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Substrate efflux propensity is the key determinant of Ca2+-independent phospholipase A-? (iPLA?)-mediated glycerophospholipid hydrolysis.

ABSTRACT: The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca(2+)-independent PLA-? (iPLA?) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLA? as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLA?-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.

SUBMITTER: Batchu KC

PROVIDER: S-EPMC4400325 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Substrate efflux propensity is the key determinant of Ca2+-independent phospholipase A-β (iPLAβ)-mediated glycerophospholipid hydrolysis.

Batchu Krishna Chaithanya KC Hokynar Kati K Jeltsch Michael M Mattonet Kenny K Somerharju Pentti P

The Journal of biological chemistry 20150223 16

The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca(2+)-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some ...[more]

PMID: 25713085

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Project description:Mitochondrial Ca2+-independent phospholipase A2γ (iPLA2γ/PNPLA8) was previously shown to be directly activated by H2O2 and release free fatty acids (FAs) for FA-dependent H+ transport mediated by the adenine nucleotide translocase (ANT) or uncoupling protein 2 (UCP2). The resulting mild mitochondrial uncoupling and consequent partial attenuation of mitochondrial superoxide production lead to an antioxidant effect. However, the antioxidant role of iPLA2γ in the brain is not completely understood. Here, using wild-type and iPLA2γ-KO mice, we demonstrate the ability of tert-butylhydroperoxide (TBHP) to activate iPLA2γ in isolated brain mitochondria, with consequent liberation of FAs and lysophospholipids. The liberated FA caused an increase in respiratory rate, which was fully inhibited by carboxyatractyloside (CATR), a specific inhibitor of ANT. Employing detailed lipidomic analysis, we also demonstrate a typical cleavage pattern for TBHP-activated iPLA2γ, reflecting cleavage of glycerophospholipids from both sn-1 and sn-2 positions releasing saturated FAs, monoenoic FAs, and predominant polyunsaturated FAs. The acute antioxidant role of iPLA2γ-released FAs is supported by monitoring both intramitochondrial superoxide and extramitochondrial H2O2 release. We also show that iPLA2γ-KO mice were more sensitive to stimulation by pro-inflammatory lipopolysaccharide, as reflected by the concomitant increase in protein carbonyls in the brain and pro-inflammatory IL-6 release in the serum. These data support the antioxidant and anti-inflammatory role of iPLA2γ in vivo. Our data also reveal a substantial decrease of several high molecular weight cardiolipin (CL) species and accumulation of low molecular weight CL species in brain mitochondria of iPLA2γ-KO mice. Collectively, our results support a key role of iPLA2γ in the remodeling of lower molecular weight immature cardiolipins with predominantly saturated acyl chains to high molecular weight mature cardiolipins with highly unsaturated PUFA acyl chains, typical for the brain.

Dataset Information

Substrate efflux propensity is the key determinant of Ca2+-independent phospholipase A-? (iPLA?)-mediated glycerophospholipid hydrolysis.

Publications

Substrate efflux propensity is the key determinant of Ca2+-independent phospholipase A-β (iPLAβ)-mediated glycerophospholipid hydrolysis.

Similar Datasets

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