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In addition to its well-known receptor-mediated function in cell survival, differentiation and growth, we report that the extracellular brain-derived neurotrophic factor (BDNF) also controls the intracellular KEAP1-NRF2 cytoprotective system by a receptor-independent pathway. Extracellular BDNF can cross the cell membrane as it possesses a protein-translocation domain, also known as cell-penetrating peptide. This membrane crossing process is energy-independent, ruling out endocytosis and receptor-dependent mechanisms. Once in the cytosol, BDNF binds to KEAP1 with a nanomolar affinity, enabling nuclear translocation of NRF2 and transcription of NRF2-target genes. BDNF is thus a major regulator of NRF2 activation. A dysfunction of this BDNF-KEAP1-NRF2 pathway may be involved in most diseases where antioxidant and cytoprotective functions are altered. This novel form of communication, whereby a receptor ligand protein exerts a biological activity by crossing the cell membrane, opens new avenues for cell signaling.

Redox homeostasis is an equilibrium between reducing and oxidizing reactions within cells. It is an essential, dynamic process, which allows proper cellular reactions and regulates biological responses. Unbalanced redox homeostasis is the hallmark of many diseases, including cancer or inflammatory responses, and can eventually lead to cell death. Specifically, disrupting redox balance, essentially by increasing pro-oxidative molecules and favouring hyperoxidation, is a smart strategy to eliminate cells and has been used for cancer treatment, for example. Selectivity between cancer and normal cells thus appears crucial to avoid toxicity as much as possible. Redox-based approaches are also employed in the case of infectious diseases to tackle the pathogens specifically, with limited impacts on host cells. In this review, we focus on recent advances in redox-based strategies to fight eukaryotic pathogens, especially fungi and eukaryotic parasites. We report molecules recently described for causing or being associated with compromising redox homeostasis in pathogens and discuss therapeutic possibilities.

International audience; We report a patient carrying a novel pathogenic variant p.(Tyr101Cys) in ISCA1 leading to MMDS type 5. He initially presented a psychomotor regression with loss of gait and language skills and a tetrapyramidal spastic syndrome. Biochemical analysis of patient fibroblasts revealed impaired lipoic acid synthesis and decreased activities of complex I and II of respiratory chain. While ISCA1 is involved in the mitochondrial machinery for iron-sulfur cluster biogenesis, these dysfunctions are secondary to impaired maturation of mitochondrial proteins containing the [4Fe-4S] clusters. Expression and purification of the human ISCA1 showed a decreased stability of the [2Fe-2S] cluster in the mutated protein.

Mitochondrial proteins carrying iron-sulfur (Fe-S) clusters are involved in essential cellular pathways such as oxidative phosphorylation, lipoic acid synthesis, and iron metabolism. NFU1, BOLA3, IBA57, ISCA2, and ISCA1 are involved in the last steps of the maturation of mitochondrial [4Fe-4S]-containing proteins. Since 2011, mutations in their genes leading to five multiple mitochondrial dysfunction syndromes (MMDS types 1 to 5) were reported. The aim of this systematic review is to describe all reported MMDS-patients. Their clinical, biological, and radiological data and associated genotype will be compared to each other. Despite certain specific clinical elements such as pulmonary hypertension or dilated cardiomyopathy in MMDS type 1 or 2, respectively, nearly all of the patients with MMDS presented with severe and early onset leukoencephalopathy. Diagnosis could be suggested by high lactate, pyruvate, and glycine levels in body fluids. Genetic analysis including large gene panels (Next Generation Sequencing) or whole exome sequencing is needed to confirm diagnosis.

The human mitochondrial protein, mitoNEET (mNT), belongs to the family of small [2Fe-2S] NEET proteins that bind their iron-sulfur clusters with a novel and characteristic 3Cys:1His coordination motif. mNT has been implicated in the regulation of lipid and glucose metabolisms, iron/reactive oxygen species homeostasis, cancer and possibly Parkinson’s disease. The geometric structure of mNT as a function of redox state and pH is critical for its function. In this study we combine (57)Fe nuclear resonance vibrational spectroscopy (NRVS) with density functional theory (DFT) calculations to understand the novel properties of this important protein.

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.

Pour la sixième année, dans le cadre du module d’enseignement « Physiopathologie de la signalisation » proposé par l’université Paris-sud, les étudiants du Master « Biologie Santé » de l’université Paris-Saclay se sont confrontés à l’écriture scientifique. Ils ont sélectionné une quinzaine d’articles scientifiques récents dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant des relations hôte-pathogène aux innovations thérapeutiques, en passant par la signalisation hépatique et le métabolisme. Après un travail préparatoire réalisé avec l’équipe pédagogique, les étudiants, organisés en binômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l’originalité de l’article étudié. Ils ont beaucoup apprécié cette initiation à l’écriture d’articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme ! Trois de ces Nouvelles sont publiées dans ce numéro, les autres le seront dans des prochains numéros.

