The overall goal of this grant over the years has been to characterize the detailed mechanism of action of various physiologically important forms of phospholipase A2 (PLA2). During the course of these studies, it has become apparent that the activity of this superfamily of enzymes depends critically on the interaction of the proteins with large lipid aggregates, 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 difficult to explore because of the fact that it represents the interaction of two large macromolecules. This has presented challenges for traditional NMR and X-ray crystallographic studies. The activity of many of these enzymes increases when the enzyme is at the lipid-water interface. This activation is due in part to changes in enzyme-lipid orientation and to conformational changes in the enzyme. This renewal application will extend our current studies on the human Ca2+-independent Group VIA iPLA2 (GVIA iPLA2) and the human lipoprotein-associated PLA2 / PAF (platelet activating factor) acetyl hydrolase Group VIIA Lp-PLA2 (GVIIA Lp-PLA2). The GVIA iPLA2 is responsible for remodeling of membrane phospholipids in cells and plays critical roles in the cellular regulation of several diseases. We will determine exactly how it interacts with membranes. The GVIIA Lp-PLA2 is found associated with lipoproteins, both LDL and HDL, and is implicated in the turnover of oxidized phospholipids, as well as PAF, and has been implicated in cardiovascular disease. Along with traditional biochemical, molecular biological, and kinetic approaches, we will employ amide hydrogen/deuterium exchange-mass spectrometry (DXMS). It is rapidly becoming clear that this technique can tackle many structural questions about how proteins act in solution that cannot be addressed easily by NMR or X-ray crystallography. We will apply the DXMS technique to explore the interactions of these enzymes with large lipid interfaces and specific potent inhibitors as we have done earlier under this grant with the GIA sPLA2 and GIVA cPLA2. We also will use surface plasmon resonance and our detailed surface dilution kinetic model to study the functional aspects of these questions. This work will generate important widely applicable information on how soluble enzymes interact with lipid-water interfaces.
Phospholipase A2 controls the biosynthesis of eicosanoids by catalyzing the release of arachidonic acid from phospholipids. Thus, this enzyme plays a critical role in controlling normal physiological functions, but also plays a critical role in the pathogenesis of inflammation which underlies most major diseases. Understanding how the activity of this important enzyme is controlled and regulated will yield a better understanding of both normal and pathological processes and ultimately will lead to the development of clinical interventions to control disease.
|Shah, Manish B; Jang, Hyun-Hee; Wilderman, P Ross et al. (2016) Effect of detergent binding on cytochrome P450 2B4 structure as analyzed by X-ray crystallography and deuterium-exchange mass spectrometry. Biophys Chem 216:1-8|
|Dennis, Edward A (2016) Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease. J Biol Chem 291:24431-24448|
|Aydin, Halil; Sultana, Azmiri; Li, Sheng et al. (2016) Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex. Nature 534:562-5|
|Thornburg, Natalie J; Zhang, Heng; Bangaru, Sandhya et al. (2016) H7N9 influenza virus neutralizing antibodies that possess few somatic mutations. J Clin Invest 126:1482-94|
|Garner, Thomas P; Reyna, Denis E; Priyadarshi, Amit et al. (2016) An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway. Mol Cell 63:485-97|
|Mouchlis, Varnavas D; Dennis, Edward A (2016) Membrane and inhibitor interactions of intracellular phospholipases A2. Adv Biol Regul 61:17-24|
|Ruan, Chun; Cui, Haochen; Lee, Chul-Hwan et al. (2016) Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex. J Biol Chem 291:5428-38|
|Gupta, Shakti; Kihara, Yasuyuki; Maurya, Mano R et al. (2016) Computational Modeling of Competitive Metabolism between Ï‰3- and Ï‰6-Polyunsaturated Fatty Acids in Inflammatory Macrophages. J Phys Chem B 120:8346-53|
|Kong, Leopold; Lee, David E; Kadam, Rameshwar U et al. (2016) Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen. Proc Natl Acad Sci U S A :|
|Mouchlis, Varnavas D; Morisseau, Christophe; Hammock, Bruce D et al. (2016) Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A2. Bioorg Med Chem 24:4801-4811|
Showing the most recent 10 out of 68 publications