The discovery of the Fe-S protein mitoNEET at the crossroad of the mitochondrial outer membrane and the cytosol has opened up new avenues for understanding maturation of extra-mitochondrial Fe-S proteins in mammalian cells. Investigations performed in the framework of this project will tackle unanswered questions regarding Fe-S transfer to cytosolic recipients. Characterizing mitoNEET-pioglitazone interactions should open up perspectives for improving efficacy of thiazole and thiophene drugs.
Biogenesis of iron-sulfur clusters (Fe-S) occurs in mitochondria where the iron-sulfur cluster (ISC) assembly machinery plays the central role in the maturation of both mitochondrial and extra-mitochondrial Fe-S proteins. In mammals, Fe-S proteins are involved in critical biological processes including DNA synthesis and repair and ribosome biogenesis. Mutations in the ISC assembly genes are associated with several human diseases such as myopathies, neurodegenerative and anemia-related diseases. However, little is known about Fe-S assembly in mammalian proteins. It is therefore important to investigate the mechanisms by which Fe-S are delivered to cytosolic and nuclear recipients in mammals. <br /> Recently, mitoNEET has been identified as the first Fe-S protein of the outer mitochondrial membrane. MitoNEET function is unknown but its location and the unusual ligation of its Fe-S makes it a likely participant in Fe-S transfer from mitochondria to the cytosol. The fact that mitoNEET is a pharmacological target of the diabetes drug pioglitazone is another spur to further understand its specific function. <br />The proposal is organized in three main tasks with the following objectives: i) in cellulo and in vivo characterization of the function of mitoNEET in extra-mitochondrial Fe-S assembly with a focus on the maturation of the Fe-S Iron Regulatory Protein-1, ii) investigation of the role of the particular Fe-S cluster of mitoNEET in Fe-S transfer process and iii) definition of the mechanism by which pioglitazone stabilizes mitoNEET Fe-S. This project is based on a multidisciplinary approach including molecular and cell biology, resonance Raman, EPR, NMR and Mössbauer spectroscopies in order to define the role of mitoNEET in Fe-S transfer and the mechanism of action of pioglitazone on mitoNEET function, and by in vivo shRNA gene knockdown in mice, to validate the results obtained in vitro in a physiological context.
The project is based on multidisciplinary and integrative research approach combining :
- biophysical methods (NMR, Mössbauer, Raman and UV-visible spectroscopies) and biochemical methods (Differential Migration on Native PAGE, enzymatic activities, colorimetric assays, site-targeted mutagenesis, protein expression in heterologous system and Fe-S protein purification in aerobiosis and anaerobiosis) to study mitoNEET conformational change and Fe-S release during the transfer process. These methodologies are useful to study protein-ligand interactions (e.g. pioglitazone, resveratrol-3-sulfate,…) with mitoNEET and the effects of mitoNEET ligands on the transfer reaction and Fe-S cluster stability.
- - Cell and molecular biology (immunoblot, RNA interference in cellulo and in vivo using associated-adenovirus in mice, yeast two-hybrid system, co-immunoprecipitation,…) to study the endogenous maturation pathway of mitoNEET and to identify protein partners as well as the impact of mitoNEET deficiency in mice on the regulation of cellular iron metabolism.
The work carried out over the last 18 months was mainly focused on the maturation pathway and the biological function of mitoNEET in cytosolic Fe-S biogenesis and/or repair. The combined in cellulo (through siRNA-induced mRNA knockdown and phenotype) and in vitro (using biochemistry and Raman, NMR, Mössbauer and UV-visible spectroscopies) studies led us to propose that:
- cellular Fe-S bioavailability ensures stability and proper folding of mitoNEET pointing to the physiological relevance of mitoNEET Fe-S cluster in living cells,
- Mitochondrial iron and inorganic sulfide, used for building Fe-S clusters into the matrix, are required for mitoNEET maturation,
- Maturation of mitoNEET Fe-S cluster is mediated by a novel specific mitochondrial Hsc20/ABCb7/ALR-branch pathway and not by the cytosolic iron-sufur cluster assembly (CIA) machinery,
- The sulfhydryl oxidase ALR is not a component of the iron sulfur cluster (ISC) export machinery as it was proposed for its yeast homolog Erv1p,
- The CIA scaffold Fe-S protein NARFL is sensitive to oxidative stress while mitoNEET is not,
- mitoNEET mediates cytosolic Fe-S cluster repair rather than cytosolic Fe-S assembly,
- the mitoNEET physiological acceptor for Fe-S reassembly after oxidative and nitrosative stress is cytosolic aconitase/IRP1, a master regulator of cellular iron homeostasis in mammals.
In parallel, we have started investigating the parameters required for triggering Fe-S transfer by mitoNEET. We provide biophysical and biochemical evidence that cluster transfer from mitoNEET to apo-FDX is triggered only when mitoNEET [2Fe-2S] is in its oxidized state. The transfer reaction rate is pH-dependent and not influenced by oxygen per se.
The work accomplished and results obtained are partly in agreement with initial plan goal since we have identified mitoNEET as having a role in cytosolic Fe-S repair rather than in cytosolic Fe-S maturation.
Provisional schedule: Knowing that most of the backbone of mitoNEET is oriented toward the cytosol, we have also started exploring interacting protein partners of mitoNEET44-108 soluble form using the yeast two-hybrid system (Hybrigenics services and collaboration with Dr L. Vernis, Curie, Orsay). This part of the project is currently being analyzed.
