臺灣博碩士論文加值系統

本論文的研究目主要利用動物與細胞模式探討膳食鐵量對鐵硫蛋白質生合成之影響與粒線體IscS蛋白質表現與鐵硫蛋白質生合成的關係。本研究成功地選殖到大鼠IscS cDNA序列與其抗體的製備。利用Western blot與Northern blot分析得知大鼠IscS的表現以肌肉與心臟最高而肝臟與大腦最少。大鼠IscS蛋白質分子量為47 kDa，主要分佈於粒線體中，但是沒有測到細胞質含有IscS蛋白質。缺鐵大鼠肌肉IscS蛋白質含量減少約53%，而其mRNA表現量不變，但是缺鐵大腦與肝臟的IscS蛋白質含量與正常大鼠沒有差異，由此可見膳食鐵量對肌肉IscS蛋白質的調控主要在後轉錄的層次，並且具有組織選別性之差異。
另外採用Wistar雄性離乳大鼠，分成對照、缺鐵對飼育（pair-fed）與缺鐵三組，並分別以35 mg Fe/kg diet正常或缺鐵飼料飼養1和2週。第一與第二週膳食缺鐵肌肉粒線體IscS蛋白質量減少，分別為對照與缺鐵對飼育組的45-50%。此外缺鐵大鼠肌肉c-aconitase，m-aconitase，NADH dehydrogenase與SDH的活性分別減少55-76%、約50%、26-32%與34-59%，而c-aconitase、m-aconitase、NADH dehydrognase之24 kDa Ip subunit、SDH之Fp與Ip subunits蛋白質量減少50%、58-64%、61-73% 與56-79%，但是這些蛋白質的mRNA量則不受缺鐵的影響。缺鐵時肌肉中IRP1與IRP2活性顯著高於正常大鼠分別為2.6-2.7與2.2-2.3倍。可見膳食鐵量對肌肉c-aconitase，m-aconitase，NADH dehydrogenase與SDH的調控在後轉錄的層次。缺鐵大鼠肌肉粒線體IscS蛋白質含量減少，造成鐵硫複合體生合成不足，因而導致鐵硫蛋白質活性下降。
利用Tet-Off系統控制PC-12轉殖細胞粒線體IscS的表現與IscS蛋白質過度表現細胞的篩選，IscS mRNA與蛋白質量分別增加為2-7倍與1.2-1.6倍。然而這些細胞株的m-aconitase活性傾向不一致的反應，兩株的m-aconitase活性減少而三株m-aconitase活性增加。所有IscS過度表現細胞株的c-aconitase活性不變。加鐵培養時導致粒線體IscS蛋白質含量與m-aconitase活性增加20%，但是c-aconitase活性不變。關於IscS與aconitase活性的關係尚需驗證。
綜合以上，本論文首次證實膳食缺鐵減少大鼠肌肉粒線體IscS蛋白質的表現主要在後轉錄的調控，同時肌肉IRP1與IRP2的活性會受到膳食鐵量的調控。

The thesis was aimed to study the effect of iron nutrition on biosynthesis of iron-sulfur proteins and the relationship between mitochondrial cysteine desulfurase iron-sulfur cluster S (IscS) and biosynthesis of iron-sulfur proteins, using a rat model and a cell culture model. Rat IscS cDNA was cloned and sequenced, and polyclonal antibody against recombinant rat IscS protein was prepared. The deduced protein sequence has several characteristic features common to those of eukaryotic IscS proteins which contain a typical mitochondrial targeting presequence and found to be 47 kDa proteins in mitochondrial fraction. Expression of IscS in rat was found most abundant in muscle and heart and less in liver and brain by Northern and Western blotting assays. Within cells, IscS existed predominantly in mitochondria and was not detectable in cytosol. In rats rendered iron-deficient anemic by feeding an iron-deficient diet, the mitochondrial IScS protein levels in skeletal muscle decreased to about 53% of the iron-adequate control levels, but those in liver and brain remained at control levels. Iron deficiency did not affect the IscS mRNA levels in the skeletal muscle. This indicates that iron deficiency affects the expression of IscS protein at post-transcriptional level in a tissue-specific manner.
Three groups of male weanling Wistar rats were used, one group was fed an iron deficient diet (D), and two other groups were paired-fed (P) or freely fed (C) a control (35 mg Fe/kg diet) diet for 1 or 2 weeks. At the end of week 1 and week 2, the mitochondrial IscS protein levels in the skeletal muscle of iron-deficient rats were decreased to 45% and 50% of those of the control and pair-fed rats, respectively. Iron deficiency reduced cytosolic aconitase (c-aconitase), mitochondrial aconitase (m-aconitase), NADH dehydrogenase and SDH activity to 55-76%, about 50%, 26-32% and 34-59%, respectively. The c-aconitase, m-aconitase, 24 kDa Ip subunit of NADH dehydrogenase activity, Fp and Ip subunit of SDH protein level in iron-deficient rats also declined to 50%, 58-64%, 61-73% and 56-79% of the control and pair-fed levels, respectively; however the mRNA level of these proteins remained unchanged. The IRE-binding activities of IRP1 in the iron-deficient group were 2.6-2.7 and 2.2-2.3 times of the control levels, respectively, while no difference existed between the control and pair-fed groups. Our results indicate that dietary iron modulates c-aconitase, m-aconitase, NADH dehydrogenase, SDH as well as IscS at posttranscriptional level, and the shortage of Fe-S cluster caused by depletion of iron and mitochondrial IscS may offer an explanation for the decline in the enzyme activity of Fe-S proteins in iron-deficient rat muscle.
Furthermore, IscS mRNA controlled by Tet-off system was over-expressed in PC-12 cells, and clones stably overexpressing IscS were selected. The IscS mRNA levels increased 2-7 folds and their corresponding protein levels increased 1.2-1.6 folds. However, the m-aconitase activity in these clones exhibited an inconsistent trend that two clones had decreased, while 3 clones had activity. The activity of c-aconitase in all the clones remained unchanged. Treatment of these clones with 100

