Abstract
Fatty acid desaturases are rate-limiting enzymes in long-chain polyunsaturated fatty acid biosynthesis. The transcription factor peroxisome proliferator-activated receptor alpha b (PPARαb) regulates lipid metabolism in mammals, however, the mechanism whereby PPARαb regulates fatty acid desaturases is largely unknown in fish. In this study, we report the full length cDNA sequence of Trachinotus ovatus fatty acid desaturase, which encodes a 380 amino acid polypeptide, possessing three characteristic histidine domains. Phylogenetic and gene exon/intron structure analyses showed typical phylogeny: the T. ovatus fatty acid desaturase contained a highly conserved exon/intron architecture. Moreover, functional characterization by heterologous expression in yeast indicated that T. ovatus desaturase was a fatty acid desaturase, with Δ4/Δ5/Δ8 Fad activity. Promoter activity assays indicated that ToFads6 desaturase transcription was positively regulated by PPARαb. Similarly, PPARαb RNA interference decreased ToPPARαb and ToFads6 expression at the mRNA and protein levels in a time-dependent manner. Mutation analyses showed that the M2 binding site of PPARαb was functionally important for protein binding, and transcriptional activity of the ToFads6 promoter was significantly decreased after targeted mutation of M2. Electrophoretic mobile shift assays confirmed that PPARαb interacted with the binding site of the ToFads6 promoter region, to regulate ToFads6 transcription. In summary, PPARαb played a vital role in ToFads6 regulation and may promote the biosynthesis of long-chain polyunsaturated fatty acids by regulating ToFads6 expression.
Highlights
Long-chain polyunsaturated fatty acids (LC-PUFA) are involved in numerous biological processes and are major components of complex lipid molecules [1]
Previous studies using yeast heterologous expression systems have indicated that Fads2 exhibits ∆6, ∆5, ∆4, and ∆8 activity in the biosynthetic pathway of LC-PUFAs [1,4,7,8,9,10,11]; little information on Fads6 has been reported
The function of marine fish Fads6 appears ambiguous during LC-PUFA biosynthesis
Summary
Long-chain polyunsaturated fatty acids (LC-PUFA) are involved in numerous biological processes and are major components of complex lipid molecules [1]. Lipid metabolism, including the inhibition of lipogenesis and the activation of fatty acid oxidation, is regulated by PUFA and their metabolites, along with transcription factors such as PPAR, hepatocyte nuclear factor-4 (HNF4), nuclear receptor subfamily 1 group H member 3 (NR1H3), sterol-regulatory element binding protein (SREBP), and nuclear factor-kappa B (NF-κ B) [17]. To investigate the underlying function of ToFads and the transcriptional regulation of PPARα during LC-PUFA biosynthesis, this study sought to clarify the importance of PPARαb in regulating Fads transcriptional activity. The role of the PPARαb M2 binding site in the Tofads promoter was investigated using the electrophoretic mobility shift assay (EMSA) These approaches helped identify Fad function in marine fish, and showed that PPARαb performed a vital function in Fads expression regulation
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