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In rat hepatocytes, myristic acid occurs through lipogenesis, palmitic acid shortening and lauric acid elongation
Abstracts / Chemistry and Physics of Lipids 149S (2007) S56–S73
PO 104
PO 105
Exploring the role of Sfh1p
A novel acyltransferase identifies a new fatty acid conjugation pathway in peroxisomes
Katarina Poloncova, Roman Holic Institute of Animal Biochemistry and Genetics, Slovak Akademy of Sciences, Slovakia Sfh1p is one of the proteins whose function is not known to date. It is more often mentioned in connection with essential Sec14p. It is because Sfh1p and Sec14p share about 64% primary sequence homology and thus they are close homologues. Despite this fact, Sfh1p fails to substitute efficiently for sec14∆ mutation. This can be caused by different subcellular localization of these two proteins. While Sec14p is localized mainly in the cytosol and in the membranes of Golgi apparatus, Sfh1p is localized mainly in the nucleus. To test this hypothesis, we decided to prepare Sfh1p with altered cellular localization. For this purpose, hybrid proteins between Sec14p and Sfh1p were constructed. Localization of these proteins was established by their fusion with GFP on C-terminus. We tested the ability of the hybrid proteins to repair growth defects of the sec14ts mutant strain in non-permissive temperature, as well as the ability to regulate phospholipase D1 mediated turnover of PC and the ability to transfer PC in vitro. The degree to which the hybrid proteins were able to substitute for Sec14p was affected by the ratio of Sec14p and Sfh1p. Although, Sfh1p is a close homologue of Sec14p its nuclear localization clearly suggests that this protein has a role of its own in the cell. Phenotype of a mutant strain often hints at the role of the protein in the cell. Although, the sfh1∆ strain was tested on a various substrates and on wide range of antimycotics, no phenotype was detected. In our approach, we decided to use data from DNA expression analysis, which shown increased SFH1 expression under various stress conditions like temperature changes, high osmolarity, heavy metal, increased salt concentrations and DNA damaging agents. We tested these stress factors and their combinations and were able to detect altered sensitivity to several tested stress factors. doi:10.1016/j.chemphyslip.2007.06.154
S69
Sarah-Jayne Reilly, Ethna O’Shea, Stefan E.H. Alexson, Mary C. Hunt Department of Laboratory Medicine, Karolinska Institutet, Sweden A wide variety of endogenous carboxylic acids are conjugated with amino acids in reactions catalyzed by different acyltransferase enzymes and this includes endogenous compounds like bile acids and fatty acids. The bile acids are amidated (or conjugated) to either glycine or taurine by an enzyme named bile acid-CoA:amino acid N-acyltransferase (BACAT). We have now identified a novel peroxisomal enzyme that shows 55% sequence identity to BACAT at the amino acid level and we hypothesised that this enzyme would function as an acyltransferase. We have named this enzyme acylCoA:amino acid N-acyltransferase 1 (ACNAT1) and confirmed it is localised in peroxisomes using green fluorescent protein studies. Recombinant ACNAT1 efficiently conjugates very long-chain and long-chain fatty acids to taurine, producing metabolites known as Nacyltaurines (NATs). Acnat1 showed highest tissue expression in liver and kidney, where these NATs have very recently been identified. Therefore, ACNAT1, in the conjugation of fatty acids to taurine may be involved in the removal of fatty acids as NATs from peroxisomes, especially under conditions where they are toxic. doi:10.1016/j.chemphyslip.2007.06.155 PO 106
In rat hepatocytes, myristic acid occurs through lipogenesis, palmitic acid shortening and lauric acid elongation Vincent Rioux, Erwan Beauchamp, Fr´ed´erique P´edrono, Daniel Catheline, Philippe Legrand Laboratoire de Biochimie, Agrocampus-INRA Rennes, France Because of its negative effects on cholesterol metabolism, at high levels, myristic acid (C14:0) has been studied when provided in the diets of animals and humans, and when added to cultured cells. Data have also been obtained by studying its specific involvement in protein N-myristoylation and its positive effect on (n − 3) polyunsaturated fatty acid bioavailability. However, none of these studies has determined the predominant origin of C14:0 in mammalian cells. Another
S70
Abstracts / Chemistry and Physics of Lipids 149S (2007) S56–S73