International audience; The human mitochondrial protein, mitoNEET (mNT), belongs to the family of small [2Fe-2S] NEET proteins that bind their iron–sulfur clusters with a novel and characteristic 3Cys:1His coordination motif. mNT has been implicated in the regulation of lipid and glucose metabolisms, iron/reactive oxygen species homeostasis, cancer, and possibly Parkinson’s disease. The geometric structure of mNT as a function of redox state and pH is critical for its function. In this study, we combine 57Fe nuclear resonance vibrational spectroscopy with density functional theory calculations to understand the novel properties of this important protein.

International audience; Malaria, caused by protozoan parasites, is a major public health issue in subtropical countries. An arsenal of antimalarial treatments is available, however, resistance is spreading, calling for the development of new antimalarial compounds. The new lead antimalarial drug plasmodione is a redox-active compound that impairs the redox balance of parasites leading to cell death. Based on extensive in vitro assays, a model of its mode of action was drawn, involving the generation of active plasmodione metabolites that act as subversive substrates of flavoproteins, initiating a redox cycling process producing reactive oxygen species. We showed that, in yeast, the mitochondrial respiratory chain NADH-dehydrogenases are the main redox-cycling target enzymes. Furthermore, our data supported the proposal that plasmodione is a pro-drug acting via its benzhydrol and benzoyl metabolites. Here, we selected plasmodione-resistant yeast mutants to further decipher plasmodione mode of action. Of the eleven mutants analysed, nine harboured a mutation in the FAD binding subunit of succinate dehydrogenase (SDH). The analysis of the SDH mutations points towards a specific role for SDH-bound FAD in plasmodione bioactivation, possibly in the first step of the process, highlighting a novel property of SDH.

International audience

International audience; Human mitoNEET (mNT) is the first identified Fe−S protein of the mammalian outer mitochondrial membrane. Recently, we demonstrated the involvement of mNT in a specific cytosolic pathway dedicated to the reactivation of oxidatively damaged cytosolic aconitase by cluster transfer. In vitro studies using apo-ferredoxin (FDX) reveal that mNT uses an Fe-based redox switch mechanism to regulate the transfer of its cluster. Using the "gold standard" cluster recipient protein, FDX, we show that this transfer is direct and that only one of the two mNT clusters is transferred when the second one is decomposed. Combining complementary biophysical and biochemical approaches, we show that pH affects both the sensitivity of the cluster to O 2 and dimer stability. Around physiological cytosolic pH, the ability of mNT to transfer its cluster is tightly regulated by the pH. Finally, mNT is extremely resistant to H 2 O 2 compared to ISCU and SufB, two other Fe−S cluster transfer proteins, which is consistent with its involvement in a repair pathway of stress-damaged Fe−S proteins. Taken together, our results suggest that the ability of mNT to transfer its cluster to recipient proteins is not only controlled by the redox state of its cluster but also tightly modulated by the pH of the cytosol. We propose that when pathophysiological conditions such as cancer and neurodegenerative diseases dysregulate cellular pH homeostasis, this pH-dependent regulation of mNT is lost, as is the regulation of cellular pathways under the control of mNT. I ron−sulfur (Fe−S) clusters are evolutionarily ancient and highly conserved prosthetic cofactors. Composed of only iron and sulfur, they are involved in many essential biological processes. 1,2 MitoNEET (mNT), also known as CISD1, is the first identified Fe−S protein of the mammalian outer mitochondrial membrane (OMM). 3,4 This is a small homodimeric protein (13 kDa for each monomer) anchored to the OMM by its 32-amino acid N-terminus with the major part of the protein, including the C-terminal Fe−S binding domain, located in the cytosol. 4 Each monomer accommodates one [2Fe-2S] cluster coordinated by three cysteines (C72, C74, and C83) and one histidine (H87) in a CDGSH domain 5−8 as other members of the NEET protein family, 9 which also includes Miner1 (or CISD2) and Miner2 (or CISD3) in mammals. 10 Although the biological activity of mNT is still debated, 11 studies have shown that it is involved in the regulation of iron/reactive oxygen species homeo-stasis, 12−14 in the regulation of lipid and glucose metabolism , 13,15 and in cell proliferation in breast cancer. 16 In vitro studies revealed that holo-mNT (the form of the protein with the cluster) is able to transfer its Fe−S cluster to very diverse apoprotein (an Fe−S protein, which has lost its cluster) recipients assembling either a [2Fe-2S] cluster as ferredoxin from various organisms, 17,18 human anamorsin 19 and CISD2, 20 or a [4Fe-4S] cluster as mammalian iron regulatory protein-1 (IRP-1)/cytosolic aconitase (c-aconi-tase). 14 On the basis of in cellulo experiments, we showed that mNT is able to repair the oxidatively damaged Fe−S cluster of human IRP-1/c-aconitase by transferring its cluster to the damaged protein. 14 Recently, we started to investigate in depth the in vitro cluster transfer reaction, focusing on the transfer from holo-mNT to [2Fe-2S] recipient protein. We unambiguously demonstrated that oxidized mNT ([2Fe-2S] 2+) triggers cluster transfer, whereas reduction of its cluster abrogates this transfer. Moreover, while O 2 significantly affects the lability of the oxidized mNT cluster, it does not interfere with the cluster

International audience; Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an iron-sulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein.

