title: NAD(P)H cytochrome b5 oxidoreductase deficiency in Leishmania major results in impaired linoleate synthesis followed by increased oxidative stress and cell death.
abstract: NAD(P)H cytochrome b(5) oxidoreductase (Ncb5or), comprising cytochrome b(5) and cytochrome b(5) reductase domains, is widely distributed in eukaryotic organisms. Although Ncb5or plays a crucial role in lipid metabolism of mice, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed, and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies show that NADH can quickly reduce the ferric state of the enzyme to the ferrous state and is able to donate an electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create NAD(P)H cytochrome b(5) oxidoreductase from L. major (LmNcb5or) knock-out mutants by targeted gene replacement technique. A free fatty acid profile in knock-out (KO) cells reveals marked deficiency in linoleate and linolenate when compared with wild type (WT) or overexpressing cells. KO culture has a higher percentage of dead cells compared with both WT and overexpressing cells. Increased O(2) uptake, uncoupling and ATP synthesis, and loss of mitochondrial membrane potential are evident in KO cells. Flow cytometric analysis reveals the presence of a higher concentration of intracellular H(2)O(2), indicative of increased oxidative stress in parasites lacking LmNcb5or. Cell death is significantly reduced when the KO cells are pretreated with BSA bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. Together these findings suggest that decreased linoleate synthesis, and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania.
authors.affiliation: Division of Structural Biology and Bioinformatics, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
authors.forename: Supratim
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: Division of Structural Biology and Bioinformatics, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
authors.forename: Sumit
authors.initials: S
authors.lastname: Sen Santara
authors.affiliation: Division of Structural Biology and Bioinformatics, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
authors.forename: Shantanabha
authors.initials: S
authors.lastname: Das
authors.affiliation: Division of Structural Biology and Bioinformatics, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
authors.forename: Moumita
authors.initials: M
authors.lastname: Bose
authors.affiliation: Division of Structural Biology and Bioinformatics, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India.
authors.forename: Jayasree
authors.initials: J
authors.lastname: Roy
authors.affiliation: Division of Structural Biology and Bioinformatics, Council of Scientific and Industrial Research, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata 700 032, India. Electronic address: adaks@iicb.res.in.
authors.forename: Subrata
authors.initials: S
authors.lastname: Adak
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D042966:Cytochrome-B(5) Reductase / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D055785:Gene Knockdown Techniques; D018320:Leishmania major / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D019787:Linoleic Acid / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D018384:Oxidative Stress / Q000502:physiology*; D010100:Oxygen; D010101:Oxygen Consumption / Q000502:physiology; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D016053:RNA, Protozoan / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D019787:Linoleic Acid; D042966:Cytochrome-B(5) Reductase; D010100:Oxygen