A yeast PAF acetylhydrolase ortholog suppresses oxidative death

Abstract

Phospholipids containing sn-2 polyunsaturated fatty acyl residues are primary targets of oxidizing radicals, producing proapoptotic and membrane perturbing fragmented phospholipids. The only known phospholipases that specifically select these oxidized and/or short-chained phospholipids as substrates are mammalian group VII phospholipases A2s that were purified and cloned as PAF acetylhydrolases. Platelet-activating factor (PAF) is a short-chained phospholipid, and whether these enzymes actually are PAF hydrolases or evolved as oxidized phospholipid phospholipases is unknown. The fission yeast Schizosaccharomyces pombe, which does not form or use PAF as a signaling molecule, contains an open-reading frame potentially homologous to mammalian group VII phospholipase A2s. We cloned this SPBC106.11c locus and expressed it in distantly related Saccharomyces cerevisiae that lack homologous sequences. The S. pombe locus encoded a functional phospholipase A2, now renamed plg7+, that hydrolyzed PAF and a synthetic oxidized phospholipid. Expression of human type II PAF acetylhydrolase or S. pombe Plg7p enhanced the viability of S. cerevisiae subjected to oxidative stress. We conclude that a single-celled organism with an exceedingly spare genome still expresses an unusually discriminating phospholipase A2, and that selective hydrolysis of phospholipid oxidation products is an early, and critical, way to overcome oxidative membrane damage and oxidant-induced cell death.

Figure 1. The S. pombe genome contains a sequence homologous to the human plasma and type 2 PAF-acetylhydrolases

Sequence alignment of the S. pombe locus SPBC106.11c, human plasma PAF acetylhydrolase (PLA2g7) and type 2 PAF-acetylhydrolase (HPAFAH2) using ClustalW, BLOSUM program. Characters highlighted in black are exact matches, characters highlighted in gray are similar in identity. Amino acids marked (*) are essential for enzyme activity.

Figure 2. Δplg7 cells have decreased but residual PAF-AH activity

(A) Gene deletion scheme to replace plg7⁺ with his7⁺ in S. pombe strain CHP428. Approximately 200 base pairs of both ends of plg7⁺ were added to a construct with the his7⁺ gene. 5’ ends of the genes are indicated with black arrows. (B) PCR analysis of plg7⁺::his7⁺ gene replacement in wild type (Wt) and Δplg7 cells using genomic and his7⁺ specific primers; (−) = negative PCR reaction. PCR detecting intact genomic plg7⁺ in wild type and Δplg7 cells. (C) PAF-AH activity assay of OD (550 nm) normalized wild type and Δplg7 cells, lysed and pre-treated with mock or 100 µM Pefabloc SC. Values are expressed relative to mock treated wild type lysates, n=2 in duplicate * = P<0.001 vs plg7⁺ (One Way Anova, Student t-test).

Figure 3. plg7⁺ encodes a protein with Ser257 dependent PLA₂ activity

(A) Western blot of lysates from empty vector, plg7⁺, or plg7-S257A-pDEST52 transformed DY 1838 cells using a combination of antibodies against the V5 epitope tag (upper band) and a diluted antibody to actin (lower band). (B) PAF-AH activity assay of cell lysates from empty vector, non-tagged plg7⁺, plg7-S257A, or human PAFAH2-pDEST52 transformed INVSc1 cells, normalized by total protein. (C) PAF-AH activity assay of cell lysates from non-tagged plg7⁺ or human PAFAH2-pDEST52 DY 1838 transformed cells, treated with 1 mM Pefabloc SC or MAFP. Results for B: n=4, in duplicate * = P<0.05 vs empty, Plg7p-S257A; results for C: n=3, in duplicate * = P<0.05 vs Pefabloc SC, MAFP (One Way ANOVA, Tukey Test).

Figure 4. S. cerevisiae supplemented with linolenic acid is sensitive to oxidation

(A) INVSc1 wildtype cells loaded with the lipophilic dye Bodipy® 581/591 C11 that fluoresces after oxidation were examined by fluorimetry using 488/20 nm bandwidth excitation, 528/20 nm bandwidth emission in a representative experiment done in duplicate. Key: Non treated (open square), copper treated (filled square), C_18:3 supplemented (open circle), and C_18:3 supplemented and copper treated (filled circle). (B) INVSc1 strain was supplemented with C_18:3, loaded with Bodipy® 581/591 C11, and treated with 50 µM CuSO₄ for 60 min. Cells were visualized by confocal microscopy using a 488 nm excitation laser. (C) fet3Δ cells supplemented with C_18:3 and loaded with Bodipy® 581/591 C11 were visualized after 60 min of mock or 50 µM CuSO₄ treatment. Scale bar = 10 µm.

Figure 5. plg7⁺ protects S. cerevisiae against oxidative stress

(A) Viability assay of INVSc1 cells −/+ C_18:3 supplementation, treated with 50 µM CuSO₄ for 0, 10 or 60 min, diluted 10-fold serially and plated on YEPD plates. (B) C_18:3 supplemented INVSc1 cells were transformed with empty vector or plg7⁺pDEST52 and challenged with 50 µM CuSO₄ for the stated times, then serially diluted and plated to test viability. (C) Viability assay using fet3Δ cells transformed with plg7⁺, plg7-S257A. (D) HPAFAH2-pDEST52, or empty vector, supplemented with 1 mM C_18:3, treated with 50 µM CuSO₄, serially diluted and grown on YEPD plates for 4 days at room temperature.