Biochemical characterization of human dihydroxyacetone phosphate acyltransferase, the first step in ether lipid biosynthesis
Date
2024-09-11
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Abstract
Ether lipids are a subclass of glycerophospholipids where an alkyl tail is attached to the sn-1 position of the glycerol backbone. They are found enriched in the central nervous system. Defects in ether lipid synthesis lead to severe human conditions including Zellweger syndrome and rhizomelic chrondrodysplasia punctata. The latter manifests with congenital cataracts, skeletal abnormalities, facial deformities, and high childhood mortality, underlining the critical role of the ether lipid biosynthesis. The first acylation step in the synthesis of ether lipids occurs in peroxisomes and is catalyzed by the enzyme dihydroxyacetonephosphate acyltransferase (DHAPAT EC2.3.1.42). DHAPAT has a peroxisomal targeting signal type 1 (PTS1) at the C-terminus, lacks predicted membrane domains or lipid binding motifs. A functional assay in yeast was devised where full length DHAPAT was able to support life of a double knockout yeast devoid of endogenous acyltransferases. This was dependent on DHAPAT activity and the presence of the residues encoded by the first three exons. Despite comparable expression levels to the wild type, a catalytically dead mutant failed to support life of the knockout yeast. Furthermore, an alternatively spliced variant lacking exon 2 (V2) also resulted in the inability to support life of double knockout yeast. Unexpectedly, full length DHAPAT localizes to both peroxisomes and lipid droplets (LDs) in yeast, while V2 localizes exclusively to peroxisomes. Interestingly, putative amphipathic helices (AHs) were identified within the region encoded by exons 2 and 3 which may aid in association with LDs. A systematic series of truncations in the first 146 amino acids revealed that the minimum sequence required for LD association relies in the region encoded by exon 3. A colorimetric DHAPAT activity assay was developed and validated by detecting activity of the full-length enzyme but not of the catalytically dead H162A mutant. Consistent with its failure to support the life of the double knockout yeast, no activity was detected for V2, at least in its soluble form. Insights from in silico modelling and non-reducing SDS-PAGE analysis support a role of the region encoded by exons 1-3 in dimerization of the enzyme. Together, these results indicate that the amino end of DHAPAT modulates its targeting priority to peroxisomes when the PTS1 signal is present and regulates its activity.
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Keywords
Lipid metabolism, Ether lipids, Dihydroxyacetone phosphate acyltransferase, DHAPAT, GNPAT, GPAT, Plasmalogen, Peroxisomes, Lipid droplets, Yeast, Glycerophospholipids
Citation
Li, V. (2024). Biochemical characterization of human dihydroxyacetone phosphate acyltransferase, the first step in ether lipid biosynthesis (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.