圖與表目錄 IV
縮寫對照表 VII
中文摘要 VIII
英文摘要 IX
緒言 XI

第一章 文獻回顧 1
第一節、生物體內鐵硫蛋白的生成 1
一、鐵硫複合體的特性與功能 1
二、動物細胞常見的鐵硫蛋白質 4
三、酵母菌鐵硫複合體的生合成 6
1. 粒線體鐵硫蛋白質的組合 7
2. 鐵硫複合體的運送 13
3. 細胞質鐵硫蛋白質的組合 14
四、動物細胞鐵硫蛋白質的生合成 15
五、鐵硫蛋白質生合成異常與人類遺傳性疾病 17
第二節、膳食鐵量對於肌肉生化與生理的影響 18
第三節、膳食鐵量對肌肉的鐵硫蛋白生合成的影響 19
第四節、實驗假說與研究架構 20

第二章 材料與方法 21
前言 21
第一節、動物飼養與飼料組成 21
第二節、分析項目與方法 22
一、血液生化分析 22
二、胞器分離 23
三、酵素活性分析 24
四、IRE-binding活性分析 26
五、RNA分析 29
六、蛋白質分析 33
七、統計分析 37

第三章 IscS在大鼠組織中的表現與細胞內的分佈 38
前言 38
第一節、IscS cDNA選殖與其抗體的製備 39
一、實驗設計 39
二、分析方法 40
三、結果 48
四、討論 56
五、結論 57

第二節 膳食缺鐵對於大鼠肌肉與大腦IscS蛋白質表現之影響 58
一、實驗設計 58
二、分析項目 58
三、結果 59
四、討論 65
五、結論 67

第四章 膳食鐵量與處理時間對大鼠肌肉IscS蛋白質表現與鐵硫蛋白生合成之影響 68
前言 68
第一節、實驗設計 68
第二節、分析項目 69
第三節、結果 70
第四節、討論 87
第五節、結論 91

第五章 IscS過度表現對aconitase活性之影響 92
前言 92
第一節、材料與方法 92
一、細胞培養 92
二、細胞培養常用之試劑 95
三、IscS cDNA質體的選殖 95
四、IscS基因表現載體的構築 95
五、細胞轉殖（Transfection） 96
六、分析項目與方法 97
第二節、結果 100
第三節、討論 113
第四節、結論 114

第六章 綜合討論與結論 115
一、鐵營養調控IscS蛋白質表現的生理意義 115
二、膳食鐵量與鐵硫蛋白質生合成的可能關係 117
（A）鐵硫脫輔基蛋白質（apoprotein）之生合成 117
（B）鐵硫複合體的受限 117
三、鐵營養影響鐵硫蛋白質活性與生化代謝的意義 118
四、結論 119