Malaria is caused by protozoan parasites and remains a major public health issue in subtropical areas. Plasmodione (3-[4-(trifluoromethyl)benzyl]-menadione) is a novel early lead compound displaying fast-acting antimalarial activity. Treatment with this redox active compound disrupts the redox balance of parasite-infected red blood cells. In vitro, the benzoyl analogue of plasmodione can act as a subversive substrate of the parasite flavoprotein NADPH-dependent glutathione reductase, initiating a redox cycling process producing ROS. Whether this is also true in vivo remains to be investigated. Here, we used the yeast model to investigate the mode of action of plasmodione and uncover enzymes and pathways involved in its activity. We showed that plasmodione is a potent inhibitor of yeast respiratory growth, that in drug-treated cells, the ROS-sensitive aconitase was impaired and that cells with a lower oxidative stress defence were highly sensitive to the drug, indicating that plasmodione may act via an oxidative stress. We found that the mitochondrial respiratory chain flavoprotein NADH-dehydrogenases play a key role in plasmodione activity. Plasmodione and metabolites act as substrates of these enzymes, the reaction resulting in ROS production. This in turn would damage ROS-sensitive enzymes leading to growth arrest. Our data further suggest that plasmodione is a pro-drug whose activity is mainly mediated by its benzhydrol and benzoyl metabolites. Our results in yeast are coherent with existing data obtained in vitro and in Plasmodium falciparum, and provide additional hypotheses that should be investigated in parasites.

Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, we demonstrated the involvement of mNT in a specific cytosolic pathway dedicated to the reactivation of oxidatively damaged cytosolic aconitase by cluster transfer. In vitro studies using apo-ferredoxin (FDX) reveal that mNT uses an Fe-based redox switch mechanism to regulate the transfer of its cluster. Using the "gold standard" cluster recipient protein, FDX, we show that this transfer is direct and that only one of the two mNT clusters is transferred when the second one is decomposed. Combining complementary biophysical and biochemical approaches, we show that pH affects both the sensitivity of the cluster to O

International audience; Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups composed of only iron and inorganic sulfur atoms with variable nuclearities. Found in all kingdoms of life, they perform numerous critical functions in fundamental processes (e.g. respiration, photosynthesis, nitrogen fixation). Organisms develop different pathways to sense their local environment such as nutrient availability, level of oxida-tive stress or of an element such as iron, and to respond and adapt to changes. The chemistry of Fe-S clusters makes them ideal for sensing various redox environmental signals and subsequently for mediating appropriate cellular responses. Fe-S cluster-containing sensors can lose their cluster, accommodate another type of cluster (e.g. interconversion between 4Fe-4S and 2Fe-2S clusters) or receive/give electrons (change in the redox state of the cluster). The present review focuses on the latter sensing mechanism, which controls the activity of Fe-S proteins in response to redox signals by changing the redox state of its cluster. Proteins using this mechanism can be found in bacteria, yeasts as well as mammals and are involved in enzyme protection (FeSII), Fe-S cluster transfer/repair (mitoNEET), DNA repair (Base Excision Repair (BER) glycosylases and helicases), and regulation of gene expression (ThnY, AirS, SoxR). In all these proteins, when the Fe-S cluster is reduced, proteins are in a "dormant state". When their cluster perceives a signal that induces its oxidation, they switch to an "active state". This sensing mechanism efficiently helps cells to turn on survival pathways quickly and recover from stressful conditions.

L’actualite scientifique vue par les etudiants du Master Biologie Sante, module physiopathologie de la signalisation, Universite Paris-Saclay Cette annee encore, dans le cadre du module d’enseignement « Physiopathologie de la signalisation » propose par l’universite Paris-sud, les etudiants du Master « Biologie Sante » de l’universite Paris-Saclay se sont confrontes a l’ecriture scientifique. Ils ont selectionne 12 articles scientifiques recents dans le domaine de la signalisation cellulaire presentant des resultats originaux, via des approches experimentales variees, sur des themes allant des interactions hote-pathogene au metabolisme en passant par la competition cellulaire et le microbiote. Apres un travail preparatoire realise avec l’equipe pedagogique, les etudiants organises en binomes/trinomes ont ensuite redige, guides par des chercheurs, une Nouvelle soulignant les resultats majeurs et l’originalite de l’article etudie. Ils ont beaucoup apprecie cette initiation a l’ecriture d’articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme !

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.

