Abstract

The objective of this proposal is to investigate the substrate binding, isoforms, associated proteins, and substrate size specificity of E.C.3.4.24.15 (EP24.15), with the goal of availing design of new therapeutic drugs and small molecules, to modulate development of neuroendocrine/neurodegenerative diseases and endocrine cancer(s). EP24.15 is the prototypic member of the metalloendopeptidase family, and is widely expressed in the brain, pituitary, and reproductive organs. Extracellularly, EP24.15 cleaves and modulates neuropeptides, and thus EP24.15 plays a significant role in neuroendocrine hormone regulation, pain perception, development of neurodegenerative diseases, and prostate cancer cell growth. The goal of this proposal is to further the understanding of the functions of EP24.15, of its isoforms, and of closely related metalloendopeptidases, with the goal of understandingthe prominence of EP24.15 in neuroendocrine/neurodegenerative diseases and cancer. Therefore, this proposal will integrate biochemical, genetic, structural, and proteomic approaches to study substrate binding, isoforms, associated proteins, and substrate size specificity of EP24.15. Therefore, the specific aims are:
SPECIFIC AIM 1. Elucidate functional sites conferring substrate binding and catalysis.
Aim la. Determine which residues comprise the substrate (inhibitor) binding site of EP24.15.
Aim I b. Determine which residues are crucial for substrate catalysis and specificity.
SPECIFIC AIM 2. Determine isoforms and protein binding partners of EP24.15 and of related enzymes.
Aim 2 a. Identify the extracellular and intracellular isoforms of EP24.15 and EP24.16.
Aim 2 b. Characterize complexes of EP24.15 with associated protein-binding partners.
SPECIFIC AIM 3. Unveil new substrates and mechanism of substrate size selectivity.
Aim 3 a. Determine what other (neuro)peptide substrates exist for EP24.15.
Aim 3 b. Elucidate structural changes upon substrate binding, focusing on domain movements.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS039892-10
Application #
7534327
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Gnadt, James W
Project Start
2000-04-20
Project End
2011-08-31
Budget Start
2008-12-01
Budget End
2011-08-31
Support Year
10
Fiscal Year
2009
Total Cost
$332,082
Indirect Cost

  Institution

Name
Rosalind Franklin University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
069501252
City
North Chicago
State
IL
Country
United States
Zip Code
60064

  Publications

Gomez, Ruben; Por, Elaine D; Berg, Kelly A et al. (2011) Metallopeptidase inhibition potentiates bradykinin-induced hyperalgesia. Pain 152:1548-54
Russo, Lilian C; Goñi, Camila N; Castro, Leandro M et al. (2009) Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation. FEBS J 276:4358-71
Bruce, Lisa A; Sigman, Jeffrey A; Randall, Danica et al. (2008) Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection. FEBS J 275:5607-17
Huang, J Y; Bruno, A M; Patel, C A et al. (2008) Human membrane metallo-endopeptidase-like protein degrades both beta-amyloid 42 and beta-amyloid 40. Neuroscience 155:258-62
Roberts, James L; Mani, Shaila K; Woller, Michael J et al. (2007) LHRH-(1-5): a bioactive peptide regulating reproduction. Trends Endocrinol Metab 18:386-92
Jeske, Nathaniel A; Berg, Kelly A; Cousins, Joanne C et al. (2006) Modulation of bradykinin signaling by EP24.15 and EP24.16 in cultured trigeminal ganglia. J Neurochem 97:13-21
Scholle, Michael D; Kriplani, Ushma; Pabon, Amanda et al. (2006) Mapping protease substrates by using a biotinylated phage substrate library. Chembiochem 7:834-8
Oliveira, Vitor; Garrido, Paula A G; Rodrigues, Claudia C et al. (2005) Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity. FEBS J 272:2978-92
Sigman, Jeffrey A; Patwa, Tasneem H; Tablante, Ana V et al. (2005) Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies. Biochem J 388:255-61
Jeske, Nathaniel A; Glucksman, Marc J; Roberts, James L (2004) Metalloendopeptidase EC3.4.24.15 is constitutively released from the exofacial leaflet of lipid rafts in GT1-7 cells. J Neurochem 90:819-28

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