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Srf1 is a novel regulator of phospholipase D activity and is essential to buffer the toxic effects of C16:0 platelet activating factor.

Kennedy MA, Kabbani N, Lambert JP, Swayne LA, Ahmed F, Figeys D, Bennett SA, Bryan J, Baetz K - PLoS Genet. (2011)

Bottom Line: As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects.Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF.Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada.

ABSTRACT
During Alzheimer's Disease, sustained exposure to amyloid-β₄₂ oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-β₄₂.

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Model of Srf1 regulation of Spo14 activity in mitotic cells.
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pgen-1001299-g008: Model of Srf1 regulation of Spo14 activity in mitotic cells.

Mentions: Our unbiased chemical genomic approach identified the deletion mutant of SRF1 as having the most significant differential sensitivity to C16:0 PAF (Figure 2B). We identified a robust interaction between Srf1-TAP and Spo14 (Figure 3), whose deletion mutant is also hypersensitive to C16:0 PAF [Figures 2B, 4 and ref. 17]. The identification of a physical interaction between Srf1 and Spo14 is striking as only two other proteins, neither with roles in PLD function, have been reported to co-purify Spo14 in high-throughput TAP studies [20]. Furthermore, biochemical assays determined that Srf1 is required for PLD activity in mitotic cells (Figures 5 and 6). A role for Srf1 in mitotic PLD activity is also supported by genome-wide synthetic lethal genetic screens which revealed that deletion mutants of both SPO14 and SRF1 display genetic interactions with the sec14-bypass mutants CKI1 and KES1 [36]. However, in contrast to Spo14 [24], [37], [38], Srf1 is not essential for sporulation (Table 1) which suggests Srf1 is not regulating PLD activity in meiosis. Our results clearly show that Srf1-TAP can co-purify Spo14 suggesting a model where Spo14 and Srf1 form a complex in mitotic cells that is required for PLD activity and to buffer the toxicity of C16:0 PAF (Figure 8).


Srf1 is a novel regulator of phospholipase D activity and is essential to buffer the toxic effects of C16:0 platelet activating factor.

Kennedy MA, Kabbani N, Lambert JP, Swayne LA, Ahmed F, Figeys D, Bennett SA, Bryan J, Baetz K - PLoS Genet. (2011)

Model of Srf1 regulation of Spo14 activity in mitotic cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037409&req=5

pgen-1001299-g008: Model of Srf1 regulation of Spo14 activity in mitotic cells.
Mentions: Our unbiased chemical genomic approach identified the deletion mutant of SRF1 as having the most significant differential sensitivity to C16:0 PAF (Figure 2B). We identified a robust interaction between Srf1-TAP and Spo14 (Figure 3), whose deletion mutant is also hypersensitive to C16:0 PAF [Figures 2B, 4 and ref. 17]. The identification of a physical interaction between Srf1 and Spo14 is striking as only two other proteins, neither with roles in PLD function, have been reported to co-purify Spo14 in high-throughput TAP studies [20]. Furthermore, biochemical assays determined that Srf1 is required for PLD activity in mitotic cells (Figures 5 and 6). A role for Srf1 in mitotic PLD activity is also supported by genome-wide synthetic lethal genetic screens which revealed that deletion mutants of both SPO14 and SRF1 display genetic interactions with the sec14-bypass mutants CKI1 and KES1 [36]. However, in contrast to Spo14 [24], [37], [38], Srf1 is not essential for sporulation (Table 1) which suggests Srf1 is not regulating PLD activity in meiosis. Our results clearly show that Srf1-TAP can co-purify Spo14 suggesting a model where Spo14 and Srf1 form a complex in mitotic cells that is required for PLD activity and to buffer the toxicity of C16:0 PAF (Figure 8).

Bottom Line: As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects.Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF.Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Canada.

ABSTRACT
During Alzheimer's Disease, sustained exposure to amyloid-β₄₂ oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-β₄₂.

Show MeSH
Related in: MedlinePlus