Abstract
Iron-sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant-negative variants of the primary Fe-S biogenesis scaffold protein iron-sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S-containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a ∼12-fold increase in intracellular citrate content in Fe-S-deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S-deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S-deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.
Highlights
Iron–sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis
The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S– deficient cells
Biological studies of the human Fe-S cluster scaffold ISCU have relied on RNAi-mediated knockdown strategies [19], genetic studies in model organisms [17], and hypomorphic human disease models [7, 8, 20, 21]
Summary
ISCU is the main scaffold protein upon which nascent Fe-S clusters are first assembled and transferred to recipient proteins [9]. Cells expressing ISCU2D71A and ISCU2C69S showed a profound misregulation of cellular iron homeostasis as assessed by increased IRE-binding activity of IRP1 and IRP2 (Fig. 2B) and loss of activity of cytosolic and mitochondrial aconitases. NMR analysis suggested that intracellular [13C6]glucose/glucose 6-phosphate levels increased in cells expressing ISCU2D71A (Fig. 4B), whereas several positions of the adenine ring [8, 2] in AXP and the ribose ring (1Ј) of AXP showed significantly less 13C signal (Fig. 4B), suggesting that purine synthesis was diminished, probably due to loss of [4Fe-4S]-containing PPAT enzyme activity (discussed further below). To directly evaluate the 13C fractional enrichment of selected lipid functional groups in [13C6]glucose-labeled cells expressing empty vector or ISCU2D71A sequences, we quantified peak areas in selected regions of 2D TOCSY NMR spectra corresponding to the glycerol subunit and the fatty acyl chains, as shown in Fig. S4 (A and B). HRCE cells are nontransformed primary cells and do not possess any known genetic abnormalities
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