Arachidonic acid (AA), which is liberated from membrane phospholipids by phospholipase A2 (PLA2), is converted to oxidized lipids collectively known as eicosanoids, including prostaglandins and leukotrienes (LT), through the cyclooxygenase (COX) and lipoxygenase (LO) pathways. Because an imbalance in the production of these potent mediators of allergic and inflammatory reactions is known to cause inflammatory diseases, cardiovascular diseases, and cancer, understanding the regulation of eicosanoid biosynthesis is an important step toward the elucidation of the pathogenesis of these serious diseases. It has been well documented that the eicosanoid biosynthetic system offers many important drug targets (e.g., aspirin and other non-steroidal anti-inflammatory drugs targeted to COXs);however, inhibition of an individual component without full understanding of this complex system may cause deleterious side effects, as seen with COX-2 inhibitors such as VIOXX. An important theme derived from our previous studies on PLA2s, including secretory group V PLA2 (gVPLA2) and cytosolic group IVA PLA2 (cPLA21), is that the spatiotemporal coordination of PLA2s and other components of the LT biosynthetic system and the cross-talk among them are important for the control of inflammatory responses. To date, however, no systematic and mechanistic studies on the spatiotemporal coordination of eicosanoid biosynthetic proteins have been reported. During the next grant period, we therefore propose to expand our investigation to other known components of LT biosynthetic system, including 5-lipoxygenase (5LO), 5LO-activating protein (FLAP) and LTC4 synthase (LTC4S). We will first determine the mechanisms underlying the unique and complex membrane binding and cellular dynamics of 5LO. In particular, we will determine how 5LO phosphorylation and AA regulate the membrane interaction, dynamics, and activity of 5LO. We will then elucidate the elusive mechanism by which FLAP regulates the cellular localization and activity of 5LO by dual-color single molecule tracking, fluorescence cross-correlation spectroscopy, and a 5LO cell activity assay. We will also determine how the spatiotemporal dynamics and activities of all potential components of LTC4 biosynthesis are orchestrated in mammalian cells and how modulation of each of these processes affects the LT biosynthesis by the same techniques.
Many inflammatory diseases, including asthma and rheumatoid arthritis, cardiovascular diseases, and cancer, are linked to defects in regulation of eicosanoid biosynthesis. Consequently, eicosanoid biosynthetic pathways are major targets for drug development (for example, aspirin and other non- steroidal anti-inflammatory drugs) but the complexity of these pathways has hampered the discovery of specific and potent drugs with minimal side effects. Therefore, understanding the mechanisms underlying complex eicosanoid biosynthetic pathways will greatly aid in developing new classes of therapeutic agents that can treat diseases caused by dysfunctional eicosanoid biosynthesis. The proposed studies will provide new mechanistic insight into the spatiotemporal coordination of eicosanoid biosynthetic proteins and the cross-talk among them. This timely and critical information will accelerate development and evaluation of new therapeutic agents for inflammatory diseases, cardiovascular diseases, and cancer.
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