title: Leishmania donovani eukaryotic initiation factor 5A: molecular characterization, localization and homology modelling studies.
abstract: Eukaryotic translation initiation factor 5A (eIF5A) is a small acidic protein highly conserved from archaea to mammals. eIF5A is the only protein which undergoes a unique lysine residue modification to hypusine. Hypusinylation is important for the function of eIF5A which is reported to be essential for cell viability. eIF5A promotes formation of the first peptide bond at the onset of protein synthesis. However, its function in Leishmania donovani is unclear. The present study focuses on the characterization and localization of L. donovani eIF5A protein. The eIF5A gene contains an ORF of 501×bp encoding 166 amino acid residues with a predicted molecular mass and isoelectric point of 17.8 kDa and 4.83 respectively. A phylogenetic tree analysis revealed its close proximity to trypanosomes however it is distantly located from Trichomonas vaginalis and Plasmodium falciparum. The L. donovani eIF5A was expressed as a 6× His tagged protein whose identity was confirmed by western blot and MALDI. Biophysical investigation by CD revealed the predominant presence of 49% β sheet structure which correlated well with secondary structure prediction. To gain insight into the role of eIF5A in L. donovani, we investigated the subcellular distribution of eIF5A. A GFP-fusion of L. donovani eIF5A was found to be localized in cytoplasm as confirmed by subcellular fractionation. Our studies indicated that eIF5A is primarily localized to cytoplasm and is undetectable in nuclear fraction. The homology model of eIF5A of L. donovani was built and the resulting model showed acceptable Ramachandran statistics. The model is reliable and can be used to study eIF5A binding with its effector molecules.
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India. Electronic address: sushmasingh@niper.ac.in.
authors.forename: Sushma
authors.initials: S
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: K
authors.initials: K
authors.lastname: Raju
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Deepika
authors.initials: D
authors.lastname: Jatekar
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Mohali 160062, Punjab, India.
authors.forename: Neeradi
authors.initials: N
authors.lastname: Dinesh
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar Mohali 160062, Punjab, India.
authors.forename: M Stanley
authors.initials: MS
authors.lastname: Paul
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar Mohali 160062, Punjab, India.
authors.forename: M E
authors.initials: ME
authors.lastname: Sobhia
mesh_terms: D002467:Cell Nucleus / Q000737:chemistry; D002942:Circular Dichroism; D003593:Cytoplasm / Q000737:chemistry; D007526:Isoelectric Point; D007893:Leishmania donovani / Q000201:enzymology*; D008958:Models, Molecular; D008970:Molecular Weight; D016366:Open Reading Frames; D010448:Peptide Initiation Factors / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D010802:Phylogeny; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry; D000097574:Eukaryotic Translation Initiation Factor 5A
chemical_list: D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A
keywords: Homology model; Hypusine; Localization; MALDI; eIF5A