International audience; Human mitoNEET (mNT) is the first identified Fe-S protein of the mammalian outer mitochondrial membrane. Recently, mNT has been implicated in cytosolic Fe-S repair of a key regulator of cellular iron homeostasis. Here, we aimed to decipher the mechanism by which mNT triggers its Fe-S repair capacity. By using tightly controlled reactions combined with complementary spectroscopic approaches, we have determined the differential roles played by both the redox state of the mNT cluster and dioxygen in cluster transfer and protein stability. We unambiguously demonstrated that only the oxidized state of the mNT cluster triggers cluster transfer to a generic acceptor protein and that dioxygen is neither required for the cluster transfer reaction nor does it affect the transfer rate. In the absence of apo-acceptors, a large fraction of the oxidized holo-mNT form is converted back to reduced holo-mNT under low oxygen tension. Reduced holo-mNT, which holds a [2Fe-2S](+)with a global protein fold similar to that of the oxidized form is, by contrast, resistant in losing its cluster or in transferring it. Our findings thus demonstrate that mNT uses an iron-based redox switch mechanism to regulate the transfer of its cluster. The oxidized state is the "active state," which reacts promptly to initiate Fe-S transfer independently of dioxygen, whereas the reduced state is a "dormant form." Finally, we propose that the redox-sensing function of mNT is a key component of the cellular adaptive response to help stress-sensitive Fe-S proteins recover from oxidative injury.

Malaria is a major health burden in tropical and subtropical countries. The antimalarial drug primaquine is extremely useful for killing the transmissible gametocyte forms of Plasmodium falciparum and the hepatic quiescent forms of P. vivax. Yet its mechanism of action is still poorly understood. In this study, we used the yeast Saccharomyces cerevisiae model to help uncover the mode of action of primaquine. We found that the growth inhibitory effect of primaquine was restricted to cells that relied on respiratory function to proliferate and that deletion of SOD2 encoding the mitochondrial superoxide dismutase severely increased its effect, which can be countered by the overexpression of AIM32 and MCR1 encoding mitochondrial enzymes involved in the response to oxidative stress. This indicated that ROS produced by respiratory activity had a key role in primaquine-induced growth defect. We observed that Δsod2 cells treated with primaquine displayed a severely decreased activity of aconitase that contains a Fe–S cluster notoriously sensitive to oxidative damage. We also showed that in vitro exposure to primaquine impaired the activity of purified aconitase and accelerated the turnover of the Fe–S cluster of the essential protein Rli1. It is suggested that ROS-labile Fe–S groups are the primary targets of primaquine. Aconitase activity is known to be essential at certain life-cycle stages of the malaria parasite. Thus primaquine-induced damage of its labile Fe–S cluster – and of other ROS-sensitive enzymes – could inhibit parasite development., Graphical abstract fx1, Highlights • The mode of action of the antimalarial drug primaquine is poorly understood. • The yeast model is used to decipher its mechanism of action. • SOD and respiratory function are key for yeast sensitivity to primaquine. • Primaquine treatment impairs Fe–S containing enzyme aconitase. • Its attack on Fe–S clusters could explain the primaquine-induced growth inhibition.

International audience; 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.

International audience; Lipid droplet metabolism and secretory pathway trafficking both require activation of the Arf1 small G protein. The spatiotemporal regulation of Arf1 activation is mediated by guanine nucleotide exchange factors (GEFs) of the GBF and BIG families, but the mechanisms of their localization to multiple sites within cells are poorly understood. Here we show that GBF1 has a lipid-binding domain (HDS1) immediately downstream of the catalytic Sec7 domain, which mediates association with both lipid droplets and Golgi membranes in cells, and with bilayer liposomes and artificial droplets in vitro. An amphipathic helix within HDS1 is necessary and sufficient for lipid binding, both in vitro and in cells. The HDS1 domain of GBF1 is stably associated with lipid droplets in cells, and the catalytic Sec7 domain inhibits this potent lipid-droplet-binding capacity. Additional sequences upstream of the Sec7 domain-HDS1 tandem are required for localization to Golgi membranes. This mechanism provides insight into crosstalk between lipid droplet function and secretory trafficking.

International audience; The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets. Citation: Ellong EN, Soni KG, Bui Q-T, Sougrat R, Golinelli-Cohen M-P, et al. (2011) Interaction between the Triglyceride Lipase ATGL and the Arf1 Activator GBF1. PLoS ONE 6(7): e21889.

International audience; The Sec7 domain ADP-ribosylation factor (Arf) guanine nucleotide exchange factors (GEFs) are found in all eukaryotes, and are involved in membrane remodeling processes throughout the cell. This review is focused on members of the GBF/Gea and BIG/Sec7 sub-families of Arf GEFs, all of which use the class I Arf proteins (Arf1-3) as substrates, and play a fundamental role in traYcking in the endoplasmic reticulum (ER)-Golgi and endosomal membrane systems. Members of the GBF/Gea and BIG/Sec7 subfamilies are large proteins on the order of 200 kDa, and they possess multiple homology domains. Phylogenetic analyses indicate that both of these subfami-lies of Arf GEFs have members in at least Wve out of the six eukaryotic supergroups, and hence were likely present very early in eukaryotic evolution. The homology domains of the large Arf1 GEFs play important functional roles, and are involved in interactions with numerous protein partners. The large Arf1 GEFs have been implicated in several human diseases. They are crucial host factors for the repli-cation of several viral pathogens, including poliovirus, cox-sackievirus, mouse hepatitis coronavirus, and hepatitis C virus. Mutations in the BIG2 Arf1 GEF have been linked to autosomal recessive periventricular heterotopia, a disorder of neuronal migration that leads to severe malformation of the cerebral cortex. Understanding the roles of the Arf1 GEFs in membrane dynamics is crucial to a full understanding of traYcking in the secretory and endosomal pathways, which in turn will provide essential insights into human diseases that arise from misregulation of these pathways.

