| Summary: | 博士 === 國立臺灣大學 === 微生物與生化學研究所 === 93 === 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
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