PROJECT SUMMARY/ABSTRACT
The overall goal of this grant in recent years has been to describe in molecular detail the mechanism of
action of physiologically important human forms of phospholipase A2 (PLA2). During the course of these
studies, we have discovered that the activity of this superfamily of enzymes depends critically on the
interaction of two large macromolecules (the protein and the large lipid aggregate), where the orientation of
the enzyme with respect to the plane of the lipid-water interface can have a dramatic effect on activity. The
nature of this interaction has been challenging to explore, but we have now shown that association of the
membrane or micelle interface with the enzyme causes an allosteric activation through a resulting
conformational change. This renewal application will extend our current studies on the pure recombinant
human cytosolic Group IVA cPLA2, secretory Group V sPLA2, Ca2+-independent Group VIA iPLA2, and
lipoprotein-associated PLA2/PAF (platelet-activating factor) acetyl hydrolase Group VIIA LpPLA2. During the
renewal period, we will focus on three new directions. First, we will explore the further role of additional
allosteric sites on iPLA2 (for ATP and calmodulin) and cPLA2 (for PIP2) for enzyme regulation and as drug
targets. Second, we will expand and apply what we learned with phospholipases to triglyceride lipases
starting with PNPLA3, which contains a patatin-like domain and is homologous to the catalytic domain of
iPLA2. PNPLA3 is of great interest because GWAS studies have shown that a natural mutation (I148M)
enriched in the Hispanic population leads to an increase in nonalcoholic steatohepatitis (NASH), the
advanced form of nonalcoholic fatty liver disease (NAFLD). Our lipidomics analysis shows that the pure
recombinant human mutant PNPLA3 has decreased triglyceride hydrolase activity and our MD studies show
that the catalytic site has adopted to a triacylglyceride substrate rather than the phospholipid substrate in
iPLA2. Third, we will explore the functioning and physiological role of the various intracellular phospholipase
A2s in relevant intact cells, where the actual specificity will depend on the proximity and availability of optimal
phospholipid molecular species. Although we have developed a novel lipidomics assay of PLA2 specificity
and function in vitro, one barrier to progress in this field is the lack of methods for determining PLA2 activity in
living cells. To address this issue, we have developed a new platform for measuring PLA2 specificity and
inhibition ex vivo in macrophage cells in culture. This work has and will generate important widely applicable
novel information on how physiologically important phospholipases and triacylglycerol lipases interact with
the lipid-water interfaces of membranes, micelles and lipid droplets to compete physiologically in selecting
their substrates. This work should enable us to fully explain and integrate at a structural level the resulting
specificity of multiple members of the PLA2 superfamily acting in vitro with the specific molecular species of
phospholipids hydrolyzed and the specific fatty acids released as well as correlating with ex vivo specificity.