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The Skaggs Institute for Chemical Biology
Scientific Report 1999-2000


Chemical Physiology


B.F. Cravatt, G. Adam, L. Crane, D. Kidd, K. Kustedjo, N. Jessani, G. Hawkins, M.H. Bracey, Y. Liu

Our laboratory is interested in understanding complex physiology and behavior at the level of chemistry and molecules. At the center of cross talk between different physiologic processes are endogenous compounds that act as a molecular mode for intersystem communication. However, many of these molecular messages remain unknown, and even in the cases in which the participating molecules have been defined, the mechanisms by which these compounds function are for the most part still a mystery.

Our current efforts focus on a family of chemical messengers termed the fatty acid amides, which affect many physiologic functions, including sleep, thermoregulation, pain sensitivity, and angiogenesis. In particular, one member of this family, oleamide, accumulates selectively in the cerebrospinal fluid of tired animals. This finding suggests that oleamide may be a molecular indicator of the organism's need for sleep, and, indeed, rats treated with oleamide fall asleep.

The in vivo levels of chemical messengers such as the fatty acid amides must be tightly regulated to maintain proper control over the influence of the messengers on brain and body physiology. We are characterizing one mechanism by which the level of fatty acid amides can be regulated in vivo. The enzyme fatty acid amide hydrolase degrades the fatty acid amides to inactive metabolites (Fig. 1). Thus, fatty acid amide hydrolase effectively terminates the signaling messages conveyed by fatty acid amides, possibly ensuring that these molecules do not generate physiologic responses in excess of their intended purpose.

We are using genetic and synthetic chemistry techniques to study the role of fatty acid amide hydrolase in the dynamic regulation of fatty acid amide levels in vivo. We are also interested in proteins responsible for both the biosynthesis of fatty acid amides and the selective uptake of the amides into cells and in determining novel molecular sites of action for these compounds.

A second major focus of the laboratory is the design and use of chemical probes for the global analysis of protein function. The evolving field of proteomics, defined as the simultaneous analysis of the complete protein content of a given cell or tissue, faces considerable conceptual and technical challenges. We hope to enhance the quality of information obtained from proteomics experiments by using chemical probes that read out the collective catalytic activities of entire classes of enzymes. These probes would thus be a means of recording variations in protein function independent of alterations in protein abundance, offering a potentially powerful and complimentary set of tools for proteome analysis.

We have succeeded in generating one such chemical reagent, FP-biotin, that targets the serine hydrolases, a large family of enzymes composed of numerous proteases, lipases, esterases, and amidases. We are using FP-biotin to explore the roles that serine hydrolases play in a variety of physiologic and pathologic processes. Additionally, in an ongoing collaboration with E.J. Sorensen, the Skaggs Institute, to expand our capacity to profile proteins in an activity-based manner, we are designing and testing chemical probes that target other enzyme families.

Publications

Adam, G.C., Cravatt, B.F., Sorensen, E.J. Profiling the specific reactivity of the proteome with nondirected activity-based probes. Chem. Biol., in press.

Cravatt. B.F., Sorensen E.J. Chemical strategies for the global analysis of protein function. Curr. Opin. Chem. Biol. 10:2613, 2000.

Kustedjo, K., Bracey, M.H., Cravatt, B.F. Torsin A and its torsion dystonia-associated mutant forms are lumenal glycoproteins that exhibit distinct subcellular localizations. J. Biol. Chem. 275:27933, 2000.

Liu, Y., Patricelli, M.P., Cravatt, B.F. Activity-based protein profiling: The serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 96:14694, 1999.

Patricelli, M.P., Cravatt, B.F. Clarifying the catalytic roles of conserved residues in the amidase signature family. J. Biol. Chem. 275:19177, 2000.

Patricelli, M.P., Cravatt, B.F. Fatty acid amide hydrolase competitively degrades bioactive amides and esters through a nonconventional catalytic mechanism. Biochemistry 38:14125, 1999.

 

 







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