The long-term goal of this project is to describe the biochemical mechanisms of the lipolytic enzymes responsible for dietary fat digestion. Dietary fats provide a major source of energy, essential fatty acids, a vehicle for fat-soluble vitamins, and components of cellular membranes. Fats eaten in excess adversely affect health increasing the risk of cardiovascular disease and obesity. Before fats can be absorbed, they must be digested. Lipases in the stomach and duodenum digest dietary fats. This proposal focuses on the lipase responsible for the majority of fat digestion, pancreatic triglyceride lipase, and another pancreatic protein, colipase, which is required, by pancreatic triglyceride lipase for activity. We propose that specific interactions among lipase, colipase, lipids, and bile salts, which are necessary for absorption of the fatty acids released by lipase, dictate the function of pancreatic triglyceride lipase. The first specific aim is to characterize the mechanisms that mediate specific interactions among lipase, colipase, bile salts, and lipids. We will substitute selected amino acids in lipase or colipase by site-specific mutagenesis to identify functionally important regions of both proteins. The second specific aim is to determine the mechanisms that trigger the conversion of lipase from an inactive to an active conformation. In aqueous solution lipase is inactive. When lipase encounters the lipid substrate its conformation changes and it becomes active. We will follow the conformational change by tryptophan fluorescence to determine the factors that influence this critical conformational change. The last specific aim is to develop an expression system for labeling recombinant colipase with stable isotopes. The ability to label colipase with stable isotopes will enable NMR studies of the conformation of colipase in aqueous solutions under different conditions. We will test various culture conditions to improve our current system for the production of 15N labeled colipase and we will develop a system for economically labeling colipase with 13C and 15N. These studies will determine the biochemical details of the interactions between lipase, colipase, lipids, and bile salts that impact lipase function. The results will ultimately permit us to manipulate these interactions for therapeutic purposes.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD033060-09
Application #
6711029
Study Section
Special Emphasis Panel (ZRG1-SSS-T (01))
Program Officer
Grave, Gilman D
Project Start
1996-08-01
Project End
2006-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
9
Fiscal Year
2004
Total Cost
$193,557
Indirect Cost
Name
Children's Hosp Pittsburgh/Upmc Health Sys
Department
Type
DUNS #
044304145
City
Pittsburgh
State
PA
Country
United States
Zip Code
15224
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Bourbon-Freie, Angela; Dub, Rachel E; Xiao, Xunjun et al. (2009) Trp-107 and trp-253 account for the increased steady state fluorescence that accompanies the conformational change in human pancreatic triglyceride lipase induced by tetrahydrolipstatin and bile salt. J Biol Chem 284:14157-64
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Alves, Bryce N; Leong, Jeff; Tamang, David L et al. (2009) Pancreatic lipase-related protein 2 (PLRP2) induction by IL-4 in cytotoxic T lymphocytes (CTLs) and reevaluation of the negative effects of its gene ablation on cytotoxicity. J Leukoc Biol 86:701-12
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Freie, Angela Bourbon; Ferrato, Francine; Carriere, Frederic et al. (2006) Val-407 and Ile-408 in the beta5'-loop of pancreatic lipase mediate lipase-colipase interactions in the presence of bile salt micelles. J Biol Chem 281:7793-800
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Lowe, Mark E (2002) The triglyceride lipases of the pancreas. J Lipid Res 43:2007-16
Crandall, W V; Lowe, M E (2001) Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles. J Biol Chem 276:12505-12

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