Dataset Information

Analyses of Calcium-Independent Phospholipase A2beta (iPLA2?) in Biological Systems.

ABSTRACT: The Ca2+-independent phospholipases A2 (iPLA2s) are part of a diverse family of PLA2s, manifest activity in the absence of Ca2+, are ubiquitous, and participate in a variety of biological processes. Among the iPLA2s, the cytosolic iPLA2? has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2? can have a profound impact on the onset and/or progression of many diseases (e.g., cardiovascular, neurological, metabolic, autoimmune). Therefore, appropriate approaches are warranted to gain a better understanding of the role of iPLA2? in vivo and its contribution to pathophysiology. Given that iPLA2? is very labile, its basal expression is low in a number of cell systems, and that crystal structure of iPLA2? is not yet available, careful and efficient protocols are needed to appropriately assess iPLA2? biochemistry, dynamics, and membrane association. Here, step-by-step details are provided to (a) measure iPLA2?-specific activity in cell lines or tissue preparations (using a simple radiolabel-based assay) and assess the impact of stimuli and inhibitors on resting- and disease-state iPLA2? activity, (b) purify the iPLA2? to near homogeneity (via sequential chromatography) from cell line or tissue preparations, enabling concentration of the enzyme for subsequent analyses (e.g., proteomics), and (c) employ hydrogen/deuterium exchange mass spectrometry analyses to probe both the structure of iPLA2? and dynamics of its association with the membranes, substrates, and inhibitors.

SUBMITTER: Barbour SE

PROVIDER: S-EPMC5771648 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Analyses of Calcium-Independent Phospholipase A2beta (iPLA2β) in Biological Systems.

Barbour S E SE Ramanadham S S

Methods in enzymology 20161018

The Ca2+-independent phospholipases A2 (iPLA2s) are part of a diverse family of PLA2s, manifest activity in the absence of Ca2+, are ubiquitous, and participate in a variety of biological processes. Among the iPLA2s, the cytosolic iPLA2β has received considerable attention and ongoing studies from various laboratories suggest that dysregulation of iPLA2β can have a profound impact on the onset and/or progress ...[more]

PMID: 28063488

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Project description:Congestive heart failure typically arises from cardiac myocyte necrosis/apoptosis, associated with the pathological opening of the mitochondrial permeability transition pore (mPTP). mPTP opening decreases the mitochondrial membrane potential leading to the activation of Ca2+-independent phospholipase A2γ (iPLA2γ) and the production of downstream toxic metabolites. However, the array of enzymatic mediators and the exact chemical mechanisms responsible for modulating myocardial mPTP opening remain unclear. Herein, we demonstrate that human heart failure activates specific myocardial mitochondrial phospholipases that increase Ca2+-dependent production of toxic hydroxyeicosatetraenoic acids (HETEs) and attenuate the activity of phospholipases that promote the synthesis of protective epoxyeicosatrienoic acids (EETs). Mechanistically, HETEs activated the Ca2+-induced opening of the mPTP in failing human myocardium, and the highly selective pharmacological blockade of either iPLA2γ or lipoxygenases attenuated mPTP opening in failing hearts. In contrast, pharmacological inhibition of cytochrome P450 epoxygenases opened the myocardial mPTP in human heart mitochondria. Remarkably, the major mitochondrial phospholipase responsible for Ca2+-activated release of arachidonic acid (AA) in mitochondria from non-failing hearts was calcium-dependent phospholipase A2ζ (cPLA2ζ) identified by sequential column chromatographies and activity-based protein profiling. In contrast, iPLA2γ predominated in failing human myocardium. Stable isotope kinetics revealed that in non-failing human hearts, cPLA2ζ metabolically channels arachidonic acid into EETs, whereas in failing hearts, increased iPLA2γ activity channels AA into toxic HETEs. These results mechanistically identify the sequelae of pathological remodeling of human mitochondrial phospholipases in failing myocardium. This remodeling metabolically channels AA into toxic HETEs promoting mPTP opening, which induces necrosis/apoptosis leading to further progression of heart failure.

Dataset Information

Analyses of Calcium-Independent Phospholipase A2beta (iPLA2?) in Biological Systems.

Publications

Analyses of Calcium-Independent Phospholipase A<sub>2</sub>beta (iPLA<sub>2</sub>β) in Biological Systems.

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