Dysfunction in the mitochondrial Fe-S assembly machinery leads to formation of the chemoresistant truncated VDAC1 isoform without HIF-1α activation.

Bernard Mari,Ioana Ferecatu,Marie-Pierre Golinelli-Cohen,Frédéric Canal,Nathalie M. Mazure,Lucilla Fabbri,Cécile Bouton

doi:10.1371/journal.pone.0194782

Abstract

Biogenesis of iron-sulfur clusters (ISC) is essential to almost all forms of life and involves complex protein machineries. This process is initiated within the mitochondrial matrix by the ISC assembly machinery. Cohort and case report studies have linked mutations in ISC assembly machinery to severe mitochondrial diseases. The voltage-dependent anion channel (VDAC) located within the mitochondrial outer membrane regulates both cell metabolism and apoptosis. Recently, the C-terminal truncation of the VDAC1 isoform, termed VDAC1-ΔC, has been observed in chemoresistant late-stage tumor cells grown under hypoxic conditions with activation of the hypoxia-response nuclear factor HIF-1α. These cells harbored atypical enlarged mitochondria. Here, we show for the first time that depletion of several proteins of the mitochondrial ISC machinery in normoxia leads to a similar enlarged mitochondria phenotype associated with accumulation of VDAC1-ΔC. This truncated form of VDAC1 accumulates in the absence of HIF-1α and HIF-2α activations and confers cell resistance to drug-induced apoptosis. Furthermore, we show that when hypoxia and siRNA knock-down of the ISC machinery core components are coupled, the cell phenotype is further accentuated, with greater accumulation of VDAC1-ΔC. Interestingly, we show that hypoxia promotes the downregulation of several proteins (ISCU, NFS1, FXN) involved in the early steps of mitochondrial Fe-S cluster biogenesis. Finally, we have identified the mitochondria-associated membrane (MAM) localized Fe-S protein CISD2 as a link between ISC machinery downregulation and accumulation of anti-apoptotic VDAC1-ΔC. Our results are the first to associate dysfunction in Fe-S cluster biogenesis with cleavage of VDAC1, a form which has previously been shown to promote tumor resistance to chemotherapy, and raise new perspectives for targets in cancer therapy.

Highlights

In mammals, iron-sulfur (Fe-S) clusters are essential cofactors for numerous proteins involved in critical cellular functions, including electron transfer for oxidative phosphorylation, ribosome biogenesis, and DNA synthesis and repair [1]
To examine the effects of iron-sulfur cluster (ISC) deficiency on mitochondrial network, we used different siRNAs to selectively deplete the iron carrier MFRN2 or proteins of the mitochondrial ISC machinery (NFS1, ISCU and HSC20) in Human epithelial carcinoma cells (HeLa) cells grown in normoxia (21% of O2)
In order to discriminate the intrinsic role of Fe-S deficiency towards hypoxia in the formation of VDAD25K, we studied whether depletion of MFRN2 and of mitochondrial ISC assembly proteins in HeLa cells in normoxia induces the stabilization of hypoxia central transcription factor hypoxia-inducible factor-1α (HIF-1α) (Fig 1C) or the expression of one of its main transcriptional targets, carbonic anhydrase IX, at both mRNA and protein levels (CAIX, Fig 1C), as compared with cells treated overnight with the hypoxia-mimetic agent desferrioxamine (DFO) [24]

Summary

Introduction

Iron-sulfur (Fe-S) clusters are essential cofactors for numerous proteins involved in critical cellular functions, including electron transfer for oxidative phosphorylation, ribosome biogenesis, and DNA synthesis and repair [1]. A [2Fe-2S] cluster is assembled on the scaffold protein ISCU with the help of frataxin (FXN) and of the ferredoxin/ferredoxin reductase reducing system This transiently bound [2Fe-2S] can be transferred to mitochondrial [2Fe-2S]-assembling recipient apo-proteins with the participation of the chaperone and co-chaperone HSPA9/HSC20 [3,4] and glutaredoxin 5 [5]. It can either serve for the synthesis of [4Fe-4S] clusters and their insertion into mitochondrial [4Fe-4S]-assembling recipients (e.g. the Fe-S-containing respiratory complex subunits, the aconitase of the Krebs cycle) or it can be exported out of mitochondria via an ill-defined ISC export machinery for the maturation of extra-mitochondrial Fe-S proteins by the cytosolic assembly machinery (CIA) [1]. Fe-S proteins including NEET proteins have recently been linked to response mechanisms to cell damage, including apoptosis and autophagy regulation [7,8,9]

Methods

Results

Conclusion