第七章 參考文獻 120

附錄一 構築實驗所需之載體 135

圖與表目錄
圖1-1 哺乳細胞常見鐵硫複合體的結構 2
表1-1哺乳細胞鐵硫蛋白質的胞器位置，鐵硫複合體之形式與其生理功能 3
表1-2 細菌與酵母菌鐵硫複合體生合成之成分對照 9
圖1-2 酵母菌鐵硫複合體生合成之模式 10
圖3-1以菜膠電泳鑑定rIscS cDNA產物 50
圖3-2大鼠IscS的cDNA與胺基酸序列 51
圖3-3 IPTG處理時間對rIscS-His•tag 重組蛋白質表現之影響 52
圖3-4 rIscS-His•tag 重組蛋白質之SDS-PAGE電泳分析 53
圖 3-5 Anti-human IscS抗體專一性之鑑定 54
圖3-6大鼠各組織中IscS mRNA（A）與其蛋白質（B）的表現 55
表3.1 膳食鐵量攝取對大鼠生長與攝食的狀況 60
表3.2 膳食鐵量攝取對大鼠血液鐵營養指標之影響 61
圖 3.10膳食鐵量攝食對大鼠大腦與肌肉IscS mRNA表現之影響 62
圖 3.11膳食鐵量攝取對大鼠大腦、肌肉與肝臟IscS蛋白質表現之影響 63
表3.3 膳食鐵量攝取對大鼠腦與肌肉c-aconitase、m-aconitase與succinate dehydrogenase活性之影響 64
表4-1 膳食鐵量與處理時間對大鼠生長與攝食狀況之影響 75
表4-2 膳食鐵量與處理時間對大鼠血液鐵營養指標的影響 76
圖4-1 膳食鐵量與處理時間對於大鼠肌肉粒線體IscS蛋白質與其mRNA表現之影響 77
圖 4-2 膳食鐵量與處理時間對大鼠肌肉細胞質c-aconitase酵素活性（A）的與其蛋白質表現量（B）之影響 78
圖4-3膳食鐵量與處理時間對大鼠肌肉細胞質c-aconitase（A）與粒線體m-aconitase （B）mRNA表現之影響 79
圖4-4 膳食鐵量與處理時間對大鼠肌肉粒線體m-aconitase酵素活性（A）與其蛋白質表現量（B）之影響 80
圖4-5 膳食鐵量與處理時間對大鼠肌肉succinate dehydrogenase酵素活性（A）與其Ip subunit蛋白質表現量（B）之影響 81
圖4-6 膳食鐵量與處理時間對大鼠肌肉NADH dehydrogenase酵素活性（A）與其24 kDa Ip subunit蛋白質表現量（B）之影響 82
圖 4-7 膳食鐵量與處理時間對大鼠肌肉NADH dehydrogenase之24 kDa Ip subunit mRNA（A）與succinate dehydrogenase之Ip subunit （B）mRNA表現量的影響 83
圖4-8膳食鐵量與處理時間對大鼠肌肉IRP1（A）、IRP2（B）與總IRPs（C）活性之影響 84
圖4-9 大鼠肌肉之粒線體IscS蛋白質含量與c-aconitase（A），m-aconitase（B），SDH（C）與NADH dehydrogenase （D）活性之相關性分析 85
圖4-10 大鼠肌肉總IRE-binding活性與m-aconitase（A），NADH dehdyrogenase之24 kDa Ip subunit（B），succinate dehydrogenase之Fp subunit（C）與Ip subunit（D）蛋白質表現量之相關性分析 86
表5-1 DMEM之組成分 94
圖5-1 IscS-pTRE2hyg質體之限制酶圖譜分析 105
圖 5-2 IscS-pTRE2hyg與pTRE2hyg質體轉殖細胞染色體IscS基因之表現 106
圖5-3 Doxycycline去除培養對pTRE2hyg與IscS-pTRE2hyg載體轉殖細胞的IscS與m-aconitase mRNA表現之影響 107
圖5-4 IscS–pTRE2hyg與pTRE2hyg質體轉殖細胞IscS mRNA之表現 108
圖5-5 IscS-pTRE2hyg與pTRE2hyg質體轉殖細胞IscS蛋白質之表現 109
圖5-6 rIscS-pTRE2hyg與pTRE2hyg質體轉殖細胞c-aconitase（A）與m-aconitase（B）之活性 110
圖5-7 加鐵處理對IscS-pTRE2hyg與pTRE2hyg質體轉殖細胞粒線體IscS蛋白質量之影響 111
圖5-8 外加鐵處理對IscS-pTRE2hyg與pTRE2hyg質體轉殖細胞c-aonitase（A）與m-aconitase（B）活性之影響 112

附錄圖 A-1 以洋菜膠電泳鑑定SDH之Ip subunit與NADH dehydrogenase之24 kDa Ip subunit cDNA產物 139
附錄圖A-2 以洋菜膠電泳鑑定限制酶處理之pGEM-T-Easy- Ip subunit of SDH與pGEM-T-Easy-24 kDa Ip of NADH dehydrogenase產物 140

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