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.

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.

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.

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

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

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

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

Quantification of Cellular Deoxyribonucleoside Triphosphates by Rolling Circle Amplification and Förster Resonance Energy Transfer.

The quantification of cellular deoxyribonucleoside triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Förster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.

Crosstalk between TAp73 and TGF-ß in fibroblast regulates iNOS expression and Nrf2-dependent gene transcription.

Inducible nitric oxide synthase (iNOS) activity produces anti-tumor and anti-microbial effects but also promotes carcinogenesis through mutagenic, immunosuppressive and pro-angiogenic mechanisms. The tumor suppressor p53 contributes to iNOS downregulation by repressing induction of the NOS2 gene encoding iNOS, thereby limiting NO-mediated DNA damages. This study focuses on the role of the p53 homologue TAp73 in the regulation of iNOS expression. Induction of iNOS by immunological stimuli was upregulated in immortalized MEFs from TAp73-/- mice, compared to TAp73+/+ fibroblasts. This overexpression resulted both from increased levels of NOS2 transcripts, and from an increased stability of the protein. Limitation of iNOS expression by TAp73 in wild-type cells is alleviated by TGF-ß receptor I inhibitors, suggesting a cooperation between TAp73 and TGF-ß in suppression of iNOS expression. Accordingly, downregulation of iNOS expression by exogenous TGF-ß1 was impaired in TAp73-/- fibroblasts. Increased NO production in these cells resulted in a stronger, NO-dependent induction of Nrf2 target genes, indicating that the Nrf2-dependent adaptive response to nitrosative stress in fibroblasts is proportional to iNOS activity. NO-dependent induction of two HIF-1 target genes was also stronger in TAp73-deficient cells. Finally, the antimicrobial action of NO against Trypanosoma musculi parasites was enhanced in TAp73-/- fibroblasts. Our data indicate that tumor suppressive TAp73 isoforms cooperate with TGF-ß to control iNOS expression, NO-dependent adaptive responses to stress, and pathogen proliferation.

Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

NME1 (nonmetastatic expressed 1) gene, which encodes nucleoside diphosphate kinase (NDPK) A [also known as nonmetastatic clone 23 (NM23)-H1 in humans and NM23-M1 in mice], is a suppressor of metastasis, but several lines of evidence-mostly from plants-also implicate it in the regulation of the oxidative stress response. Here, our aim was to investigate the physiologic relevance of NDPK A with respect to the oxidative stress response in mammals and to study its molecular basis. NME1-knockout mice died sooner, suffered greater hepatocyte injury, and had lower superoxide dismutase activity than did wild-type (WT) mice in response to paraquat-induced acute oxidative stress. Deletion of NME1 reduced total NDPK activity and exacerbated activation of the stress-related MAPK, JNK, in the liver in response to paraquat. In a mouse transformed hepatocyte cell line and in primary cultures of normal human keratinocytes, MAPK activation in response to H2O2 and UVB, respectively, was dampened by expression of NM23-M1/NM23-H1, dependent on its NDPK catalytic activity. Furthermore, excess or depletion of NM23-M1/NM23-H1 NDPK activity did not affect the intracellular bulk concentration of nucleoside di- and triphosphates. NME1-deficient mouse embryo fibroblasts grew poorly in culture, were more sensitive to stress than WT fibroblasts, and did not immortalize, which suggested that they senesce earlier than do WT fibroblasts. Collectively, these results indicate that the NDPK activity of NM23-M1/NM23-H1 protects cells from acute oxidative stress by inhibiting activation of JNK in mammal models.-Peuchant, E., Bats, M.-L., Moranvillier, I., Lepoivre, M., Guitton, J., Wendum, D., Lacombe, M.-L., Moreau-Gaudry, F., Boissan, M., Dabernat, S. Metastasis suppressor NM23 limits oxidative stress in mammals by preventing activation of stress-activated protein kinases/JNKs through its nucleoside diphosphate kinase activity.

Apoptosis induction by combination of drugs or a conjugated molecule associating non-steroidal anti-inflammatory and nitric oxide donor effects in medullary thyroid cancer models: implication of the tumor suppressor p73.

