Mammalian Frataxin: An Essential Function for Cellular Viability through an Interaction with a Preformed ISCU/NFS1/ISD11 Iron-Sulfur Assembly Complex

Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis.. Fe-S cluster biosynthesis

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo

Structural bases for the interaction of frataxin with the central components of iron–sulphur cluster assembly

Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreich's ataxia. Independent reports have linked frataxin to iron–sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics

The function of yeast frataxin in iron-sulfur cluster biogenesis : a systematic mutagenesis of solvent-exposed side chains of the beta-sheet platform

Friedreich's ataxia is a neurodegenerative disorder caused by the low expression of a mitochondrial protein called frataxin. Studies in the yeast Saccharomyces cerevisiae have unraveled a role for the frataxin homologue (Yfh1p) in iron-sulfur cluster (Fe/S) biosynthesis, probably by interacting with the scaffold protein, Isu1p, and providing iron to the machinery. Yfh1p possesses a large â-sheet platform that may be involved in the interaction with other proteins through conserved residues at its surface. We have used directed mutagenesis associated with polymerase chain reaction (PCR) to study conserved residues localizing either at the surface of the protein, Thr110, Thr118, Val120, Asn122, Gln124, Gln129, Trp131, Ser137 and Arg141, or buried in the core of the protein, Ile130 and Leu132. Mutants T110A, T118A, V120A, N122A, Q124A, Q129A, I130A, W131A, L132A, S137A and R141A were generated in yeast. Growth on iron- or copper-containing medium was severely impaired for mutants Q129A, I130A, W131A and R141A. Others were roughly growing as well as the wild-type strain. We assessed the efficiency of Fe/S biosynthesis by measuring aconitase activity. The results confirmed those obtained on metal-containing medium: mutants Q129A, I130A, W131A and R141A showed a high decrease in their aconitase activity that dropped to the deleted strain level. Moreover, S137A showed also a decreased aconitase activity. We monitored the interaction between Yfh1p and Isu1p by co-immunoprecipitation and it turned out that only the W131A mutation affects directly this interaction. Even if the amount of Yfh1p determined by western blot analysis was highly decreased for several mutants, it is not sufficient to explain the phenotypes as they were poorly restored by overexpression of the mutant proteins to wild-type levels, except for W131F. We have concluded that Gln129, Trp131, and Arg141 are important for Yfh1p function, while Ile130 and Ser137 are required for the folding of the protein. All these residues cluster to the 4th and 5th â-strand of the protein. Our work has demonstrated for the first time the importance of this area for Yfh1p function and shows that Trp131 is involved in the interaction with Isu1p.(BIOL 3) -- UCL, 200

In situ and in vitro profiling of brain tumour initiating cells of high-grade gliomas

High-grade gliomas, especially glioblastomas, are highly complex and heterogeneous primary brain tumours. Glioblastoma (GBM) is one of the most aggressive cancers with poor overall survival prognosis. Fast and widespread invasion of the brain parenchyma by a subpopulation of progenitor tumour cells is a main pathophysiological feature of these tumours. Invasion renders localised therapies ineffective and is a primary cause of tumour recurrence as well as associated morbidity. Identification of factors related to invasion of brain tumour initiating cells (BTIC) into the surrounding parenchyma may provide strategic targets for early and sustained anti-invasion therapies. The dependency and interaction of infiltrative progeny cells with the tumour microenvironment as well as their adaptive development from the tumour cell fraction have not been adequately investigated. Organotypic brain slice cultures (OBSC) were used as a valuable in situ model to analyse migration, as well as invasion, in conditions that simulate normal brain tissue and its respective microenvironmental influences. Unlike common animal models, this allowed monitoring of live tumour cell invasion in real time. Invasion of glioblastoma cells was induced by inoculation of fluorescence tagged human BTICs in the hippocampal region of OBSCs. During this analysis, morphologically distinct invasive cells emerged from the initial cell population, which drove invasion into the surrounding tissue. Differences in cell morphology and behaviour allowed categorisation of three populations of cells into leader, follower, and stationary which were otherwise not possible to observe in 2D migration assays. To assess cellular expression markers specific for each group, a micromanipulator adapter was developed to isolate the separate cell subpopulations from OBSCs. After cDNA library generation and subsequent microarray analysis, a detailed comparison between stationary, follower, and leader cells was conducted. Invasive cells exhibited a markedly distinct expression pattern in comparison to follower and stationary cells, which allowed designation of a leader cell-specific gene expression signature. Unravelling the dynamics and complexity of leader cell invasion and induction by microenvironmental cues may provide new targets for future development of effective glioma treatments. Deciphering the roles of leader, follower, and stationary cells in heterogeneous GBM is a long-term goal. However, an important short-term objective should be the improvement of existing standard therapy. Combined adjuvant therapies may prolong the currently very short median survival rate of patients with glioma. It was previously demonstrated that ibuprofen and diclofenac exhibit significant anti-tumourigenic effects on GBM cell lines in vitro. Although it was not conclusively proven here, restriction of signal transducer and activator of transcription number 3 (STAT3) phosphorylation and c-myc modulation could constitute mechanisms of their GBM restricting capacities. In parallel, the unknown characteristics of primary BTICs and their differentiated pairs were analysed with regard to selected transcription factor expression as well as their motility and proliferation. Correlation of proliferation and migration allowed for clustering of the cells into separate subgroups. Subsequently, separate, well-characterised cell lines were analysed for sensitivity to STAT3-specific inhibition. This analysis revealed a remarkable restriction of both proliferation and migration in in vitro as well as in situ analyses. The findings of the present study suggest that STAT3 is an important target for further therapy approaches of high-grade gliomas, and is supported by the identification of STAT3 as part of the leader cell gene expression signature in this work. The results presented here indicate that there may be value in the investigation of Stattic or other STAT3 specific inhibitors in clinical trials for glioma treatment

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