The objectives of this program are to discover the origin of the unique specificities observed in protein cleavage reactions catalyzed by the aspartic proteinase family. Examples of specificity range from broad, for digestive enzymes, to limited, for enzymes that participate in activation of a single prohormone. As all members of this family have similar three-dimensional structures and catalytic mechanisms, design of selective inhibitors must be based on details of the differences within the active sites. By combining the use of synthetic oligopeptide substrates for kinetic studies, site-directed mutagenesis of residues within the active site clefts contributing to binding and specificity, and comparisons within the family, the precise rationale for tight, specific binding will be discovered. Efforts will focus on the following specific aims: 1. Expansion of the study of active site interactions into the prime region of the cleft. 2. Comparison of the structure and properties of human cathepsin E with the other mammalian enzymes, pepsin and cathepsin D. 3. Generation and analysis of chimeric constructs in which N-terminal and C-terminal domains are interchanged to evaluate the effect of long-range electrostatic effects. 4. Evaluation of the relationship between the three-dimensional structure of the Ascaris pepsin inhibitor and the unique specificity of inhibition.
Aim 1 will continue the examination of the extended active site cleft by making new mutations in pepsin, cathepsin D, and a new objective, cathepsin E, which will be studied in detail in aim 2 of the new proposal.
Aim 3 proposes an expansion of a mutagenesis strategy through the creation of new protein species where the role of the overall electrostatic environment and mixed backgrounds for point mutations will be explored.
In aim 4, the intent is to expand investigation at the protein-protein level by obtaining the structure of a specific natural protein inhibitor. The molecular origins of the strong pepsin directed specificity of the Ascaris inhibitor may be found.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK018865-22
Application #
2856714
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
May, Michael K
Project Start
1978-06-01
Project End
2001-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
22
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Florida
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073130411
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Janka, Linda; Clemente, Jose; Vaiana, N et al. (2008) Targeting the plasmepsin 4 orthologs of Plasmodium sp. with ""double drug"" inhibitors. Protein Pept Lett 15:868-73
Moose, Rebecca E; Clemente, Jose C; Jackson, Larry R et al. (2007) Analysis of binding interactions of pepsin inhibitor-3 to mammalian and malarial aspartic proteases. Biochemistry 46:14198-205
Clemente, Jose C; Govindasamy, Lakshmanan; Madabushi, Amrita et al. (2006) Structure of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae bound to an allophenylnorstatine-based inhibitor. Acta Crystallogr D Biol Crystallogr 62:246-52
Goldfarb, Nathan E; Lam, Minh T; Bose, Arjo K et al. (2005) Electrostatic switches that mediate the pH-dependent conformational change of ""short"" recombinant human pseudocathepsin D. Biochemistry 44:15725-33
Beyer, Bret B; Johnson, Jodie V; Chung, Alfred Y et al. (2005) Active-site specificity of digestive aspartic peptidases from the four species of Plasmodium that infect humans using chromogenic combinatorial peptide libraries. Biochemistry 44:1768-79
Madabushi, Amrita; Chakraborty, Sibani; Fisher, S Zoe et al. (2005) Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae. Acta Crystallogr Sect F Struct Biol Cryst Commun 61:228-31
Wlodawer, Alexander; Li, Mi; Gustchina, Alla et al. (2004) Two inhibitor molecules bound in the active site of Pseudomonas sedolisin: a model for the bi-product complex following cleavage of a peptide substrate. Biochem Biophys Res Commun 314:638-45
Li, Tang; Yowell, Charles A; Beyer, Bret B et al. (2004) Recombinant expression and enzymatic subsite characterization of plasmepsin 4 from the four Plasmodium species infecting man. Mol Biochem Parasitol 135:101-9
Wlodawer, Alexander; Durell, Stewart R; Li, Mi et al. (2003) A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases. BMC Struct Biol 3:8
Wlodawer, Alexander; Li, Mi; Gustchina, Alla et al. (2003) Structural and enzymatic properties of the sedolisin family of serine-carboxyl peptidases. Acta Biochim Pol 50:81-102

Showing the most recent 10 out of 31 publications