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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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Characterization of a genetic interaction between SPO14 and SRF1 revealed by C16:0 PAF.(A) Sensitivity of srf1Δ to C16:0 PAF is suppressed by spo14Δ. Wild type (YPH500), spo14Δ (YKB2076), srf1Δ (YKB1164) and srf1Δspo14Δ (YKB2080) cells were plated in five-fold serial dilution onto YPD or YPD supplemented with C16:0 PAF as indicated. The plates were incubated for 3 days at 30°C. (B) Overexpression of SPO14 does not alleviate the sensitivity of srf1Δ cells to C16:0 PAF. Wild type (YPH500), srf1Δ (YKB1164), and spo14Δ (YKB2076) cells transformed with pRS415 (vector), pME962 (SPO14), pME1096 (GFP-SPO14) or pME1130 (GFP-SPO14K–H)[24] were plated in five-fold serial dilution onto SD-Leu or SD-Leu supplemented with C16:0 PAF as indicated. The plates were incubated for 2 days at 30oC. The growth of all strains tested was moderately improved on minimal media compared with YPD at equivalent concentrations of C16:0 PAF. This is reflected by the observed differences in growth for both the wild type and srf1Δ strain when comparing panels (A) and (B).
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pgen-1001299-g004: Characterization of a genetic interaction between SPO14 and SRF1 revealed by C16:0 PAF.(A) Sensitivity of srf1Δ to C16:0 PAF is suppressed by spo14Δ. Wild type (YPH500), spo14Δ (YKB2076), srf1Δ (YKB1164) and srf1Δspo14Δ (YKB2080) cells were plated in five-fold serial dilution onto YPD or YPD supplemented with C16:0 PAF as indicated. The plates were incubated for 3 days at 30°C. (B) Overexpression of SPO14 does not alleviate the sensitivity of srf1Δ cells to C16:0 PAF. Wild type (YPH500), srf1Δ (YKB1164), and spo14Δ (YKB2076) cells transformed with pRS415 (vector), pME962 (SPO14), pME1096 (GFP-SPO14) or pME1130 (GFP-SPO14K–H)[24] were plated in five-fold serial dilution onto SD-Leu or SD-Leu supplemented with C16:0 PAF as indicated. The plates were incubated for 2 days at 30oC. The growth of all strains tested was moderately improved on minimal media compared with YPD at equivalent concentrations of C16:0 PAF. This is reflected by the observed differences in growth for both the wild type and srf1Δ strain when comparing panels (A) and (B).

Mentions: Our results indicate that at the 40 µM treatment level, Srf1 is pivotal for buffering the effects of C16:0 PAF. The biological function(s) of Srf1 is unknown but it is predicted to be a transmembrane protein. Therefore we sought to decipher its cellular function by identifying proteins that interact with Srf1. As traditional tandem affinity purification (TAP) protocols were not successful in purifying Srf1-TAP [data not shown and ref. 20], [21], we utilized a less stringent single step affinity purification approach based on the modified chromatin immunopurification (mChIP) technique [22]. Though this technique was developed for improving the purification of insoluble chromatin associated proteins, it is also applicable to other subclasses, including membrane associated proteins [23]. Using mChIP we successfully purified Srf1-TAP and identified five co-purifying proteins by mass spectrometry, of which the largest number of peptides correspond to Spo14 (Figure 3). The physical interaction between Srf1 and Spo14, combined with the sensitivity of the corresponding deletion mutants to C16:0 PAF [Figures 2, 4 and ref. 17] suggest Srf1 may work in a complex with Spo14 to regulate PA metabolism.


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)

Characterization of a genetic interaction between SPO14 and SRF1 revealed by C16:0 PAF.(A) Sensitivity of srf1Δ to C16:0 PAF is suppressed by spo14Δ. Wild type (YPH500), spo14Δ (YKB2076), srf1Δ (YKB1164) and srf1Δspo14Δ (YKB2080) cells were plated in five-fold serial dilution onto YPD or YPD supplemented with C16:0 PAF as indicated. The plates were incubated for 3 days at 30°C. (B) Overexpression of SPO14 does not alleviate the sensitivity of srf1Δ cells to C16:0 PAF. Wild type (YPH500), srf1Δ (YKB1164), and spo14Δ (YKB2076) cells transformed with pRS415 (vector), pME962 (SPO14), pME1096 (GFP-SPO14) or pME1130 (GFP-SPO14K–H)[24] were plated in five-fold serial dilution onto SD-Leu or SD-Leu supplemented with C16:0 PAF as indicated. The plates were incubated for 2 days at 30oC. The growth of all strains tested was moderately improved on minimal media compared with YPD at equivalent concentrations of C16:0 PAF. This is reflected by the observed differences in growth for both the wild type and srf1Δ strain when comparing panels (A) and (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037409&req=5

pgen-1001299-g004: Characterization of a genetic interaction between SPO14 and SRF1 revealed by C16:0 PAF.(A) Sensitivity of srf1Δ to C16:0 PAF is suppressed by spo14Δ. Wild type (YPH500), spo14Δ (YKB2076), srf1Δ (YKB1164) and srf1Δspo14Δ (YKB2080) cells were plated in five-fold serial dilution onto YPD or YPD supplemented with C16:0 PAF as indicated. The plates were incubated for 3 days at 30°C. (B) Overexpression of SPO14 does not alleviate the sensitivity of srf1Δ cells to C16:0 PAF. Wild type (YPH500), srf1Δ (YKB1164), and spo14Δ (YKB2076) cells transformed with pRS415 (vector), pME962 (SPO14), pME1096 (GFP-SPO14) or pME1130 (GFP-SPO14K–H)[24] were plated in five-fold serial dilution onto SD-Leu or SD-Leu supplemented with C16:0 PAF as indicated. The plates were incubated for 2 days at 30oC. The growth of all strains tested was moderately improved on minimal media compared with YPD at equivalent concentrations of C16:0 PAF. This is reflected by the observed differences in growth for both the wild type and srf1Δ strain when comparing panels (A) and (B).
Mentions: Our results indicate that at the 40 µM treatment level, Srf1 is pivotal for buffering the effects of C16:0 PAF. The biological function(s) of Srf1 is unknown but it is predicted to be a transmembrane protein. Therefore we sought to decipher its cellular function by identifying proteins that interact with Srf1. As traditional tandem affinity purification (TAP) protocols were not successful in purifying Srf1-TAP [data not shown and ref. 20], [21], we utilized a less stringent single step affinity purification approach based on the modified chromatin immunopurification (mChIP) technique [22]. Though this technique was developed for improving the purification of insoluble chromatin associated proteins, it is also applicable to other subclasses, including membrane associated proteins [23]. Using mChIP we successfully purified Srf1-TAP and identified five co-purifying proteins by mass spectrometry, of which the largest number of peptides correspond to Spo14 (Figure 3). The physical interaction between Srf1 and Spo14, combined with the sensitivity of the corresponding deletion mutants to C16:0 PAF [Figures 2, 4 and ref. 17] suggest Srf1 may work in a complex with Spo14 to regulate PA metabolism.

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