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Genetic manipulation of palmitoylethanolamide production and inactivation in Saccharomyces cerevisiae.

Muccioli GG, Sia A, Muchowski PJ, Stella N - PLoS ONE (2009)

Bottom Line: Lipids can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions.The signaling lipid N-palmitoylethanolamine (PEA) is known to activate intracellular and membrane-associated receptors and regulate physiological functions, but little is known about the enzymes involved in its production and inactivation.Accordingly, using single gene deletion mutants, we identified yeast genes that control PEA metabolism, including SPO14 (a yeast homologue of the mammalian phospholipase D) that controls PEA production and YJU3 (a yeast homologue of the mammalian monoacylglycerol lipase) that controls PEA inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Lipids can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. To understand how a newly discovered signaling lipid functions, it is necessary to identify and characterize the enzymes involved in their production and inactivation. The signaling lipid N-palmitoylethanolamine (PEA) is known to activate intracellular and membrane-associated receptors and regulate physiological functions, but little is known about the enzymes involved in its production and inactivation.

Principal findings: Here we show that Saccharomyces cerevisiae produce and inactivate PEA, suggesting that genetic manipulations of this lower eukaryote may be used to identify the enzymes involved in PEA metabolism. Accordingly, using single gene deletion mutants, we identified yeast genes that control PEA metabolism, including SPO14 (a yeast homologue of the mammalian phospholipase D) that controls PEA production and YJU3 (a yeast homologue of the mammalian monoacylglycerol lipase) that controls PEA inactivation. We also found that PEA metabolism is affected by heterologous expression of two mammalian proteins involved in neurodegenerative diseases, namely huntingtin and alpha-synuclein.

Significance: Together these findings show that forward and reverse genetics in S. cerevisiae can be used to identify proteins involved in PEA production and inactivation, and suggest that mutated proteins causing neurodegenerative diseases might affect the metabolism of this important signaling lipid.

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Related in: MedlinePlus

Htt fragments and α-synuclein reduce PEA hydrolysis in yeast.[3H]-PEA hydrolysis measured in BY4741 yeast strains expressing a 25Q and 103Q huntingtin fragment and in yeast strain expressing α-synuclein (BY4741) were compared to BY4741 strains expressing an empty vector (dotted line). Similarly [3H]-PEA hydrolysis found in YJU3 gene-deleted yeast strains expressing the same proteins (yju3Δ) were compared to yju3Δ strains expressing an empty vector (dotted line) (A–C). Values are the mean±SEM of three to five experiments performed in duplicate and are expressed as percentage of corresponding control (empty vector expressing BY4741 strain and empty vector expressing yju3Δ strain, respectively). ** P<0.05 and ** P<0.01 compared to respective control (unpaired two-tailed Student's t-test).
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pone-0005942-g007: Htt fragments and α-synuclein reduce PEA hydrolysis in yeast.[3H]-PEA hydrolysis measured in BY4741 yeast strains expressing a 25Q and 103Q huntingtin fragment and in yeast strain expressing α-synuclein (BY4741) were compared to BY4741 strains expressing an empty vector (dotted line). Similarly [3H]-PEA hydrolysis found in YJU3 gene-deleted yeast strains expressing the same proteins (yju3Δ) were compared to yju3Δ strains expressing an empty vector (dotted line) (A–C). Values are the mean±SEM of three to five experiments performed in duplicate and are expressed as percentage of corresponding control (empty vector expressing BY4741 strain and empty vector expressing yju3Δ strain, respectively). ** P<0.05 and ** P<0.01 compared to respective control (unpaired two-tailed Student's t-test).

Mentions: With regard to PEA hydrolysis, Htt25Q reduced it by 65%, Htt103Q had a small (17%), but significant effect, and α-synuclein reduced this activity by 32% (Figure 7A–C). The empty vector had no significant effect (data not shown). To determine if this decrease in PEA hydrolysis involves Yju3, we expressed Htt fragments and α-synuclein in the yju3Δ strain. Here too, PEA hydrolysis was similarly decreased by these pathogenic fragments and proteins (Figure 7A–C), suggesting that they reduce the expression of another PEA hydrolyzing enzyme.


