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A mutation of EPT1 ( SELENOI ) underlies a new disorder of Kennedy pathway phospholipid biosynthesis

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ABSTRACT

EPT1 encodes an enzyme involved in the Kennedy pathway of phospholipid biosynthesis, important for cell membrane integrity. Ahmed et al. identify an EPT1 mutation that impairs enzyme activity and causes complex motor neuron degenerative disease. This is the first human disorder shown to arise through Kennedy pathway dysfunction.

No MeSH data available.


Related in: MedlinePlus

Outcomes of the p.Arg112Pro EPT1 sequence alteration. Yeast cells devoid of endogenous ethanolaminephosphotransferase activity were transformed with plasmids bearing wild-type human EPT1 (hEPT1) or EPT1 containing the p.Arg112Pro mutation (hEPT1*) each tagged with a DDK epitope. (A) Schematic representation of the CDP-ethanolamine branch of Kennedy pathway showing the role of EPT1 in PE formation. CK = choline kinase; CPT = choline phosphotransferase; CT = phosphocholine cytidylyltransferase; EK = ethanolamine kinase; ET = phosphoethanolamine cytidylyltransferase; Etn = ethanolamine; LPEAT = lyso-PE acyltransferase; PSD = phosphatidyl serine decarboxylase; PSS = phophatidyl serine synthase. (B) Western blot versus whole cell extracts (WCE), which were fractionated into soluble (S100) and membrane (P100) fractions, and probed using anti-DDK antibodies. Pgk1 and Dpm1 are soluble and membrane fraction loading and fractionation purification controls, respectively. EV = empty vector control. (C) Mid-log phase cells were radiolabelled with 14C-ethanolamine for 1 h. As a positive control yeast strain HJ001 (cpt1::LEU2) transformed with an empty vector was also radiolabelled; this strain possess the wild-type genomic allele of yeast EPT1 (yEPT1). Cells were processed for lipid extraction and the radioactivity associated with PE and PC was determined.
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aww318-F2: Outcomes of the p.Arg112Pro EPT1 sequence alteration. Yeast cells devoid of endogenous ethanolaminephosphotransferase activity were transformed with plasmids bearing wild-type human EPT1 (hEPT1) or EPT1 containing the p.Arg112Pro mutation (hEPT1*) each tagged with a DDK epitope. (A) Schematic representation of the CDP-ethanolamine branch of Kennedy pathway showing the role of EPT1 in PE formation. CK = choline kinase; CPT = choline phosphotransferase; CT = phosphocholine cytidylyltransferase; EK = ethanolamine kinase; ET = phosphoethanolamine cytidylyltransferase; Etn = ethanolamine; LPEAT = lyso-PE acyltransferase; PSD = phosphatidyl serine decarboxylase; PSS = phophatidyl serine synthase. (B) Western blot versus whole cell extracts (WCE), which were fractionated into soluble (S100) and membrane (P100) fractions, and probed using anti-DDK antibodies. Pgk1 and Dpm1 are soluble and membrane fraction loading and fractionation purification controls, respectively. EV = empty vector control. (C) Mid-log phase cells were radiolabelled with 14C-ethanolamine for 1 h. As a positive control yeast strain HJ001 (cpt1::LEU2) transformed with an empty vector was also radiolabelled; this strain possess the wild-type genomic allele of yeast EPT1 (yEPT1). Cells were processed for lipid extraction and the radioactivity associated with PE and PC was determined.

Mentions: To determine the likely pathogenicity of the variant, we next investigated the effect of the p.Arg112Pro mutation on EPT1 activity. SELENOI/EPT1 encodes a CDP-ethanolamine specific enzyme that catalyses the final step in the synthesis of PE via the Kennedy pathway (Fig. 2A) (Horibata and Hirabayashi, 2007). EPT1 belongs to a superfamily of integral membrane phospholipid synthesising enzymes that catalyse displacement of CMP from a CDP-alcohol by a second alcohol with formation of a phosphodiester bond to synthesize a phospholipid. This family of enzymes contains a highly conserved catalytic motif, the CDP-alcohol phosphotransferase motif DG(X2)AR(X8)G(X3)D(X3)D (Williams and McMaster, 1998), which for EPT1 is found between amino acid residues 107–129 (107DGKQAR112RTNSSTPLGELFDHGLD129). As the sequence alteration described here affects the highly conserved arginine residue (p.Arg112Pro) within this CDP-alcohol phosphotransferase motif, we examined whether the alteration affects EPT1 catalytic activity. In order to determine this, human EPT1 and mutant EPT1Arg112Pro were expressed from a constitutive promoter in a S. cerevisiae strain devoid of endogenous ethanolaminephosphotransferase activity, and their capacity to synthesize PE was determined by metabolic labelling studies. Western blots demonstrated that both human SELENOI/EPT1 alleles were expressed in yeast at comparable levels indicating that protein stability was not affected, were associated with the membrane fraction as would be expected for an integral membrane protein, and exhibited their projected molecular weight of 46 kDa (Fig. 2B).Figure 2


