New insights into the regulation of steroidogenesis: P450 side-chain cleavage enzyme (Cyp11a2) and ferredoxin (Fdx1b) specifically regulate interrenal steroid synthesis in zebrafish

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 358-380-Steroid Hormone Biosynthesis & Metabolism
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-380
Aliesha Griffin*, Silvia Parajes, Angela E Taylor, Cedric H Shackleton, Ferenc Müller and Nils Krone
University of Birmingham, Birmingham, United Kingdom
Cytochrome P450 (CYP) enzymes rely on redox cofactors for their catalytic activity. The mitochondrial side-chain cleavage enzyme (CYP11A1) is dependent on electron transfer from ferredoxin (FDX1) to convert cholesterol to pregnenolone in the first and rate limiting step of steroidogenesis. In vitro data suggests redox cofactors can modulate CYP enzyme activity, and are likely crucial to the modulation of steroidogenic capacity. The mechanistic in vivo consequences are unknown. Therefore, we characterised the function and specificity of zebrafish Fdx and Cyp11a to establish the impact of redox regulation on steroidogenesis in vivo. In contrast to humans, zebrafish have two FDX1 genes, fdx1 and fdx1b. Our studies show fdx1 has maternal expression which is maintained during development. Expression of its paralog, fdx1b begins at 24 hours post-fertilisation. Importantly, this mirrors the expression of zebrafish P450 side-chain cleavage enzyme, cyp11a2, and coincides with the development of the zebrafish interrenal (the mammalian adrenal counterpart). While fdx1 is ubiquitously expressed in adult zebrafish, fdx1b expression is restricted to the main steroidogenic tissues: interrenal, gonads and brain. We also identified the same tissue expression for cyp11a2. This suggests Fdx1b is the redox partner of Cyp11a2 and together they regulate interrenal steroidogenesis in zebrafish. The in vivo function of the Fdx paralogs and Cyp11a2 was investigated using morpholino knockdown. Loss of Fdx1 showed early morphological defects during zebrafish development. Alternatively, Fdx1b morphants developed late onset metabolic abnormalities, consisting of an enlarged yolk and delayed inflation of their swim bladder. A similar phenotype was observed in our Cyp11a2 morphants suggesting Fdx1b can modify Cyp11a2 activity in vivo. Steroid hormone measurements during development by liquid chromatography tandem mass spectrometry confirmed that Fdx1b is vital for interrenal steroid synthesis; despite that co-expression of Fdx1 was observed. Likewise, transient loss of Cyp11a2 also prevented de novo steroidogenesis during development. We identified definite roles for the zebrafish Fdx paralogs. Specifically that Fdx1b is the main redox partner of Cyp11a2 and they regulate steroid synthesis in zebrafish. This study gives insights into mechanisms of mitochondrial redox regulation and establishes zebrafish as a model to study inborn errors of adrenal steroidogenesis and adrenal physiology.

Nothing to Disclose: AG, SP, AET, CHS, FM, NK

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