BACKGROUND: Medullary thyroid cancer (MTC) is a C-cell neoplasm. Surgery remains its main treatment. Promising therapies based on tyrosine kinase inhibitors demand careful patient selection. We previously observed that two non-steroidal anti-inflammatory drugs (NSAID), indomethacin, celecoxib, and nitric oxide (NO) prevented tumor growth in a model of human MTC cell line (TT) in nude mice. METHODS: In the present study, we tested the NO donor: glyceryl trinitrate (GTN), at pharmacological dose, alone and in combination with each of the two NSAIDs on TT cells. We also assessed the anti-proliferative potential of NO-indomethacin, an indomethacin molecule chemically conjugated with a NO moiety (NCX 530, Nicox SA) on TT cells and indomethacin/GTN association in rMTC 6-23 cells. The anti-tumoral action of the combined sc. injections of GTN with oral delivery of indomethacin was also studied on subcutaneous TT tumors in nude mice. Apoptosis mechanisms were assessed by expression of caspase-3, TAp73α, TAp73α inhibition by siRNA or Annexin V externalisation. RESULTS: The two NSAIDs and GTN reduced mitotic activity in TT cells versus control (cell number and PCNA protein expression). The combined treatments amplified the anti-tumor effect of single agents in the two tested cell lines and promoted cell death. Moreover, indomethacin/GTN association stopped the growth of established TT tumors in nude mice. We observed a significant cleavage of full length PARP, a caspase-3 substrate. The cell death appearance was correlated with a two-fold increase in TAp73α expression, with inhibition of apoptosis after TAp73α siRNA addition, demonstrating its crucial role in apoptosis. CONCLUSION: Association of NO with NSAID exhibited amplified anti-tumoral effects on in vitro and in vivo MTC models by inducing p73-dependent apoptotic cell death.

Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis.

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp(342) and Asn(343). Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.

Comparative study of the antitumor effect of natural monoterpenes: relationship to cell cycle analysis

Monoterpenes have been identified as responsible of important therapeutic effects of plant-extracts. In this work, we try to compare the cytotoxic effect of six monoterpenes (carvacrol, thymol, carveol, carvone, eugenol and isopulegol) as well as their molecular mechanisms. The in vitro antitumor activity of the tested products, evaluated against five tumor cell lines, show that the carvacrol is the most cytotoxic monoterpene. The investigation of an eventual synergistic effect of the six natural monoterpenes with two anticancer drugs revealed that there is a significant synergy between them (p<5%). On the other hand, the effect of the tested products on cell cycle progression was examined by flow cytometry after DNA staining in order to investigate the molecular mechanism of their cytotoxic activity. The results revealed that carvacrol and carveol stopped the cell cycle progression in S phase; however, thymol and isopulegol stopped it in G0/G1 phase. Regarding carvone and eugenol, no effect on cell cycle was observed.

TAp73 induction by nitric oxide: regulation by checkpoint kinase 1 (CHK1) and protection against apoptosis.

Nitric oxide (NO) is a potent activator of the p53 tumor suppressor protein, thereby inducing cell cycle arrest and apoptosis. However, little is known about the regulation of the two other p53-family members, p63 and p73, by nitrogen oxides. We report here an up-regulation of p73 by NO in p53-null K-562 leukemia cells. Chemical NO prodrugs or macrophage iNOS activity induced an accumulation of the TAp73α isoform in these cells, whereas macrophages from iNOS(-/-) mice did not. NO also up-regulated TAp73 mRNA expression, suggesting a transcriptional regulation. The checkpoint kinases Chk1 and Chk2 can regulate TAp73 induction after DNA damage. We show that these kinases were rapidly phosphorylated upon NO treatment. Genetic silencing or pharmacological inhibition of Chk1 impaired NO-mediated accumulation of TAp73α. Because NO is known to block DNA synthesis through ribonucleotide reductase inhibition, the up-regulation of TAp73α might be caused by DNA damage induced by an arrest of DNA replication forks. In support of this hypothesis, DNA replication inhibitors such as hydroxyurea and aphidicolin similarly enhanced TAp73α expression and Chk1 phosphorylation. Moreover, inhibition of Chk1 also prevented TAp73α accumulation in response to replication inhibitors. The knockdown of TAp73 with siRNA sensitized K-562 cells to apoptosis induced by a nitrosative (NO) or oxidative (H(2)O(2)) injury. Therefore, TAp73α has an unusual cytoprotective role in K-562 cells, contrasting with its pro-apoptotic functions in many other cell models. In conclusion, NO up-regulates several p53 family members displaying pro- and anti-apoptotic effects, suggesting a complex network of interactions and cross-regulations between NO production and p53-related proteins.