intriguing question is the potential metabolic properties of endogenous C14:0 as compared to exogenous C14:0. Myristic acid usually accounts for less than 1% of fatty acids in mammalian cells. Comparatively, it is particularly abundant (7–14% of FAs) in milk fat. In rat mammary glands, this high level has been related to the presence of a cytosolic thioesterase II that releases FAs shorter than the predominant palmitic acid usually produced by the fatty acid synthase complex (FAS), which is associated with thioesterase I. In tissues other than the mammary gland, however, the origin of C14:0 and the regulation of its low concentration are not known. In this study, we demonstrate that, in liver cells, in addition to the usual FAS pathway that produces predominantly palmitic acid and minor amounts of myristic acid, part of cellular C14:0 also comes from a shortening of palmitic acid by peroxisomal -oxidation and from lauric acid by elongation. From a nutritional point of view, C16:0 is universally found in natural fats and its shortening to myristic acid could contribute to a non-negligible source of this fatty acid. Then, we measured the distribution of endogenously synthesized myristic acid in lipid species and compared it with that of exogenous myristic acid. Our results do not support the hypothesis of different metabolic fates of endogenous and exogenous myristic acid, and suggest that whatever the origin of C14:0, its cellular concentration and lipid distribution are highly regulated. doi:10.1016/j.chemphyslip.2007.06.156 PO 107
Expression of active forms of endoglycoceramidase II and its activator protein in Rhodococcus erythropolis Keishi Sakaguchi 1 , Tomohiro Tamura 2 , Isao Tanaka 3 , Nozomu Okino 1 , Makoto Ito 4 1 Department
of Bioscience and Biotechnology, Kyushu University, Japan; 2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, Japan; 3 Division of Biological Sciences, Hokkaido University, Japan; 4 Department of Bioscience and Biotechnology, Kyushu University Bio-Architecture Center, Kyushu University, Japan Endoglycoceramidase II (EGCase II, EC 3.2.1.123), an enzyme capable of degrading various glycosphingolipids (GSLs), was found in Rhodococcus equi. To express enzyme activity in vitro, EGCase II requires detergents, possibly due to the hydrophobic nature of
both substrates and the enzyme. Interestingly, EGCase activity is stimulated by activator protein (ACT) in the absence of detergents, which was found to be produced simultaneously by R. equi. Remarkably, with the assistance of ACT, native EGCase II can hydrolyze cellsurface GSLs in physiological conditions. Thus, native EGCase II and ACT are valuable for elucidating the biological functions of cell-surface GSLs; however, rACT expressed in E. coli was found to be almost inactive. Similarly, rEGCase II expressed in E. coli was not able to hydrolyze cell-surface GSLs in the presence of native ACT, although the recombinant enzyme is fully active in vitro in the presence of detergents. Thus, we tried to express the recombinant forms of EGCase II and ACT using a new expression system with Rhodococcus erythropolis as the host cells, which are closely related to R. equi. rACT was further converted to a 27.9 kDa form possessing full activity by treatment with trypsin. It was found that rEGCase II can hydrolyze cell-surface GM3 of horse erythrocytes in the presence of 27.9 kDa rACT. Furthermore, we found that the N-terminal of native and recombinant Rhodococcus EGCase II was modified while rEGCase II expressed in E. coli was not, suggesting that modification of the N-terminus of the enzyme is responsible for the activity toward GSLs of intact cells but not GSL in detergent micelles. This result may indicate that N-terminal modification affects the interaction of the enzyme with intact cell membranes. In summary, we successfully prepared fully active forms of recombinant EGCase II as well as ACT when they are expressed in R. erythropolis. doi:10.1016/j.chemphyslip.2007.06.157 PO 108
Oxidized LDL promotes, similar to the cationic amphiphiles and NPC1-mimetic U-18666A, endolysosomal phospholipidosis with extension of cell surface ceramide rafts Gerd Schmitz, Margot Grandl Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany Although the in vitro atherogenic property of oxidized LDL (oxLDL) is well documented, the cellular processing of ingested oxLDL is still rudimentary investigated. Monocyte-derived macrophages from human apolipoprotein E3/E3 homozygous donors were incubated with two atherogenic lipoproteins, oxLDL and enzymatic-modified LDL (eLDL). The impaired lysosomal degradation of oxLDL load in macrophages leads to a higher induction of ABCA1/ABCG1 mRNA and