International audience; Arf (ADP-ribosylation factor) family small G proteins are crucial regulators of intracellular transport. The active GTP-bound form of Arf interacts with a set of proteins-effectors-which mediate the downstream signalling events of Arf activation. A well-studied class of Arf1 effectors comprises the coat complexes, such as the cis-Golgi-localized COPI (coat protein complex I) coat, and trans-Golgi network-endosomal clathrin coats. At least five different coats require Arf1-GTP to localize to organelle membranes. How a single Arf protein recruits different coat complexes to distinct membrane sites raises the question of how specificity is achieved. Here, we propose a molecular mechanism of this specificity for the COPI coat by showing a direct and specific interaction between a COPI subunit and a cis-Golgi localized subfamily of Arf guanine nucleotide exchange factors (GEFs) that takes place independently of Arf1 activation. In this way, a specific output on Arf1 activation can be programmed before the exchange reaction by the GEF itself.

International audience; Guanine nucleotide exchange factors carrying a Sec7 domain (ArfGEFs) activate the small GTP-binding protein Arf, a major regulator of membrane remodeling and protein trafficking in eukaryotic cells. Only two of the seven subfamilies of ArfGEFs-GBF and BIG-are found in all eukaryotes. In addition to the Sec7 domain that catalyzes GDP/GTP exchange on Arf, the GBF and BIG ArfGEFs have five common homology domains. Very little is known about the functions of these non-catalytic domains, but it is likely that they serve to integrate upstream signals that define the conditions of Arf activation. Here we describe interactions between two conserved domains upstream of the Sec7 domain (DCB and HUS) that determine the architecture of the N-terminal regions of the GBF and BIG ArfGEFs using a combination of biochemical, yeast two-hybrid and cellular assays. Our data demonstrate a strong interaction between DCB domains of GBF1, BIG1 and BIG2 to maintain homodimers, and an interaction between DCB and HUS domains within each homodimer. The DCB/HUS interaction is mediated by the HUS box, the most conserved motif in large ArfGEFs after the Sec7 catalytic domain. In support of the in vitro data, we show that both the DCB and the HUS domains are necessary for GBF1 dimerization in mammalian cells, and that the DCB domain is essential for yeast viability. We propose that the dimeric DCB-HUS structural unit exists in all members of the GBF and BIG ArfGEF groups and in the related Mon2p family, and likely serves an important regulatory role in Arf activation.

In yeast, Arc1p interacts with ScMetRS and ScGluRS and operates as a tRNA-Interacting Factor (tIF) in trans of these two synthetases. Its N-terminal domain (N-Arc1p) binds the two synthetases and its C-terminal domain is an EMAPII-like domain organized around an OB-fold-based tIF. ARC1 is not an essential gene but its deletion (arc1 − cells) is accompanied by a growth retardation phenotype. Here, we show that expression of N-Arc1p or of C-Arc1p alone palliates the growth defect of arc1 − cells, and that bacterial Trbp111 or human p43, two proteins containing EMAPII-like domains, also improve the growth of an arc1 − strain. The synthetic lethality of an arc1 − los1 − strain can be complemented with either ARC1 or LOS1. Expression of N-Arc1p or C-Arc1p alone does not complement an arc1 − los1 − phenotype, but coexpression of the two domains does. Our data demonstrate that Trbp111 or p43 may replace C-Arc1p to complement an arc1 − los1 − strain. The two functional domains of Arc1p (N-Arc1p and C-Arc1p) are required to get rid of the synthetic lethal phenotype but do not need to be physically linked. To get some clues to the discrete functions of N-Arc1p and C-Arc1p, we targeted ScMetRS or tIF domains to the nuclear compartment and analyzed their cellular localization by using GFP fusions, and their ability to sustain growth. Our results are consistent with a model according to which Arc1p is a bifunctional protein involved in the subcellular localization of ScMetRS and ScGluRS via its N-terminal domain and of tRNA via its C-terminal domain.

