No abstract

The phospholipase A superfamily of enzymes plays a significant role in the development and progression of numerous inflammatory diseases. Through their catalytic action on membrane phospholipids, phospholipases are the upstream regulators of the eicosanoid pathway releasing free fatty acids for cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes which produce various well-known inflammatory mediators including leukotrienes, thromboxanes and prostaglandins. Elucidating the association of phospholipases A with the membrane, the extraction and binding of phospholipid substrates, and their interactions with small-molecule inhibitors is crucial for the development of new anti-inflammatory therapeutics. Studying phospholipases has been challenging because they act on the surface of cellular membranes and micelles. Multidisciplinary approaches including hydrogen/deuterium exchange mass spectrometry, molecular dynamics simulations, and other computer-aided drug design techniques have been successfully employed by our laboratory to study interactions of phospholipases with membranes, phospholipid substrates and inhibitors. This review summarizes the application of these techniques to study four human recombinant phospholipases A.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein’s phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme’s activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2‘s specificity toward oxidized phospholipids.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has been far more difficult to achieve. Instead of relying on unpredictable ion manipulations or slow photoactivatable toxins at axon terminals, we took a different approach to leverage the natural ability of inhibitory presynaptic GPCRs to silence synaptic transmission. Here we characterize parapinopsin (PPO), a photoswitchable non-visual opsin from lamprey pineal gland that couples to Gi/o-signaling cascades. PPO can be rapidly activated by pulsed blue light, switched off with amber light, and is effective for repeated or prolonged inhibition. We developed viral vectors for cell-specific expression of PPO, which traffics very effectively in numerous neuron types. At presynaptic terminals, PPO can silence glutamate release and suppress dopamine-dependent reward and cocaine place preference behaviors in vivo. PPO immediately fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition, and has broader utility for achieving spatiotemporal control of inhibitory GPCR signaling cascades in other biological and pharmacological applications.

No abstract