PO 104
PO 105
Exploring the role of Sfh1p
A novel acyltransferase identifies a new fatty acid conjugation pathway in peroxisomes
Katarina Poloncova, Roman Holic Institute of Animal Biochemistry and Genetics, Slovak Akademy of Sciences, Slovakia Sfh1p is one of the proteins whose function is not known to date. It is more often mentioned in connection with essential Sec14p. It is because Sfh1p and Sec14p share about 64% primary sequence homology and thus they are close homologues. Despite this fact, Sfh1p fails to substitute efficiently for sec14∆ mutation. This can be caused by different subcellular localization of these two proteins. While Sec14p is localized mainly in the cytosol and in the membranes of Golgi apparatus, Sfh1p is localized mainly in the nucleus. To test this hypothesis, we decided to prepare Sfh1p with altered cellular localization. For this purpose, hybrid proteins between Sec14p and Sfh1p were constructed. Localization of these proteins was established by their fusion with GFP on C-terminus. We tested the ability of the hybrid proteins to repair growth defects of the sec14ts mutant strain in non-permissive temperature, as well as the ability to regulate phospholipase D1 mediated turnover of PC and the ability to transfer PC in vitro. The degree to which the hybrid proteins were able to substitute for Sec14p was affected by the ratio of Sec14p and Sfh1p. Although, Sfh1p is a close homologue of Sec14p its nuclear localization clearly suggests that this protein has a role of its own in the cell. Phenotype of a mutant strain often hints at the role of the protein in the cell. Although, the sfh1∆ strain was tested on a various substrates and on wide range of antimycotics, no phenotype was detected. In our approach, we decided to use data from DNA expression analysis, which shown increased SFH1 expression under various stress conditions like temperature changes, high osmolarity, heavy metal, increased salt concentrations and DNA damaging agents. We tested these stress factors and their combinations and were able to detect altered sensitivity to several tested stress factors. doi:10.1016/j.chemphyslip.2007.06.154
S69
Sarah-Jayne Reilly, Ethna O’Shea, Stefan E.H. Alexson, Mary C. Hunt Department of Laboratory Medicine, Karolinska Institutet, Sweden A wide variety of endogenous carboxylic acids are conjugated with amino acids in reactions catalyzed by different acyltransferase enzymes and this includes endogenous compounds like bile acids and fatty acids. The bile acids are amidated (or conjugated) to either glycine or taurine by an enzyme named bile acid-CoA:amino acid N-acyltransferase (BACAT). We have now identified a novel peroxisomal enzyme that shows 55% sequence identity to BACAT at the amino acid level and we hypothesised that this enzyme would function as an acyltransferase. We have named this enzyme acylCoA:amino acid N-acyltransferase 1 (ACNAT1) and confirmed it is localised in peroxisomes using green fluorescent protein studies. Recombinant ACNAT1 efficiently conjugates very long-chain and long-chain fatty acids to taurine, producing metabolites known as Nacyltaurines (NATs). Acnat1 showed highest tissue expression in liver and kidney, where these NATs have very recently been identified. Therefore, ACNAT1, in the conjugation of fatty acids to taurine may be involved in the removal of fatty acids as NATs from peroxisomes, especially under conditions where they are toxic. doi:10.1016/j.chemphyslip.2007.06.155 PO 106
In rat hepatocytes, myristic acid occurs through lipogenesis, palmitic acid shortening and lauric acid elongation Vincent Rioux, Erwan Beauchamp, Fr´ed´erique P´edrono, Daniel Catheline, Philippe Legrand Laboratoire de Biochimie, Agrocampus-INRA Rennes, France Because of its negative effects on cholesterol metabolism, at high levels, myristic acid (C14:0) has been studied when provided in the diets of animals and humans, and when added to cultured cells. Data have also been obtained by studying its specific involvement in protein N-myristoylation and its positive effect on (n − 3) polyunsaturated fatty acid bioavailability. However, none of these studies has determined the predominant origin of C14:0 in mammalian cells. Another
S70
Abstracts / Chemistry and Physics of Lipids 149S (2007) S56–S73