International audience; Yeast Arc1p, human p43 and plant methionyl-tRNA synthetase (MetRS) possess an EMAPII-like domain capable of non-specific interactions with tRNA. Arc1p interacts with MetRS (MES1) and GluRS and operates as a tRNA-interacting factor (tIF) in trans of these two synthetases. In plant MetRS, the EMAPII-like domain is fused to the catalytic core of the synthetase and acts as a cis-acting tIF for aminoacylation. We observed that the catalytic core of plant MetRS expressed from a centromeric plasmid cannot complement a yeast arc1 2 mes1 2 strain. Overexpression of the mutant enzyme from a high-copy number plasmid restored cell growth, suggesting that deletion of its C-terminal tIF domain was responsible for the poor aminoacylation efficiency of that enzyme in vivo. Accordingly, expression of full-size plant MetRS from a centromeric plasmid, but also of fusion proteins between its catalytic core and the EMAPII-like domains of yeast Arc1p or of human p43 restored cell viability. These data showed that homologous tIF domains from different origins are interchangeable and may act indifferently in trans or in cis of the catalytic domain of a synthetase. Unexpectedly, co-expression of Arc1p with the catalytic core of plant MetRS restored cell viability as well, even though Arc1p did not associate with plant MetRS. Because Arc1p also interacts with yeast GluRS, restoration of cell growth could be due at least in part to its role of cofactor for that enzyme. However, co-expression of human p43, a tIF that did not associate with plant MetRS or with yeast GluRS and MetRS, also restored cell viability of a yeast strain that expressed the catalytic core of plant MetRS. These results show that p43 and Arc1p are able to facilitate tRNA aminoacylation in vivo even if they do not interact physically with the synthetases. We propose that p43/Arc1p may be involved in sequestering tRNAs in the cytoplasm of eukaryotic cells, thereby increasing their availability for protein synthesis.

MutY is an adenine glycosylase in the base excision repair (BER) superfamily that is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. MutY contains a [4Fe-4S]2+ cluster that is part of a novel DNA binding motif, referred to as the iron-sulfur cluster loop (FCL) motif. This motif is found in a subset of members of the BER glycosylase superfamily, defining the endonuclease III-like subfamily. Site-specific cross-linking was successfully employed to investigate the DNA-protein interface of MutY. The photoreactive nucleotide 4-thiothymidine (4ST) incorporated adjacent to the OG:A mismatch formed a specific cross-link between the substrate DNA and MutY. The amino acid participating in the cross-linking reaction was characterized by positive ion electrospray ionization (ESI) tandem mass spectrometry. This analysis revealed Arg 143 as the site of modification in MutY. Arg 143 and nearby Arg 147 are conserved throughout the endo III-like subfamily. Replacement of Arg 143 and Arg 147 with alanine by site-directed mutagenesis reduces adenine glycosylase activity of MutY toward OG:A and G:A mispairs. In addition, the R143A and R147A enzymes exhibit a reduced affinity for duplexes containing the substrate analogue 2'-deoxy-2'-fluoroadenosine opposite OG and G. Modeling of MutY bound to DNA using an endonuclease III-DNA complex structure shows that these two conserved arginines are located within close proximity to the DNA backbone. The insight from mass spectrometry experiments combined with functional mutagenesis results indicate that these two amino acids in the [4Fe-4S]2+ cluster-containing subfamily play an important role in recognition of the damaged DNA substrate.

CISD2 belongs to the NEET family of proteins characterized by an atypical coordination of their [2Fe-2S] center by three cysteines and a histidine, and their ability to transfer their [Fe-S] center in vitro to an acceptor protein. CISD2 is a homodimeric protein anchored at contact sites between the endoplasmic reticulum and mitochondria with one [2Fe-2S] cluster per protomer. Most of CISD2, including its [Fe-S] cluster coordination domain, is cytosolic. The latter is very close in sequence and structure to that of mitoNEET, another member of the NEET family.I first expressed the soluble part of human CISD2 in E. coli and then purified it. I studied the stability of its cluster and its ability to be transferred in vitro. I showed that its lability and ability to be transfered increases at acidic pH. However, the dependence of these reactions on pH is much less than for mitoNEET. Moreover, unlike mitoNEET, I have not identified conditions under which the transfer of the Fe-S cluster would be fast. I then showed that glutathione reduces the stability of the CISD2 cluster but in a more moderate way than for mitoNEET. Finally, like mitoNEET, CISD2 is extremely resistant to H2O2 at neutral pH but the lability of its center increases significantly at slightly acidic pH. Next, I was interested in point mutations of CISD2 encountered in patients with Wolfram syndrome type 2 (N72S and S104P). I found that these two mutations significantly affected the stability of the Fe-S cluster. Finally, I focused on the CISD2-BCL2 interaction. I purified the cytosolic domain of BCL-2, and confirmed the interaction between purified CISD2 and BCL-2 in vitro.; CISD2 appartient à la famille de protéines NEET caractérisée par une coordination atypique de leur centre [2Fe-2S] par trois cystéines et une histidine, et leur capacité à transférer leur centre [Fe-S] in vitro vers une protéine acceptrice. CISD2 est une protéine homodimérique ancrée au niveau des sites de contact entre le réticulum endoplasmique et la mitochondrie avec un centre [2Fe-2S] par protomère. La majeure partie de CISD2, dont son domaine de coordination du centre [Fe-S], est cytosolique. Ce dernier est très proche en séquence et en structure de celui de mitoNEET, autre membre de la famille NEET.En premier lieu, j’ai exprimé la partie soluble de CISD2 humaine chez E. coli puis je l’ai purifiée. J’ai ensuite étudié la stabilité de son centre et sa capacité à être transféré in vitro. J’ai montré que sa labilité et sa capacité à être transféré augmente à pH acide. Cependant, la dépendance de ces réactions vis-à-vis du pH est nettement moins importante que pour mitoNEET. De plus, contrairement à mitoNEET, je n’ai pas identifié de conditions pour laquelle le transfert du centre Fe-S serait rapide in vitro.J’avais ensuite montré que le glutathion réduit la stabilité du centre de CISD2 mais de manière plus modérée que pour mitoNEET. Pour finir, tout comme mitoNEET, CISD2 est extrêmement résistante à H2O2 à pH neutre mais la labilité de son centre augmente de manière significative à pH légèrement acide. Ensuite, je me suis intéressée à des mutations ponctuelles de CISD2 rencontrées chez des patients atteints par le syndrome de Wolfram de type 2 (N72S et S104P). J’ai alors constaté que ces deux mutations affectaient de manière significative la stabilité du centre Fe-S. Pour finir, je me suis focalisée sur l’interaction CISD2-BCL2. J’ai purifiée le domaine cytosolique de BCL-2, et j’ai confirmé l’interaction entre CISD2 et BCL-2 purifiées in vitro.

