Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype.

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.

The bactericidal effect of human secreted group IID phospholipase A2 results from both hydrolytic and non-hydrolytic activities.

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca(2+)-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10µg/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca(2+)-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca(2+)-independent damaging activity may play a role in the bactericidal function of the protein.

Inhibitory effects of surfactant protein A on surfactant phospholipid hydrolysis by secreted phospholipases A2.

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.

Inhibition of secreted phospholipases A2 by annexin V. Competition for anionic phospholipid interfaces allows an assessment of the relative interfacial affinities of secreted phospholipases A2.

The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. In this investigation we have examined the substrate depletion mechanism of annexin V using a variety of phospholipid substrates and secreted PLA2's (sPLA2). The results suggest that the term interfacial competition best describes the inhibitory effect of annexin V although the overall inhibitory process remains one of substrate sequestration by the annexin. We have utilised the competitive nature of the interaction of enzyme and annexin V for a phospholipid interface as a means of quantifying the relative affinity of sPLA2's for anionic phospholipid vesicles. The results highlight the very high affinity of the human non-pancreatic sPLA2 for such vesicles (Kd<<10-(10) M) while the Naja naja venom PLA2 and porcine pancreatic sPLA2 showed lower affinities. Hydrolysis of mixed vesicles containing phosphatidylserine and phosphatidylcholine by the venom and pancreatic enzymes were differentially inhibited by annexin V. This difference must reflect the preference of both annexin V and the pancreatic enzyme for an anionic phospholipid interface. In contrast, the venom enzyme is able to readily hydrolyse phosphatidylcholine domains that would be minimally affected by annexin V. Annexin V was an effective inhibitor of cardiolipin hydrolysis by the pancreatic PLA2, however the inhibition was of a more complex nature than seen with other phospholipids tested. Overall the results highlight the ability of annexin V to inhibit phospholipid hydrolysis by sPLA2's by an interfacial competition (substrate depletion) mechanism. The effectiveness of annexin V as an apparent inhibitor depends on the nature of the enzyme and the phospholipid substrate.