title: Interaction of frataxin, an iron binding protein, with IscU of Fe-S clusters biogenesis pathway and its upregulation in AmpB resistant Leishmania donovani.
abstract: 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.
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India.
authors.forename: Amir
authors.initials: A
authors.lastname: Zaidi
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India.
authors.forename: Krishn Pratap
authors.initials: KP
authors.lastname: Singh
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India.
authors.forename: Shadab
authors.initials: S
authors.lastname: Anwar
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India.
authors.forename: Shashi S
authors.initials: SS
authors.lastname: Suman
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India.
authors.forename: Asif
authors.initials: A
authors.lastname: Equbal
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India.
authors.forename: Kuljit
authors.initials: K
authors.lastname: Singh
authors.affiliation: Biomedical Informatic Centre, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna, India.
authors.forename: Manas R
authors.initials: MR
authors.lastname: Dikhit
authors.affiliation: Department of Immunology, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna, India.
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Bimal
authors.affiliation: Department of Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna, India.
authors.forename: Krishna
authors.initials: K
authors.lastname: Pandey
authors.affiliation: Department of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna, India.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
authors.affiliation: Laboratory of Molecular Biochemistry and Cell Biology, Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Agamkuan, Patna 800007, India. Electronic address: vahab_ali@yahoo.com.
authors.forename: Vahab
authors.initials: V
authors.lastname: Ali
mesh_terms: D000595:Amino Acid Sequence; D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000378:metabolism; D003001:Cloning, Molecular; D004351:Drug Resistance; D005260:Female; D006801:Humans; D007501:Iron / Q000378:metabolism; D033862:Iron-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D007893:Leishmania donovani / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D015854:Up-Regulation; D000096923:Frataxin
chemical_list: D000666:Amphotericin B; D013437:Carbon-Sulfur Lyases; D007501:Iron; D033862:Iron-Binding Proteins; D007506:Iron-Sulfur Proteins; D015800:Protozoan Proteins; D000096923:Frataxin; C080546:cysteine desulfurase
keywords: Frataxin; ISC machinery; Ld-frataxin; Leishmania donovani; Protein–protein interaction; [Fe–S] clusters