The objective of Project I is to understand the molecular and cellular mechanisms of action of fatty acid amide hydrolase (FAAH), a mammalian membrane-bound enzyme responsible for the catabolism of the fatty acid amide (FAA) family of endogenous signaling lipids. FAAs represent an emerging class of chemical messengers that influence a variety of behavioral processes, including, pain sensation, anxiety, sleep, and feeding. Recent genetic and pharmacological studies have demonstrated that FAAH is a principal regulator of FAA-based signaling events in vivo, suggesting that this enzyme may represent an attractive target for the treatment of neurological disorders like pain and anxiety. Nonetheless, many questions remain regarding the function of FAAH and its suitability as a therapeutic target. For example, among the numerous hydrolases present in mammalian proteomes, how does FAAH exert such extraordinary control over the levels and activity of FAAs in vivo? Might FAAH recruit these hydrophobic substrates directly from cellular membranes, thereby giving the enzyme privileged access to these lipid messengers? Likewise, does FAAH operate alone in neurons, or might this enzyme participate in higher-order protein complexes that regulate its localization and/or activity? Our recent determination of the FAAH structure by x-ray crystallography has inspired several new hypotheses to explain this enzyme's mode of action. In this project, we aim to test these hypotheses using a multidisciplinary approach, employing synthetic chemistry, enzymology, cell biology, and proteomic techniques towards the goals of: 1) determining the roles that FAAH channels play in catalysis, 2) characterizing the molecular interactions between FAAH and cell membranes, 3) identifying FAAH-associated proteins in brain and liver, and 4) comparing the cellular stability of WT-FAAH and its natural P129T variant associated with problem drug use. We anticipate that these studies will not only enhance our understanding of the molecular and cellular mechanism of action of FAAH, but also uncover novel ways to target this enzyme with inhibitors for research and therapeutic purposes.
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