Interaction of frataxin, an iron binding protein, with IscU of Fe–S clusters biogenesis pathway and its upregulation in AmpB resistant Leishmania donovani
| Autor: | Vahab Ali, Krishna Pandey, Amir Zaidi, S. Bimal, Krishn Pratap Singh, Shashi S. Suman, Asif Equbal, Manas Ranjan Dikhit, Shadab Anwar, Kuljit Singh, Pradeep Das |
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| Rok vydání: | 2015 |
| Předmět: |
Iron-Sulfur Proteins
Models Molecular Scaffold protein Protein Conformation Iron Molecular Sequence Data Drug Resistance Protozoan Proteins Leishmania donovani Respiratory chain Biochemistry Mice Amphotericin B Iron-Binding Proteins Animals Humans Amino Acid Sequence Cloning Molecular Phylogeny Binding Sites biology Iron-binding proteins General Medicine biology.organism_classification Leishmania Up-Regulation Carbon-Sulfur Lyases Protein Transport Frataxin biology.protein Female ISCU Sequence Analysis Biogenesis Protein Binding |
| Zdroj: | Biochimie. 115:120-135 |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/j.biochi.2015.05.016 |
| Popis: | Leishmania donovani is a unicellular protozoon parasite that causes visceral leishmaniasis (VL), which is a fatal disease if left untreated. Certain Fe-S proteins of the TCA cycle and respiratory chain have been found in the Leishmania parasite but the precise mechanisms for their biogenesis and the maturation of Fe-S clusters remains unknown. Fe-S clusters are ubiquitous cofactors of proteins that perform critical cellular functions. The clusters are biosynthesized by the mitochondrial Iron-Sulphur Cluster (ISC) machinery with core protein components that include the catalytic cysteine desulphurase IscS, the scaffold proteins IscU and IscA, and frataxin as an iron carrier/donor. However, no information regarding frataxin, its regulation, or its role in drug resistance is available for the Leishmania parasite. In this study, we characterized Ld-frataxin to investigate its role in the ISC machinery of L. donovani. We expressed and purified the recombinant Ld-frataxin protein and observed its interaction with Ld-IscU by co-purification and pull-down assay. Furthermore, we observed that the cysteine desulphurase activity of the purified Ld-IscS protein was stimulated in the presence of Ld-frataxin and Ld-IscU, particularly in the presence of iron; neither Ld-frataxin nor Ld-IscU alone had significant effects on Ld-IscS activity. Interestingly, RT-PCR and western blotting showed that Ld-frataxin is upregulated in AmpB-resistant isolates compared to sensitive strains, which may support higher Fe-S protein activity in AmpB-resistant L. donovani. Additionally, Ld-frataxin was localized in the mitochondria, as revealed by digitonin fractionation and indirect immunofluorescence. Thus, our results suggest the role of Ld-frataxin as an iron binding/carrier protein for Fe-S cluster biogenesis that physically interacts with other core components of the ISC machinery within the mitochondria. |
| Databáze: | OpenAIRE |
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