Genetic manipulation of palmitoylethanolamide production and inactivation in Saccharomyces cerevisiae.

Muccioli GG, Sia A, Muchowski PJ, Stella N - PLoS ONE (2009)

Htt fragments and α-synuclein reduce PEA hydrolysis in yeast.[3H]-PEA hydrolysis measured in BY4741 yeast strains expressing a 25Q and 103Q huntingtin fragment and in yeast strain expressing α-synuclein (BY4741) were compared to BY4741 strains expressing an empty vector (dotted line). Similarly [3H]-PEA hydrolysis found in YJU3 gene-deleted yeast strains expressing the same proteins (yju3Δ) were compared to yju3Δ strains expressing an empty vector (dotted line) (A–C). Values are the mean±SEM of three to five experiments performed in duplicate and are expressed as percentage of corresponding control (empty vector expressing BY4741 strain and empty vector expressing yju3Δ strain, respectively). ** P<0.05 and ** P<0.01 compared to respective control (unpaired two-tailed Student's t-test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691958&req=5

pone-0005942-g007: Htt fragments and α-synuclein reduce PEA hydrolysis in yeast.[3H]-PEA hydrolysis measured in BY4741 yeast strains expressing a 25Q and 103Q huntingtin fragment and in yeast strain expressing α-synuclein (BY4741) were compared to BY4741 strains expressing an empty vector (dotted line). Similarly [3H]-PEA hydrolysis found in YJU3 gene-deleted yeast strains expressing the same proteins (yju3Δ) were compared to yju3Δ strains expressing an empty vector (dotted line) (A–C). Values are the mean±SEM of three to five experiments performed in duplicate and are expressed as percentage of corresponding control (empty vector expressing BY4741 strain and empty vector expressing yju3Δ strain, respectively). ** P<0.05 and ** P<0.01 compared to respective control (unpaired two-tailed Student's t-test).
Mentions: With regard to PEA hydrolysis, Htt25Q reduced it by 65%, Htt103Q had a small (17%), but significant effect, and α-synuclein reduced this activity by 32% (Figure 7A–C). The empty vector had no significant effect (data not shown). To determine if this decrease in PEA hydrolysis involves Yju3, we expressed Htt fragments and α-synuclein in the yju3Δ strain. Here too, PEA hydrolysis was similarly decreased by these pathogenic fragments and proteins (Figure 7A–C), suggesting that they reduce the expression of another PEA hydrolyzing enzyme.

Bottom Line: Lipids can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions.The signaling lipid N-palmitoylethanolamine (PEA) is known to activate intracellular and membrane-associated receptors and regulate physiological functions, but little is known about the enzymes involved in its production and inactivation.Accordingly, using single gene deletion mutants, we identified yeast genes that control PEA metabolism, including SPO14 (a yeast homologue of the mammalian phospholipase D) that controls PEA production and YJU3 (a yeast homologue of the mammalian monoacylglycerol lipase) that controls PEA inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Lipids can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. To understand how a newly discovered signaling lipid functions, it is necessary to identify and characterize the enzymes involved in their production and inactivation. The signaling lipid N-palmitoylethanolamine (PEA) is known to activate intracellular and membrane-associated receptors and regulate physiological functions, but little is known about the enzymes involved in its production and inactivation.

Principal findings: Here we show that Saccharomyces cerevisiae produce and inactivate PEA, suggesting that genetic manipulations of this lower eukaryote may be used to identify the enzymes involved in PEA metabolism. Accordingly, using single gene deletion mutants, we identified yeast genes that control PEA metabolism, including SPO14 (a yeast homologue of the mammalian phospholipase D) that controls PEA production and YJU3 (a yeast homologue of the mammalian monoacylglycerol lipase) that controls PEA inactivation. We also found that PEA metabolism is affected by heterologous expression of two mammalian proteins involved in neurodegenerative diseases, namely huntingtin and alpha-synuclein.

Significance: Together these findings show that forward and reverse genetics in S. cerevisiae can be used to identify proteins involved in PEA production and inactivation, and suggest that mutated proteins causing neurodegenerative diseases might affect the metabolism of this important signaling lipid.

Show MeSH
Related in: MedlinePlus