The study of the impact of mitoNEET deficiency on cellular iron metabolism is planned for the next support period. A screen of IRP1 activation into its trans-regulatory form in several tissues (liver, heart, muscle,..) will be performed in AAV-mediated knockdown of mitoNEET expression in mice in order to investigate in further details the regulation of intracellular iron homeostasis in relevant tissues. The efficacy of three shRNA constructs targeting mitoNEET mRNA, have already been validated in living cells.
The work done will be useful to study, in the next support period, redox molecules (e.g. resveratrol-3-sulfate, pioglitazone, glutathione,…) that may regulate the Fe-S transfer reaction as well as mitoNEET protein stability. Identified parameters required for Fe-S transfer reaction to FDX will be then validated using apo-IRP1 as a recipient protein.
1. Ferecatu I., Gonçalvez S, Golinelli-Cohen M-P., Clémancey M., Martelli A., Riquier S., Guittet E., Latour J-M., Puccio H ;, Drapier J-C., Lescop E. and C. Bouton, «The Diabetes Drug Target MitoNEET Governs a Novel Trafficking Pathway to Rebuild an fe-S Cluster into Cytosolic Aconitase/Iron Regulatory Protein 1«, J. Biol. Chem., 2014, 289 :28070-86.
1. C. Bouton-Invited lecture: Role of the mitochondrial protein mitoNEET in cytosolic Fe-S cluster repair (IUBMB Symposium-Fe-S2015-Iron Sulfur Cluster Biogenesis and Regulation-June 23-26 2015, Bergamo, Italy)
1. M-P. Golinelli-Cohen-Invited lecture: Functional studies of mitoNEET, an Fe-S protein targeted by antioxidant molecules (10èmes journées de Biologie Cellulaire, 18-20 mai 2015, UPS-Orsay)
2. I. Ferecatu- Invited lecture : Dysfunction of the mitochondrial Fe-S cluster assembly machinery leads to the formation of the chemoresistant truncated VDAC1 isoform (10èmes journées de Biologie Cellulaire, 18-20 mai 2015, UPS-Orsay)
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1. C. Bouton- Seminar: Rôle de la protéine mitoNEET, une nouvelle cible de l’antidiabétique pioglitazone, dans la régulation du fer intracellulaire (Hôpital Saint-Antoine, 8/12/2014)
2. C. Mons- Poster : Functional studies of mitoNEET, an Fe-S protein targeted by antioxidant molecules (10èmes journées de Biologie du Grand Campus, 18-20 mai 2015, UPS-Orsay)
3. M.-P. Golinelli- Poster : Functional studies of mitoNEET, an Fe-S protein targeted by antioxidant molecules (XIVième symposium ICSN, 18-19 juin 2015, Gif-sur-Yvette).
The mitochondrial Fe-S cluster assembly machinery plays a central role in the maturation of both mitochondrial and extra-mitochondrial Fe-S proteins. In mammals, Fe-S cluster proteins are involved in crucial biological processes including enzymatic catalysis, DNA synthesis and repair, ribosome biogenesis, iron homeostasis and heme synthesis. Mutation in the iron-sulfur cluster assembly genes is associated with severe human diseases such as infantile encephalopathy, myopathies, neurodegenerative and anemia-related diseases. It is therefore particularly relevant to investigate the mechanisms by which Fe-S are delivered to cytosolic and nuclear recipient in mammals. Recently, mitoNEET has been identified as the first Fe-S protein of the outer mitochondrial membrane in mammalian cells. mitoNEET function is unknown but its location and the pH-lability of its Fe-S, which is coordinated to three cysteines and one histidine, makes it a high priority candidate to participate in Fe-S transfer from mitochondria to the cytosol in mammalian cells. Very recently, in vitro studies demonstrated that human mitoNEET can deliver its Fe-S to the bacterial acceptor ferredoxin. Our preliminary data, showing that mitoNEET deficiency affects the maturation of the Iron Regulatory Protein-1 (IRP1), a cytosolic Fe-S protein involved in the control of iron homeostasis, support a role of mitoNEET in Fe-S biogenesis. The fact that mitoNEET is a diabetes drug target is another spur to identify its specific function. Recent studies indicate that pioglitazone, a member of the thiazolidinedione class, stabilizes mitoNEET Fe-S, implying a direct action of this diabetes drug on the protein. The proposal is organized in three main work packages with the following specific objectives: i) specifying mitoNEET function in cytosolic Fe-S biogenesis and its impact on cellular iron metabolism controlled by IRP1, ii) characterizing the mitoNEET function in a Fe-S transfer process using physiological acceptor proteins, in particular IRP1, and iii) defining the mechanism by which pioglitazone prevents Fe-S release from mitoNEET (or stabilizes mitoNEET Fe-S). This project is based on a multidisciplinary approach including biophysics (Raman, NMR, EPR and Mössbauer spectroscopies) and biochemistry, molecular and cell biology as well as studies in living cells and animal models, to validate the in vitro approach in a physiological context.
Madame Cécile BOUTON (Institut de Chimie des Susbtances Naturelles)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IGBMC Institut de Génétique et de Biologie Moléculaire et Cellulaire
CEA/iRTSV/LCBM/pmb Laboratoire de Chimie et Biologie des Métaux
CNRS-ICSN Institut de Chimie des Susbtances Naturelles
Help of the ANR 350,000 euros
Beginning and duration of the scientific project: December 2013 - 36 Months