria and restore the MTP function by replacing the mutated protein. After transient transfection of CPP-β-subunit fusion cDNA, the CPP-fusion proteins was expressed and targeted to the mitochondria. To deliver the CPP-fusion protein itself, we expressed the recombinant α- and β-subunit in E. coli system, however, the expressed protein was inactive, presumably due to high molecular weight of the protein and presence of multiple disulfide bridges. To test protein delivery, sufficient amounts of soluble and denatured form of CPP-conjugated proteins are required. Our data provided preliminary evidence that β-subunit replacement could restore the MTP protein stability. Enzyme assay and acylcarnitines profile will be analyzed to confirm whether the biochemical phenotypes are corrected. Further study will be needed to test the feasibility of CPP-fusion protein for the enzyme replacement therapy for MTP deficiency.1 Introduction 1-1 β-oxidation 1-2 Mitochondrial trifunctional protein 2-3 Mitochondrial trifunctional protein defects 3-4 Mouse model of complete MTP deficiency 5-5 Cell penetrating peptide 6-6 Specific aim and hypothesis 8 Materials and methods 10-1 Cell culture 10-2 Cloning the expression constructs of Hadha and Hadhb 10-2-1 Constructs of CPP conjugated Hadha and Hadhb……………………...10-2-2 Constructs for recombinant retroviruses carrying Hadha and Hadhb cDNA 12-2-3 Constructs for recombinant HADHA and HADHB…………………….13-3 Production of recombinant retroviruses carrying Hadha and Hadhb cDNA 16-4 Western blotting 18-5 Enzyme assay 18-6 Confocal microscopy 19-7 Recombinant protein expression and purification 20-7-1 Recombinant HADHA and HADHB with C-terminal His tag…………20-7-2 Recombinant HADHA and HADHB with N-terminus mistic………….22-7-3 Recombinant CPP-conjugated proteins with C-terminal His tag………23-8 Refolding HADHA and HADHB from inclusion bodies 24-9 Delivery of CPP-conjugated proteins into cells 24 Results 26-1 Hadhb cDNA expression in fibroblasts isolated from Hadhb-/- mutant mice 26-2 CPP did not affect the MTP targeting to the mitochondria 27-3 Recombinant HADHA and HADHB expressed in E. coli had no enzyme activities 28-3-1 Recombinant HADHA and HADHB with C-terminus His tag………...28-3-1 Recombinant HADHA and HADHB with N-terminus mistic……….…29-3 Recombinant CPP-conjugated HADHA and HADHB were not detected in NIH3T3 cells……………………………………………….……………….30 Discussions 32-1 The M404K mutation may affect the interaction domain of α2β2-α2β2….…32-2 Why normal Hadhb cDNA could not be expressed in Hadhb-/- mutant fibroblasts?.....................................................................................................33-3 The reasons which may inactivate HADHA and HADHB from bacterial expressing system 35-4 Why the exogenous CPP-conjugated HADHA and HADHB proteins could not be detected in NIH3T3 cells? 36-5 Limitations of CPP-mediated protein delivery 38 Figures 40 Table 54 References 55 Appendixes 5