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NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death

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Title NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death
 
Creator Mukherjee, Supratim
Sen Santara, Sumit
Das, Shantanabha
Bose, Moumita
Roy, Jayasree
Adak, Subrata
 
Subject Structural Biology & Bioinformatics
 
Description NAD(P)H cytochrome b5 oxidoreductase (Ncb5or), comprising
cytochrome b5 and cytochrome b5 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 b5 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 O2 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 H2O2, indicative
of increased oxidative stress in parasites lacking
LmNcb5or. Cell death is significantly reduced when theKOcells
are pretreated with BSA bound linoleate. Real time PCR studies
demonstrate a higher �12 desaturase, superoxide dismutase,
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
mRNA with a concomitant fall in �9 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
 
Publisher American Society for Biochemistry and Molecular Biology
 
Date 2012
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/1665/1/JBC_2012.pdf
Mukherjee, Supratim and Sen Santara, Sumit and Das, Shantanabha and Bose, Moumita and Roy, Jayasree and Adak, Subrata (2012) NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death. The Journal of Biological Chemistry, 287 (42). pp. 34992-35003.
 
Relation http://dx.doi.org/10.1074/jbc.M112.389338
http://www.eprints.iicb.res.in/1665/