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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM020501-37
Application #
8294319
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Barski, Oleg
Project Start
1977-06-01
Project End
2016-11-30
Budget Start
2012-12-15
Budget End
2013-11-30
Support Year
37
Fiscal Year
2013
Total Cost
$364,250
Indirect Cost
$129,250
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha et al. (2017) Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry. J Biol Chem 292:6108-6122
Brown, Charles R; Dennis, Edward A (2017) Borrelia burgdorferi infection induces lipid mediator production during Lyme arthritis. Biochimie 141:86-90
Bruhn, Jessica F; Kirchdoerfer, Robert N; Urata, Sarah M et al. (2017) Crystal Structure of the Marburg Virus VP35 Oligomerization Domain. J Virol 91:
Bowden, John A; Heckert, Alan; Ulmer, Candice Z et al. (2017) Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma. J Lipid Res 58:2275-2288
Vasquez, Alexis M; Mouchlis, Varnavas D; Dennis, Edward A (2017) Review of four major distinct types of human phospholipase A2. Adv Biol Regul :
Chandra, Vikas; Wu, Dalei; Li, Sheng et al. (2017) The quaternary architecture of RAR?-RXR? heterodimer facilitates domain-domain signal transmission. Nat Commun 8:868
Kokotou, Maroula G; Galiatsatou, Gerasimia; Magrioti, Victoria et al. (2017) 2-Oxoesters: A Novel Class of Potent and Selective Inhibitors of Cytosolic Group IVA Phospholipase A2. Sci Rep 7:7025
Smyrniotou, Anneta; Kokotou, Maroula G; Mouchlis, Varnavas D et al. (2017) 2-Oxoamides based on dipeptides as selective calcium-independent phospholipase A2 inhibitors. Bioorg Med Chem 25:926-940
Tseng, Roger; Goularte, Nicolette F; Chavan, Archana et al. (2017) Structural basis of the day-night transition in a bacterial circadian clock. Science 355:1174-1180
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

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