Targeting the redox vulnerability of acute myeloid leukaemia cells with a combination of auranofin and vitamin C.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by complex molecular and cytogenetic abnormalities. Pro-oxidant cellular redox status is a common hallmark of AML cells, providing a rationale for redox-based anticancer strategy. We previously discovered that auranofin (AUF), initially used for the treatment of rheumatoid arthritis and repositioned for its anticancer activity, can synergize with a pharmacological concentration of vitamin C (VC) against breast cancer cell line models. In this study, we observed that this drug combination synergistically and efficiently killed cells of leukaemic cell lines established from different myeloid subtypes. In addition to an induced elevation of reactive oxygen species and ATP depletion, a rapid dephosphorylation of 4E-BP1 and p70S6K, together with a strong inhibition of protein synthesis were early events in response to AUF/VC treatment, suggesting their implication in AUF/VC-induced cytotoxicity. Importantly, a study on 22 primary AML specimens from various AML subtypes showed that AUF/VC combinations at pharmacologically achievable concentrations were effective to eradicate primary leukaemic CD34+ cells from the majority of these samples, while being less toxic to normal cord blood CD34+ cells. Our findings indicate that targeting the redox vulnerability of AML with AUF/VC combinations could present a potential anti-AML therapeutic approach.

Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can also be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro, and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

[The glutathionylation of the human mitochondrial protein MIA40 regulates ROS homeostasis]. / La glutathionylation de la protéine mitochondriale humaine MIA40 régule l'homéostasie des ROS.

Title: La glutathionylation de la protéine mitochondriale humaine MIA40 régule l'homéostasie des ROS. Abstract: Dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-Saclay, les étudiants du Master « Biologie Santé ¼ se sont confrontés à l'écriture scientifique. Ils ont sélectionné des articles scientifiques dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant de l'exploration des sites de contacts membranaires aux mécanismes moléculaires de la ferroptose, en passant par la signalisation hépatique et tumorale. Après un travail préparatoire réalisé avec l'équipe pédagogique, les étudiants, organisés en binômes/trinômes, 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 !

Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide.

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

Viruses traverse the human proteome through peptide interfaces that can be biomimetically leveraged for drug discovery.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

Redox-Based Strategies against Infections by Eukaryotic Pathogens.

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.

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.

NRVS and DFT of MitoNEET: Understanding the Special Vibrational Structure of a [2Fe-2S] Cluster with (Cys)3(His)1 Ligation.

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.

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.

[MDM2 promotes cell death by interacting with mitochondrial bioenergetics]. / La protéine MDM2 favorise la mort cellulaire en affectant la bioénergétique mitochondriale.

TITLE: La protéine MDM2 favorise la mort cellulaire en affectant la bioénergétique mitochondriale. ABSTRACT: Pour la sixième année, dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-sud, les étudiants du Master « Biologie Santé ¼ 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.

The H2O2-Resistant Fe-S Redox Switch MitoNEET Acts as a pH Sensor To Repair Stress-Damaged Fe-S Protein.

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 O2 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 H2O2 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.

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

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.

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.

Redox Control of the Human Iron-Sulfur Repair Protein MitoNEET Activity via Its Iron-Sulfur Cluster.

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.

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.

Arc1p is required for cytoplasmic confinement of synthetases and tRNA.

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.

Complementation of yeast Arc1p by the p43 component of the human multisynthetase complex does not require its association with yeast MetRS and GluRS.

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(-) mes1(-) 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.