Characterization of NAD(P)H Cytochrome b5 Oxidoreductase in Leishmania Major
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
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| Title |
Characterization of NAD(P)H Cytochrome b5 Oxidoreductase in
Leishmania Major
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| Creator |
Mukherjee, Supratim
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| Subject |
Structural Biology & Bioinformatics
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| Description |
NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) belongs to the cytochrome b5 family of flavohemoprotein that is widely expressed in eukaryotic organisms. Although, Ncb5or is an endoplasmic reticulum-bound protein that has been implicated in lipid metabolism and diabetes, 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 showed that NADH can quickly reduce the ferric state of enzyme to ferrous state and able to donate electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create LmNcb5or knock out (KO) mutants by targeted gene replacement technique. Free fatty acid profile in knock out cells revealed marked deficiency in linoleate and linolenate when compared to wild type (WT) or overexpressing (OE) cells. We have shown that Ncb5or deletion results in dramatic increments in O2 consumption, ROS generation, ATP production, and apoptosis in Leishmania. Wild type characteristics are restored in KO cells on pre-treatment with BSA-bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase and GAPDH mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. We report LmNcb5or to be the redox partner of Δ12 fatty acid desaturase in linoleate biosynthesis of Leishmania and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmania
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| Date |
2013
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| Type |
Thesis
NonPeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/1872/1/thesis.pdf
Mukherjee, Supratim (2013) Characterization of NAD(P)H Cytochrome b5 Oxidoreductase in Leishmania Major. PhD thesis, Jadavpur University. |
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| Relation |
http://www.eprints.iicb.res.in/1872/
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