Dataset Information

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

ABSTRACT: This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co²⁺ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe²⁺, as it is customarily done, Co²⁺ is most often used in experiments because Fe²⁺ is extremely unstable owing to the fast oxidation reaction Fe²⁺ → Fe³⁺. Protein stability is monitored following the conformational changes induced by Co²⁺ binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements. The stability ranking among the wild-type frataxin and its variants obtained in this way is confirmed by a detailed comparative analysis of the XAS spectra of the metal-protein complex at the Co K-edge. In particular, a fit to the EXAFS region of the spectrum allows positively identifying the frataxin acidic ridge as the most likely location of the metal-binding sites. Furthermore, we can explain the surprising feature emerging from a detailed analysis of the XANES region of the spectrum, showing that the longer 81-210 frataxin fragment has a smaller propensity for Co²⁺ binding than the shorter 90-210 one. This fact is explained by the peculiar role of the N-terminal disordered tail in modulating the protein ability to interact with the metal.

SUBMITTER: Morante S

PROVIDER: S-EPMC9195147 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Publications

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

Morante S S Botticelli S S Chiaraluce R R Consalvi V V La Penna G G Novak L L Pasquo A A Petrosino M M Proux O O Rossi G G Salina G G Stellato F F

Frontiers in molecular biosciences 20220531

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co2+ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe2+, as it is customarily done, Co2+ is most often used in experiments because Fe2+ is extremely unstable owing to the fast oxidation reaction Fe2+ → Fe3+. Protein stability is monitored following the conformational changes i ...[more]

PMID: 35712353

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Project description:Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic enzyme in human malignancy. A heterozygous genetic alteration, arginine 132, promotes the conversion of α-ketoglutarate to D-2-hydroxyglutarate (2-HG). Although pharmacologic inhibitors of mutant IDH1 are promising, resistance mechanisms to targeted therapy are not understood. Additionally, the role of wild-type IDH1 (WT.IDH1) in cancer requires further study. Recently, it was observed that the regulatory RNA-binding protein, HuR (ELAVL1), protects nutrient-deprived cancer cells without IDH1 mutations, by stabilizing WT.IDH1 transcripts. In the present study, a similar regulatory effect on both mutant (Mut.IDH1) and WT.IDH1 transcripts in heterozygous IDH1-mutant tumors is observed. In ribonucleoprotein immunoprecipitation assays of IDH1-mutant cell lines, wild-type and mutant IDH1 mRNAs each bound to HuR. Both isoforms were profoundly downregulated at the mRNA and protein levels after genetic suppression of HuR (siRNAs or CRISPR deletion) in HT1080 (R132C IDH1 mutation) and BT054 cells (R132H). Proliferation and invasion were adversely affected after HuR suppression and metabolomic studies revealed a reduction in Pentose Phosphate Pathway metabolites, nucleotide precursors, and 2-HG levels. HuR-deficient cells were especially sensitive to stress, including low glucose conditions or a mutant IDH1 inhibitor (AGI-5198). IDH1-mutant cancer cells were rescued by WT.IDH1 overexpression to a greater extent than Mut.IDH1 overexpression under these conditions. This study reveals the importance of HuR's regulation of both mutant and wild-type IDH1 in tumors harboring a heterozygous IDH1 mutation with implications for therapy. IMPLICATIONS: This study highlights the HuR-IDH1 (mutant and wild-type IDH1) regulatory axis as a critical, actionable therapeutic target in IDH1-mutated cancer, and incomplete blockade of the entire HuR-IDH1 survival axis would likely diminish the efficacy of drugs that selectively target only the mutant isoenzyme.

Dataset Information

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

Publications

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

Similar Datasets

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