A mutation of EPT1 ( SELENOI ) underlies a new disorder of Kennedy pathway phospholipid biosynthesis
Outcomes of the p.Arg112Pro EPT1 sequence alteration. Yeast cells devoid of endogenous ethanolaminephosphotransferase activity were transformed with plasmids bearing wild-type human EPT1 (hEPT1) or EPT1 containing the p.Arg112Pro mutation (hEPT1*) each tagged with a DDK epitope. (A) Schematic representation of the CDP-ethanolamine branch of Kennedy pathway showing the role of EPT1 in PE formation. CK = choline kinase; CPT = choline phosphotransferase; CT = phosphocholine cytidylyltransferase; EK = ethanolamine kinase; ET = phosphoethanolamine cytidylyltransferase; Etn = ethanolamine; LPEAT = lyso-PE acyltransferase; PSD = phosphatidyl serine decarboxylase; PSS = phophatidyl serine synthase. (B) Western blot versus whole cell extracts (WCE), which were fractionated into soluble (S100) and membrane (P100) fractions, and probed using anti-DDK antibodies. Pgk1 and Dpm1 are soluble and membrane fraction loading and fractionation purification controls, respectively. EV = empty vector control. (C) Mid-log phase cells were radiolabelled with 14C-ethanolamine for 1 h. As a positive control yeast strain HJ001 (cpt1::LEU2) transformed with an empty vector was also radiolabelled; this strain possess the wild-type genomic allele of yeast EPT1 (yEPT1). Cells were processed for lipid extraction and the radioactivity associated with PE and PC was determined.
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aww318-F2: Outcomes of the p.Arg112Pro EPT1 sequence alteration. Yeast cells devoid of endogenous ethanolaminephosphotransferase activity were transformed with plasmids bearing wild-type human EPT1 (hEPT1) or EPT1 containing the p.Arg112Pro mutation (hEPT1*) each tagged with a DDK epitope. (A) Schematic representation of the CDP-ethanolamine branch of Kennedy pathway showing the role of EPT1 in PE formation. CK = choline kinase; CPT = choline phosphotransferase; CT = phosphocholine cytidylyltransferase; EK = ethanolamine kinase; ET = phosphoethanolamine cytidylyltransferase; Etn = ethanolamine; LPEAT = lyso-PE acyltransferase; PSD = phosphatidyl serine decarboxylase; PSS = phophatidyl serine synthase. (B) Western blot versus whole cell extracts (WCE), which were fractionated into soluble (S100) and membrane (P100) fractions, and probed using anti-DDK antibodies. Pgk1 and Dpm1 are soluble and membrane fraction loading and fractionation purification controls, respectively. EV = empty vector control. (C) Mid-log phase cells were radiolabelled with 14C-ethanolamine for 1 h. As a positive control yeast strain HJ001 (cpt1::LEU2) transformed with an empty vector was also radiolabelled; this strain possess the wild-type genomic allele of yeast EPT1 (yEPT1). Cells were processed for lipid extraction and the radioactivity associated with PE and PC was determined.
Mentions: To determine the likely pathogenicity of the variant, we next investigated the effect of the p.Arg112Pro mutation on EPT1 activity. SELENOI/EPT1 encodes a CDP-ethanolamine specific enzyme that catalyses the final step in the synthesis of PE via the Kennedy pathway (Fig. 2A) (Horibata and Hirabayashi, 2007). EPT1 belongs to a superfamily of integral membrane phospholipid synthesising enzymes that catalyse displacement of CMP from a CDP-alcohol by a second alcohol with formation of a phosphodiester bond to synthesize a phospholipid. This family of enzymes contains a highly conserved catalytic motif, the CDP-alcohol phosphotransferase motif DG(X2)AR(X8)G(X3)D(X3)D (Williams and McMaster, 1998), which for EPT1 is found between amino acid residues 107–129 (107DGKQAR112RTNSSTPLGELFDHGLD129). As the sequence alteration described here affects the highly conserved arginine residue (p.Arg112Pro) within this CDP-alcohol phosphotransferase motif, we examined whether the alteration affects EPT1 catalytic activity. In order to determine this, human EPT1 and mutant EPT1Arg112Pro were expressed from a constitutive promoter in a S. cerevisiae strain devoid of endogenous ethanolaminephosphotransferase activity, and their capacity to synthesize PE was determined by metabolic labelling studies. Western blots demonstrated that both human SELENOI/EPT1 alleles were expressed in yeast at comparable levels indicating that protein stability was not affected, were associated with the membrane fraction as would be expected for an integral membrane protein, and exhibited their projected molecular weight of 46 kDa (Fig. 2B).Figure 2

View Article: PubMed Central - PubMed

ABSTRACT

EPT1 encodes an enzyme involved in the Kennedy pathway of phospholipid biosynthesis, important for cell membrane integrity. Ahmed et al. identify an EPT1 mutation that impairs enzyme activity and causes complex motor neuron degenerative disease. This is the first human disorder shown to arise through Kennedy pathway dysfunction.

No MeSH data available.


Related in: MedlinePlus