Novel Insights into Redox-Based Mechanisms for Auranofin-Induced Rapid Cancer Cell Death.

Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.

Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae.

B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here, we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET.

Human CISD2 and mitoNEET are two NEET proteins anchored in the endoplasmic reticulum and mitochondria membranes respectively, with an Fe-S containing domain stretching out in the cytosol. Their cytosolic domains are close in sequence and structure. In the present study, combining cellular and biochemical approaches, we compared both proteins in order to possibly identify specific roles and mechanisms of action in the cell. We show that both proteins exhibit a high intrinsic stability and a sensitivity of their cluster to oxygen. In contrast, they differ in according to expression profiles in tissues and intracellular half-life. The stability of their Fe-S cluster and its ability to be transferred in vitro are affected differently by pH variations in a physiological and pathological range for cytosolic pH. Finally, we question a possible role for CISD2 in cellular Fe-S cluster trafficking. In conclusion, our work highlights unexpected major differences in the cellular and biochemical features between these two structurally close NEET proteins.

Combined Biochemical, Biophysical, and Cellular Methods to Study Fe-S Cluster Transfer and Cytosolic Aconitase Repair by MitoNEET.

MitoNEET is the first identified Fe-S protein anchored to mammalian outer mitochondrial membranes with the vast majority of the protein polypeptide located in the cytosol, including its [2Fe-2S] cluster-binding domain. The coordination of the cluster is unusual and involves three cysteines and one histidine. MitoNEET is capable of transferring its redox-active Fe-S cluster to a bacterial apo-ferredoxin in vitro even under aerobic conditions, unlike other Fe-S transfer proteins such as ISCU. This specificity suggests its possible involvement in Fe-S repair after oxidative and/or nitrosative stress. Recently, we identified cytosolic aconitase/iron regulatory protein 1 (IRP1) as the first physiological protein acceptor of the mitoNEET Fe-S cluster in an Fe-S repair process. This chapter describes methods to study in vitro mitoNEET Fe-S cluster transfer/repair to a bacterial ferredoxin used as a model aporeceptor and in a more comprehensive manner to cytosolic aconitase/IRP1 after a nitrosative stress using in vitro, in cellulo, and in vivo methods.

The diabetes drug target MitoNEET governs a novel trafficking pathway to rebuild an Fe-S cluster into cytosolic aconitase/iron regulatory protein 1.

In eukaryotes, mitochondrial iron-sulfur cluster (ISC), export and cytosolic iron-sulfur cluster assembly (CIA) machineries carry out biogenesis of iron-sulfur (Fe-S) clusters, which are critical for multiple essential cellular pathways. However, little is known about their export out of mitochondria. Here we show that Fe-S assembly of mitoNEET, the first identified Fe-S protein anchored in the mitochondrial outer membrane, strictly depends on ISC machineries and not on the CIA or CIAPIN1. We identify a dedicated ISC/export pathway in which augmenter of liver regeneration, a mitochondrial Mia40-dependent protein, is specific to mitoNEET maturation. When inserted, the Fe-S cluster confers mitoNEET folding and stability in vitro and in vivo. The holo-form of mitoNEET is resistant to NO and H2O2 and is capable of repairing oxidatively damaged Fe-S of iron regulatory protein 1 (IRP1), a master regulator of cellular iron that has recently been involved in the mitochondrial iron supply. Therefore, our findings point to IRP1 as the missing link to explain the function of mitoNEET in the control of mitochondrial iron homeostasis.

Peroxiredoxin post-translational modifications by redox messengers.

Peroxiredoxins (Prxs) are a family of thiol peroxidases that participate in hydroperoxide detoxification and regulates H2O2 signaling. In mammals, the four typical 2-Cys Prxs (Prxs 1, 2, 3 and 4) are known to regulate H2O2-mediated intracellular signaling. The 2 catalytic cysteines of 2-Cys Prxs, the so-called peroxidatic and resolving cysteines, are regulatory switches that are prone to react with redox signaling molecules. We investigated the respective modifications induced by H2O2, NO and H2S in the murine macrophage cell line RAW264.7 by mass spectrometry and immunoblotting after separating 2-Cys Prxs by one-dimensional or two-dimensional PAGE. We found that H2S, unlike NO, does not prevent H2O2-mediated sulfinylation of 2-Cys Prxs and that Prx2 is more sensitive to NO-mediated protection against sulfinylation by peroxides. We also observed that cells exposed to exogenous NO, released by Cys-SNO or DETA-NO, or producing NO upon stimulation by IFN-γ and LPS, present an acidic form of Prx1 whose modification is consistent with S-homocysteinylation of its peroxidatic cysteine.

Peroxiredoxins and sulfiredoxin at the crossroads of the NO and H2O2 signaling pathways.

Peroxiredoxins (Prxs) are a family of peroxidases that maintain thiol homeostasis by catalyzing the reduction of organic hydroperoxides, H2O2, and peroxynitrite. Eukaryotic 2-Cys-Prxs, also referred to as typical Prxs, can be inactivated by oxidation of the catalytic cysteine to sulfinic acid, which may regulate the intracellular messenger function of H2O2. A small redox protein, sulfiredoxin (Srx), has been shown to reduce sulfinylated 2-Cys-Prxs and thus to regenerate active 2-Cys-Prxs. We previously reported that cytokine-induced nitric oxide (NO) intervenes in this pathway by decreasing the level of 2-Cys overoxidation and by upregulating Srx through the activation of the transcription factor nuclear factor erythroid 2-related factor (Nrf2). Here, we describe the methods used to monitor the interplay between NO and H2O2 in the regulation of the Prx/Srx system in immunostimulated macrophages, which produce both reactive oxygen species and NO.

Nitric oxide activates an Nrf2/sulfiredoxin antioxidant pathway in macrophages.

Peroxiredoxins (Prx's) are a family of peroxidases that maintain thiol homeostasis by catalyzing the reduction of organic hydroperoxides, H2O2, and peroxynitrite. Under conditions of oxidative stress, eukaryotic Prx's can be inactivated by the substrate-dependent oxidation of the catalytic cysteine to sulfinic acid, which may regulate the intracellular messenger function of H2O2. A small redox protein, sulfiredoxin (Srx), conserved only in eukaryotes, has been shown to reduce sulfinylated 2-Cys Prx's, adding to the complexity of the H2O2 signaling network. In this study, we addressed the regulation of Srx expression in immunostimulated primary macrophages that produce both reactive oxygen species (ROS) and nitric oxide (NO(•)). We present genetic evidence that NO-mediated Srx up-regulation is mediated by the transcription factor nuclear factor erythroid 2-related factor (Nrf2). We also show that the NO(•)/Srx pathway inhibits generation of ROS. These results reveal a link between innate immunity and H2O2 signaling. We propose that an NO(•)/Nrf2/Srx pathway participates in the maintenance of redox homeostasis in cytokine-activated macrophages and other inflammatory settings.

Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.