膳食鐵量調控大鼠IscS蛋白質表現與鐵硫蛋白質生合成

The Expression of Rat IscS Protein is Modulated by Dietary Iron and the Biosynthesis of Iron-Sulfur Protein

劉奕方(YIH-FONG LIEW)

指導教授 ： 蕭寧馨

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.02545

摘要

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。

關鍵字

膳食鐵量 ； IscS蛋白質 ； 鐵硫蛋白質 ； 鐵硫複合體 ； 粒線體 ； 肌肉 ； 大鼠

並列摘要

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

並列關鍵字

dietary iron ； IscS ； iron-sulfur protein ； iron-sulfur cluster ； muscle ； rat

參考文獻

黃惠玲，康碩芬與蕭寧馨 (2000) 膳食缺鐵與補鐵影響大鼠肝臟和肌肉IRPs與Aconitase活性。中華營誌 25: 47-61.

Schalinske, K. L. M., Chen, O. S. and Eisenstein, R. S. (1998) Iron deferrentially stimulates translation of mitochondrial aconitase and ferritin mRNAs in mammalian cells. J. Biol. Chem. 273: 3740-3746.

Cartier, L. J., Ohira, Y., Chen, M., Cuddiheee, R. W. and Holloszy, J. O. (1986) Perturbation of mitochondrial composition in skeletal muscle by iron deficiency. J. Biol. Chem. 261: 13827-113832.

Adinolfi, S., Rizzo, F., Masino, L., Nair, M., Martin, S. R., Pastore, A. and Temussi, P. A. (2004) Bacterial IscU is a well folded and functional single domain protein. Eur. J. Biochem. 271: 2093-2100.

Allikmets, R., Raskind, W. H., Hutchinson, A., SChueck, N. D., Dean, M. and Koellar, D. M. (1999) Mutation of putative mitochondrial iron transporter gene (ABC7) in X-linked sideroblastic anemia and ataxia (XLSA/A). Hum. Mol. Genet. 8: 743-749.