intriguing question is the potential metabolic properties of endogenous C14:0 as compared to exogenous C14:0. Myristic acid usually accounts for less than 1% of fatty acids in mammalian cells. Comparatively, it is particularly abundant (7–14% of FAs) in milk fat. In rat mammary glands, this high level has been related to the presence of a cytosolic thioesterase II that releases FAs shorter than the predominant palmitic acid usually produced by the fatty acid synthase complex (FAS), which is associated with thioesterase I. In tissues other than the mammary gland, however, the origin of C14:0 and the regulation of its low concentration are not known. In this study, we demonstrate that, in liver cells, in addition to the usual FAS pathway that produces predominantly palmitic acid and minor amounts of myristic acid, part of cellular C14:0 also comes from a shortening of palmitic acid by peroxisomal -oxidation and from lauric acid by elongation. From a nutritional point of view, C16:0 is universally found in natural fats and its shortening to myristic acid could contribute to a non-negligible source of this fatty acid. Then, we measured the distribution of endogenously synthesized myristic acid in lipid species and compared it with that of exogenous myristic acid. Our results do not support the hypothesis of different metabolic fates of endogenous and exogenous myristic acid, and suggest that whatever the origin of C14:0, its cellular concentration and lipid distribution are highly regulated. doi:10.1016/j.chemphyslip.2007.06.156 PO 107
Expression of active forms of endoglycoceramidase II and its activator protein in Rhodococcus erythropolis Keishi Sakaguchi 1 , Tomohiro Tamura 2 , Isao Tanaka 3 , Nozomu Okino 1 , Makoto Ito 4 1 Department
of Bioscience and Biotechnology, Kyushu University, Japan; 2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, Japan; 3 Division of Biological Sciences, Hokkaido University, Japan; 4 Department of Bioscience and Biotechnology, Kyushu University Bio-Architecture Center, Kyushu University, Japan Endoglycoceramidase II (EGCase II, EC 3.2.1.123), an enzyme capable of degrading various glycosphingolipids (GSLs), was found in Rhodococcus equi. To express enzyme activity in vitro, EGCase II requires detergents, possibly due to the hydrophobic nature of
both substrates and the enzyme. Interestingly, EGCase activity is stimulated by activator protein (ACT) in the absence of detergents, which was found to be produced simultaneously by R. equi. Remarkably, with the assistance of ACT, native EGCase II can hydrolyze cellsurface GSLs in physiological conditions. Thus, native EGCase II and ACT are valuable for elucidating the biological functions of cell-surface GSLs; however, rACT expressed in E. coli was found to be almost inactive. Similarly, rEGCase II expressed in E. coli was not able to hydrolyze cell-surface GSLs in the presence of native ACT, although the recombinant enzyme is fully active in vitro in the presence of detergents. Thus, we tried to express the recombinant forms of EGCase II and ACT using a new expression system with Rhodococcus erythropolis as the host cells, which are closely related to R. equi. rACT was further converted to a 27.9 kDa form possessing full activity by treatment with trypsin. It was found that rEGCase II can hydrolyze cell-surface GM3 of horse erythrocytes in the presence of 27.9 kDa rACT. Furthermore, we found that the N-terminal of native and recombinant Rhodococcus EGCase II was modified while rEGCase II expressed in E. coli was not, suggesting that modification of the N-terminus of the enzyme is responsible for the activity toward GSLs of intact cells but not GSL in detergent micelles. This result may indicate that N-terminal modification affects the interaction of the enzyme with intact cell membranes. In summary, we successfully prepared fully active forms of recombinant EGCase II as well as ACT when they are expressed in R. erythropolis. doi:10.1016/j.chemphyslip.2007.06.157 PO 108
Oxidized LDL promotes, similar to the cationic amphiphiles and NPC1-mimetic U-18666A, endolysosomal phospholipidosis with extension of cell surface ceramide rafts Gerd Schmitz, Margot Grandl Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany Although the in vitro atherogenic property of oxidized LDL (oxLDL) is well documented, the cellular processing of ingested oxLDL is still rudimentary investigated. Monocyte-derived macrophages from human apolipoprotein E3/E3 homozygous donors were incubated with two atherogenic lipoproteins, oxLDL and enzymatic-modified LDL (eLDL). The impaired lysosomal degradation of oxLDL load in macrophages leads to a higher induction of ABCA1/ABCG1 mRNA and