Compartment-specific Protection of Iron-Sulfur Proteins by Superoxide Dismutase

Abstract

Iron and oxygen are essential but potentially toxic constituents of most organisms, and their transport is meticulously regulated both at the cellular and systemic levels. Compartmentalization may be a homeostatic mechanism for isolating these biological reactants in cells. To investigate this hypothesis, we have undertaken a genetic analysis of the interaction between iron and oxygen metabolism in Drosophila. We show that Drosophila iron regulatory protein-1 (IRP1) registers cytosolic iron and oxidative stress through its labile iron sulfur cluster by switching between cytosolic aconitase and RNA-binding functions. IRP1 is strongly activated by silencing and genetic mutation of the cytosolic superoxide dismutase (Sod1), but is unaffected by silencing of mitochondrial Sod2. Conversely, mitochondrial aconitase activity is relatively insensitive to loss of Sod1 function, but drops dramatically if Sod2 activity is impaired. This strongly suggests that the mitochondrial boundary limits the range of superoxide reactivity in vivo. We also find that exposure of adults to paraquat converts cytosolic aconitase to IRP1 but has no affect on mitochondrial aconitase, indicating that paraquat generates superoxide in the cytosol but not in mitochondria. Accordingly, we find that transgene-mediated overexpression of Sod2 neither enhances paraquat resistance in Sod1 ؉ flies nor compensates for lack of SOD1 activity in Sod1-null mutants. We conclude that in vivo, superoxide is confined to the subcellular compartment in which it is formed, and that the mitochondrial and cytosolic SODs provide independent protection to compartment-specific protein iron-sulfur clusters against attack by superoxide generated under oxidative stress within those compartments.Iron and oxygen are indispensable but potentially harmful elements of aerobic life. Individually, their reactivity has been harnessed through association with a variety of proteins and the regulation of iron and oxygen metabolism constitutes one of the major triumphs of molecular evolution (1). Iron sulfur cluster proteins function in electron transport during oxidative phosphorylation and metabolism, but can also serve as iron and oxygen sensors (2). For instance, iron regulatory protein-1 (IRP1) 1 exerts its dual activities through the reciprocal use or dissasembly of its cubane iron sulfur [4Fe-4S] cluster; the holoprotein functions as a cytosolic aconitase, whereas the apoprotein is an RNA-binding translational regulator (1, 3). The stability and functionality of IRP1 as a translation regulator is affected not only by iron levels, but also by oxidative stress, which induces IRP1 to bind iron responsive elements (IREs) located on the 5Ј and 3Ј untranslated regions of target genes (4, 5). Although it is established that [4Fe-4S] cluster proteins can be specifically inactivated by superoxide (O 2 . ) (6 -8), the questions of whether the IRP1 [4Fe-4S] cluster reacts with O 2

Of Two Cytosolic Aconitases Expressed in Drosophila, Only One Functions as an Iron-regulatory Protein

“…6B). Separation of cellular aconitase activities by electrophoresis (10,31) confirmed that the modest increase in total activity was due to specific increase from the overexpressed cytosolic aconitases (Fig. 6C).…”

“…Together with an alternatively spliced transcript of the ferritin 1 heavy chain homolog (Fer1HCH), these are the only two genes in the Drosophila genome that were shown to possess an IRE (28 -30). Although electrophoretic analysis of fly extracts suggests the presence of a single protein binding to IRE and a single protein possessing cytosolic aconitase activity (31), Drosophila actually contains two highly homologous IRP1-like proteins (IRP-1A and IRP-1B) encoded by different genes (17,32). Herein, we have purified heterologously expressed IRP-1A, IRP-1B, and hybrid IRP-1A/IRP-1B proteins and determined that although both IRPs are functional aconitases, only IRP-1A binds IREs.…”

Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia

“…The aconitases are subject to reversible inactivation by ROS (38,39). The vulnerability of the solvent-exposed Fe-S cluster of the these enzymes to ROS attack provides a sensitive indicator of changes in ROS flux in vivo (38,39).…”

“…The aconitases are subject to reversible inactivation by ROS (38,39). The vulnerability of the solvent-exposed Fe-S cluster of the these enzymes to ROS attack provides a sensitive indicator of changes in ROS flux in vivo (38,39). To determine further whether ectopically expressed CAT actually reduces the ROS burden in DFH-deficient flies, we used the activities of mACON and cytoplasmic aconitase (cACON) to assess the level of ROS flux in DFH-deficient flies expressing mitoCAT.…”

CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life

“…However, recent studies in Drosophila lacking SOD1 revealed an increase in IRP1 RNA binding activity, suggesting an ability of O 2 À to promote complete loss of the [4Fe-4S] (Missirlis et al, 2003). An equally plausible scenario is that O 2 À also interferes with the pathway of cytosolic Fe-S assembly.…”