Tan Guolin, Napoli Eleonora, Taroni Franco, Cortopassi Gino
Department of Molecular Biosciences, 1311 Haring Hall, University of California, Davis, CA 95616, USA.
Hum Mol Genet. 2003 Jul 15;12(14):1699-711. doi: 10.1093/hmg/ddg187.
Inherited deficiency of the mitochondrial protein frataxin causes neural and cardiac cell degeneration, and Friedreich's ataxia. Five hypotheses for frataxin's mitochondrial function have been generated, largely from work in non-human cells: iron transporter, iron-sulfur cluster assembler, iron-storage protein, antioxidant and stimulator of oxidative phosphorylation. We analyzed gene expression in three human cell types using microarrays, and identified just 48 transcripts whose expression was significantly frataxin-dependent in at least two cell types. Significant decreases in seven transcripts occurred in the sulfur amino acid (SAA) biosynthetic pathway and the iron-sulfur cluster (ISC) biosynthetic pathway to which it is connected. By contrast, we did not observe a single frataxin-dependent transcript that fits with the other four current hypotheses. Quantitative reverse-transcriptase PCR analysis of ISC-S and rhodanese transcripts confirmed that the expression of these genes involved in ISC metabolism was lower in mutants. Amino acid analysis confirmed the defect in SAA metabolism: homocystine, cysteine, cystathionine and serine were significantly decreased in frataxin-deficient cell extracts and mitochondria. An ISC defect was further confirmed by observing decreases in succinate dehydrogenase and aconitase activities, whose activities require ISCs. The ISC-U scaffold protein was specifically decreased in frataxin-deficient cells, suggesting a role for frataxin in its expression or maintenance, and sodium sulfide partially rescued the oxidant-sensitivity of the FRDA cells. Also, multiple transcripts involved in the Fas/TNF/INF apoptosis pathway were up-regulated in frataxin-deficient cells, consistent with a multi-step mechanism of Friedreich's ataxia pathophysiology, and suggesting alternative possibilities for therapeutic intervention.
线粒体蛋白frataxin的遗传性缺陷会导致神经和心脏细胞退化以及弗里德赖希共济失调。关于frataxin的线粒体功能，已经提出了五种假说，这些假说主要来自非人类细胞的研究：铁转运蛋白、铁硫簇组装蛋白、铁储存蛋白、抗氧化剂以及氧化磷酸化刺激剂。我们使用微阵列分析了三种人类细胞类型中的基因表达，仅鉴定出48个转录本，其表达在至少两种细胞类型中显著依赖于frataxin。硫氨基酸（SAA）生物合成途径以及与之相连的铁硫簇（ISC）生物合成途径中的七个转录本显著减少。相比之下，我们没有观察到与当前其他四种假说相符的单个依赖于frataxin的转录本。对ISC-S和硫氰酸酶转录本进行定量逆转录酶PCR分析证实，参与ISC代谢的这些基因在突变体中的表达较低。氨基酸分析证实了SAA代谢存在缺陷：在缺乏frataxin的细胞提取物和线粒体中，同型半胱氨酸、半胱氨酸、胱硫醚和丝氨酸显著减少。通过观察琥珀酸脱氢酶和乌头酸酶活性的降低进一步证实了ISC缺陷，这两种酶的活性需要ISC。ISC-U支架蛋白在缺乏frataxin的细胞中特异性降低，表明frataxin在其表达或维持中起作用，并且硫化钠部分挽救了FRDA细胞的氧化敏感性。此外，参与Fas/TNF/INF凋亡途径的多个转录本在缺乏frataxin的细胞中上调，这与弗里德赖希共济失调病理生理学的多步骤机制一致，并提示了治疗干预的其他可能性。