Anti-tumoral effect of a celecoxib low dose on a model of human medullary thyroid cancer in nude mice.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a C cell neoplasm secreting calcitonin (CT). Surgery remains the only treatment as MTC is resistant to radio- and chemotherapies. Anti-tumoral effects of nonsteroidal anti-inflammatory drugs have been observed in various cancers. Thus, we tested the anti-tumoral action of an nonsteroidal anti-inflammatory drug, celecoxib, on MTC development. METHODS: We studied the expression of prostaglandin (PG) metabolism enzymes in our in vitro (TT cells) and in vivo (TT tumors) models and in human MTCs by Western blot. We checked the effect of celecoxib on xenografted subcutaneous tumors in nude mice. Celecoxib was administrated in powder food during 9 weeks from day 1 after TT cell injection. At the end of the experiment plasma CT was measured by radioimmunoassay, the number of proliferating cells in tumor tissues was detected by Ki67 immunocytochemistry and apoptotic nuclei by caspase 3 ad Bcl-2 expression and terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. PGE(2) concentrations in TT cell medium were evaluated by an enzyme immunoassay kit. RESULTS: Our in vitro and in vivo models were validated: the status of PG metabolism enzymes was comparable in these models and in human MTCs. A very low dose of celecoxib, 120 ppm in food, inhibited tumor volume by 71% and reduced plasma CT level. Although no proapoptotic effect was detectable in tumors, a decrease of proliferating cells was revealed. The inducible PG synthesis enzyme, cyclooxygenase 2, was only detectable in rare stromal cells. The expression of the constitutive PG synthesis enzyme, cyclooxygenase 1, was diminished, while the level of the catabolism enzyme, 15-PG dehydrogenase, was decreased. In vitro, TT cells treated for 12 days with 25 muM celecoxib reproduced these changes, and PGE(2) secretion was not significantly modified by the treatment, in these conditions. CONCLUSION: Celecoxib has a good therapeutic potential for MTC to prevent metastasis growth, and its anti-tumoral effect is, at least in part, independent of PGE(2).

Synthesis and evaluation of pyrido[1,2-a]pyrimidines as inhibitors of nitric oxide synthases.

A series of new 3-aroylpyrido[1,2-a]pyrimidines were synthesized from aryl methyl ketones in a simple two-step procedure and evaluated as nitric oxide synthases (NOS) inhibitors. In order to perform a structure-activity relationship study, different aroyl groups were introduced in 3-position and methyl groups were introduced at different positions of the pyrimidine heterocycle. Compounds with a biphenyloyl, benzyloxybenzoyl or naphthoyl group displayed the highest inhibitory effects which were further increased by introduction of a methyl group in position 8 of the pyrido[1,2-a]pyrimidine moiety. Some of the compounds exhibited promising inhibitory effects with selectivity toward the purified inducible NOS (iNOS) and were also active against iNOS expressed in stimulated RAW 264.7 cells.

Glutathione depletion reveals impairment of antigen processing and inhibition of cathepsin activity by nitric oxide in antigen-presenting cells.

Nitric oxide has been shown to induce immunosuppression by inhibiting class II MHC molecule expression and T-lymphocyte proliferation. However, much less is known about the ability of NO to interfere with antigen processing and presentation. So we questioned whether B lymphoma cells exposed to NO could be impaired in their ability to process lysozyme and to stimulate proliferation of a syngeneic T-cell hybridoma. As immunosuppressive pathological conditions are often associated with a pro-oxidative milieu, we also examined the influence of intracellular GSH levels on NO responsiveness. Exposure of GSH-depleted B cells to NO-releasing compounds lowered their capacity to present a reduced and alkylated lysozyme (TAP-HEL), although presentation of a lysozyme-derived peptide was unaffected. Cells with a normal GSH content were protected from this inhibition. Fluid phase endocytosis, protein synthesis and expression of class II molecules remained normal in GSH-depleted cells. However, proteolysis of a dye conjugate of ovalbumin was strongly inhibited, suggesting that protease inhibition might be involved. Cathepsin B activity, which was necessary to TAP-HEL processing, was inhibited by the NO-donors. The inhibition was higher in GSH-depleted cells and reproduced by treatment of A20 B cells by two cathepsin inhibitors. These results show that, in addition to cytostasis and reduction in class II expression, NO-induced immunosuppression could also implicate inhibition of antigen processing under oxidative stress conditions.