CISD2 belongs to the NEET family of proteins characterized by an atypical coordination of their [2Fe-2S] center by three cysteines and a histidine, and their ability to transfer their [Fe-S] center in vitro to an acceptor protein. CISD2 is a homodimeric protein anchored at contact sites between the endoplasmic reticulum and mitochondria with one [2Fe-2S] cluster per protomer. Most of CISD2, including its [Fe-S] cluster coordination domain, is cytosolic. The latter is very close in sequence and structure to that of mitoNEET, another member of the NEET family.I first expressed the soluble part of human CISD2 in E. coli and then purified it. I studied the stability of its cluster and its ability to be transferred in vitro. I showed that its lability and ability to be transfered increases at acidic pH. However, the dependence of these reactions on pH is much less than for mitoNEET. Moreover, unlike mitoNEET, I have not identified conditions under which the transfer of the Fe-S cluster would be fast. I then showed that glutathione reduces the stability of the CISD2 cluster but in a more moderate way than for mitoNEET. Finally, like mitoNEET, CISD2 is extremely resistant to H2O2 at neutral pH but the lability of its center increases significantly at slightly acidic pH. Next, I was interested in point mutations of CISD2 encountered in patients with Wolfram syndrome type 2 (N72S and S104P). I found that these two mutations significantly affected the stability of the Fe-S cluster. Finally, I focused on the CISD2-BCL2 interaction. I purified the cytosolic domain of BCL-2, and confirmed the interaction between purified CISD2 and BCL-2 in vitro., CISD2 appartient à la famille de protéines NEET caractérisée par une coordination atypique de leur centre [2Fe-2S] par trois cystéines et une histidine, et leur capacité à transférer leur centre [Fe-S] in vitro vers une protéine acceptrice. CISD2 est une protéine homodimérique ancrée au niveau des sites de contact entre le réticulum endoplasmique et la mitochondrie avec un centre [2Fe-2S] par protomère. La majeure partie de CISD2, dont son domaine de coordination du centre [Fe-S], est cytosolique. Ce dernier est très proche en séquence et en structure de celui de mitoNEET, autre membre de la famille NEET.En premier lieu, j’ai exprimé la partie soluble de CISD2 humaine chez E. coli puis je l’ai purifiée. J’ai ensuite étudié la stabilité de son centre et sa capacité à être transféré in vitro. J’ai montré que sa labilité et sa capacité à être transféré augmente à pH acide. Cependant, la dépendance de ces réactions vis-à-vis du pH est nettement moins importante que pour mitoNEET. De plus, contrairement à mitoNEET, je n’ai pas identifié de conditions pour laquelle le transfert du centre Fe-S serait rapide in vitro.J’avais ensuite montré que le glutathion réduit la stabilité du centre de CISD2 mais de manière plus modérée que pour mitoNEET. Pour finir, tout comme mitoNEET, CISD2 est extrêmement résistante à H2O2 à pH neutre mais la labilité de son centre augmente de manière significative à pH légèrement acide. Ensuite, je me suis intéressée à des mutations ponctuelles de CISD2 rencontrées chez des patients atteints par le syndrome de Wolfram de type 2 (N72S et S104P). J’ai alors constaté que ces deux mutations affectaient de manière significative la stabilité du centre Fe-S. Pour finir, je me suis focalisée sur l’interaction CISD2-BCL2. J’ai purifiée le domaine cytosolique de BCL-2, et j’ai confirmé l’interaction entre CISD2 et BCL-2 purifiées in vitro.

