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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

Further characterization of PEA Hydrolysis by Yeast Homogenates.The effect of increasing concentrations of PEA on [3H]-PEA hydrolysis by yeast (A) and HEK293 cells (D) homogenates and the influence of the pH on [3H]-PEA hydrolysis by yeast (B) and HEK293 cells (E) homogenates were determined. The following buffers were used (100 mM): NaAcetate (pH 3–5), HEPES (pH 6–7), Tris (pH 8–9) and Na2B4O7 (pH 10–11). The serine hydrolase inhibitors PMSF and MAFP and the specific Faah inhibitor URB597 differentially affected [3H]-PEA hydrolysis by yeast (C) and HEK cells (F) homogenates. Data points represent the mean±SEM of 3 to 5 experiments performed in duplicate and are expressed as percentage of control. * P<0.05 and ** P<0.01 compared to control homogenates (ANOVA one-way, Dunnett's post test).
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pone-0005942-g004: Further characterization of PEA Hydrolysis by Yeast Homogenates.The effect of increasing concentrations of PEA on [3H]-PEA hydrolysis by yeast (A) and HEK293 cells (D) homogenates and the influence of the pH on [3H]-PEA hydrolysis by yeast (B) and HEK293 cells (E) homogenates were determined. The following buffers were used (100 mM): NaAcetate (pH 3–5), HEPES (pH 6–7), Tris (pH 8–9) and Na2B4O7 (pH 10–11). The serine hydrolase inhibitors PMSF and MAFP and the specific Faah inhibitor URB597 differentially affected [3H]-PEA hydrolysis by yeast (C) and HEK cells (F) homogenates. Data points represent the mean±SEM of 3 to 5 experiments performed in duplicate and are expressed as percentage of control. * P<0.05 and ** P<0.01 compared to control homogenates (ANOVA one-way, Dunnett's post test).

Mentions: We performed an initial biochemical characterization of the enzymatic activity responsible for [3H]-PEA hydrolysis. In yeast, unlabelled PEA inhibited this activity in dose-dependent manner, reaching ∼50% inhibition at 100 µM PEA (Figure 4A). In HEK293 homogenates, unlabelled PEA competed for [3H]-PEA hydrolysis, reaching 50% inhibition at 3.1 µM and 90% inhibition at 100 µM PEA (Figure 4D). [3H]-PEA hydrolysis by yeast homogenates was marginally affected by changes in pH (Figure 4B), suggesting that several yeast enzymes can hydrolyze PEA, each at their optimal pH value. In contrast, hydrolysis by HEK293 cells was clearly pH dependent, increasing by 10-fold between pH 3 and 9 (Figure 4E), suggesting hydrolysis by a single enzyme with optimal activity at pH 9. One such enzyme is Faah [26], [27], [37].


Genetic manipulation of palmitoylethanolamide production and inactivation in Saccharomyces cerevisiae.

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

Further characterization of PEA Hydrolysis by Yeast Homogenates.The effect of increasing concentrations of PEA on [3H]-PEA hydrolysis by yeast (A) and HEK293 cells (D) homogenates and the influence of the pH on [3H]-PEA hydrolysis by yeast (B) and HEK293 cells (E) homogenates were determined. The following buffers were used (100 mM): NaAcetate (pH 3–5), HEPES (pH 6–7), Tris (pH 8–9) and Na2B4O7 (pH 10–11). The serine hydrolase inhibitors PMSF and MAFP and the specific Faah inhibitor URB597 differentially affected [3H]-PEA hydrolysis by yeast (C) and HEK cells (F) homogenates. Data points represent the mean±SEM of 3 to 5 experiments performed in duplicate and are expressed as percentage of control. * P<0.05 and ** P<0.01 compared to control homogenates (ANOVA one-way, Dunnett's post test).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005942-g004: Further characterization of PEA Hydrolysis by Yeast Homogenates.The effect of increasing concentrations of PEA on [3H]-PEA hydrolysis by yeast (A) and HEK293 cells (D) homogenates and the influence of the pH on [3H]-PEA hydrolysis by yeast (B) and HEK293 cells (E) homogenates were determined. The following buffers were used (100 mM): NaAcetate (pH 3–5), HEPES (pH 6–7), Tris (pH 8–9) and Na2B4O7 (pH 10–11). The serine hydrolase inhibitors PMSF and MAFP and the specific Faah inhibitor URB597 differentially affected [3H]-PEA hydrolysis by yeast (C) and HEK cells (F) homogenates. Data points represent the mean±SEM of 3 to 5 experiments performed in duplicate and are expressed as percentage of control. * P<0.05 and ** P<0.01 compared to control homogenates (ANOVA one-way, Dunnett's post test).
Mentions: We performed an initial biochemical characterization of the enzymatic activity responsible for [3H]-PEA hydrolysis. In yeast, unlabelled PEA inhibited this activity in dose-dependent manner, reaching ∼50% inhibition at 100 µM PEA (Figure 4A). In HEK293 homogenates, unlabelled PEA competed for [3H]-PEA hydrolysis, reaching 50% inhibition at 3.1 µM and 90% inhibition at 100 µM PEA (Figure 4D). [3H]-PEA hydrolysis by yeast homogenates was marginally affected by changes in pH (Figure 4B), suggesting that several yeast enzymes can hydrolyze PEA, each at their optimal pH value. In contrast, hydrolysis by HEK293 cells was clearly pH dependent, increasing by 10-fold between pH 3 and 9 (Figure 4E), suggesting hydrolysis by a single enzyme with optimal activity at pH 9. One such enzyme is Faah [26], [27], [37].

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