The aim of this work is to study mitochondrial dysfunctions related to a defect of protein lipoylation in fibroblasts of 14 patients. These patients carry a point mutation in a gene encoding for a protein involved either in lipoic acid biosynthesis (LIPT1 or LIPT2) or in the mitochondrial pathway devoted to iron-sulfur cluster biogenesis (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3) essential for maturation of mitochondrial Fe-S proteins such as lipoic acid synthase (LIAS). This work describes the second case of FDX1L deficiency and a patient with a new mutation in ISCA1 gene.We found that mitochondrial [4Fe-4S] proteins (mitochondrial aconitase, complexes I and II of the respiratory chain and LIAS) are mainly affected in fibroblasts of patients with defect in the mitochondrial Fe-S maturation pathway. Secondary, LIAS dysfunction leads to decreased lipoylation of PDHc and KGDHc, complexes involved in energy metabolism. Neither mitochondrial network nor oxidative stress biomarkers was modified in our study. Addition of exogenous lipoic acid did not rescue the mitochondrial deficiency.Protein expression profiles obtained in fibroblasts of patients suggest that NFU1, BOLA3 and IBA57 and also ISCA1, ISCA2 and IBA57 could function and interact together to form protein complexes., Le but de cette thèse est d’étudier les modifications biochimiques mitochondriales liées à un défaut de lipoylation des protéines dans les fibroblastes de 14 patients. Ces patients sont porteurs d’une mutation dans un gène codant une des protéines impliquées soit dans la synthèse de l’acide lipoïque (LIPT1, LIPT2) soit dans la voie de biogenèse des centres Fe-S mitochondriale (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3). La voie de biogenèse des centres Fe-S est nécessaire à la maturation des protéines Fe-S mitochondriales, dont la lipoic acid synthase (LIAS).Ces travaux ont permis d’étudier notamment un deuxième cas de déficit en FDX1L ainsi qu’un patient porteur d’une nouvelle mutation dans ISCA1. Les déficits dans la voie de la biogenèse des centres Fe-S observés chez les patients étudiés affectent principalement la maturation des protéines mitochondriales à centre [4Fe-4S] dont l’aconitase mitochondriale, les complexes I et II de la chaîne respiratoire et la LIAS, induisant ainsi un défaut de lipoylation d’enzymes clés du métabolisme énergétique (PDHc, KGDHc). Aucune atteinte du réseau mitochondrial ni de variations du stress oxydatif n’ont pu être mises en évidence. Finalement, l’ajout d’acide lipoïque exogène n’améliore pas les déficits observés.Les profils d’expression des protéines dans les fibroblastes des patients suggèrent que les protéines NFU1, BOLA3 et IBA57 ainsi que ISCA1, ISCA2 et IBA57 coopèrent entre elles de manière complexe.

The aim of this work is to study mitochondrial dysfunctions related to a defect of protein lipoylation in fibroblasts of 14 patients. These patients carry a point mutation in a gene encoding for a protein involved either in lipoic acid biosynthesis (LIPT1 or LIPT2) or in the mitochondrial pathway devoted to iron-sulfur cluster biogenesis (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3) essential for maturation of mitochondrial Fe-S proteins such as lipoic acid synthase (LIAS). This work describes the second case of FDX1L deficiency and a patient with a new mutation in ISCA1 gene.We found that mitochondrial [4Fe-4S] proteins (mitochondrial aconitase, complexes I and II of the respiratory chain and LIAS) are mainly affected in fibroblasts of patients with defect in the mitochondrial Fe-S maturation pathway. Secondary, LIAS dysfunction leads to decreased lipoylation of PDHc and KGDHc, complexes involved in energy metabolism. Neither mitochondrial network nor oxidative stress biomarkers was modified in our study. Addition of exogenous lipoic acid did not rescue the mitochondrial deficiency.Protein expression profiles obtained in fibroblasts of patients suggest that NFU1, BOLA3 and IBA57 and also ISCA1, ISCA2 and IBA57 could function and interact together to form protein complexes.; Le but de cette thèse est d’étudier les modifications biochimiques mitochondriales liées à un défaut de lipoylation des protéines dans les fibroblastes de 14 patients. Ces patients sont porteurs d’une mutation dans un gène codant une des protéines impliquées soit dans la synthèse de l’acide lipoïque (LIPT1, LIPT2) soit dans la voie de biogenèse des centres Fe-S mitochondriale (FDX1L, ISCA1, ISCA2, IBA57, NFU1, BOLA3). La voie de biogenèse des centres Fe-S est nécessaire à la maturation des protéines Fe-S mitochondriales, dont la lipoic acid synthase (LIAS).Ces travaux ont permis d’étudier notamment un deuxième cas de déficit en FDX1L ainsi qu’un patient porteur d’une nouvelle mutation dans ISCA1. Les déficits dans la voie de la biogenèse des centres Fe-S observés chez les patients étudiés affectent principalement la maturation des protéines mitochondriales à centre [4Fe-4S] dont l’aconitase mitochondriale, les complexes I et II de la chaîne respiratoire et la LIAS, induisant ainsi un défaut de lipoylation d’enzymes clés du métabolisme énergétique (PDHc, KGDHc). Aucune atteinte du réseau mitochondrial ni de variations du stress oxydatif n’ont pu être mises en évidence. Finalement, l’ajout d’acide lipoïque exogène n’améliore pas les déficits observés.Les profils d’expression des protéines dans les fibroblastes des patients suggèrent que les protéines NFU1, BOLA3 et IBA57 ainsi que ISCA1, ISCA2 et IBA57 coopèrent entre elles de manière complexe.