Abstract

The overall objective of this application is to elucidate the biochemical functions of E.C.3.4.24.15 [EP24.15], the prototypical member of the mammalian family of endopeptidases that process/modulate neuropeptides. EP24.15 is present in brain, pituitary, and gonads, and has been directly implicated in critical physiologic pathways and neuroendocrine/neurodegenerative disorders and cancer. This enzyme processes neuropeptides extracellularly and thus is crucial in regulating signal transduction events essential to cell growth and differentiation. Understanding the function of EP24. 15 and its interaction with substrates and inhibitors will provide unique insight into aspects of normal and disease states and will aid in the design of new therapeutic agents for a variety of disorders. Experiments in this application will integrate biochemical, genetic, theoretical, structural and cell biological approaches to studying the enzyme-ligand interactions of this metalloenzyme. These strategies are also applicable to other macromolecules. Therefore, the interrelated specific aims of this project are:
Specific Aim 1. Determinatlon of elements responsible for substrate binding and catalvsis in EP24.15 EP24. 15 protein will be functionally characterized utilizing structural genetics and kinetic determinations.
Aim la. What amino acid deterrninants are responsible for the catalysis of neuropeptides? Aim lb. Which residues comprise the substrate [and inhibitor] binding subsites of the enzyme? Specific Aim 2. Define components modulatingr EP24. 15 activitv. localization. and substrate size selectivity. 4.15 is phosphorylated in vitro/in vivo, present in the nucleus, and though an endooligopeptidase, can cleave some large substrates.
Aim 2 a. Determine the effect of phosphorylation upon EP24. 15 enzyme activity.
Aim 2 b. What are the structural determinants of the enzyme which translocate it to the nucleus? Aim 2c. EP24. 15 is defined as an 'endooligopeptidase'. What is the mechanism of size selectivity? Specific Aim3. Modeling and higher resolution studies of EP24. 15 Structural biological approaches: molecular modeling of homologous domains and diffraction analyses.
Aim 3 a. Build a prototype from bacterial homologues for this class of mammalian enzymes .
Aim 3 b. Execute multiwavelength anomalous diffraction analyses of EP24. 15.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS039892-01
Application #
6086663
Study Section
Special Emphasis Panel (ZRG1-MDCN-5 (01))
Program Officer
Marler, John R
Project Start
2000-04-20
Project End
2005-03-31
Budget Start
2000-04-20
Budget End
2001-03-31
Support Year
1
Fiscal Year
2000
Total Cost
$279,598
Indirect Cost

  Institution

Name
Mount Sinai School of Medicine
Department
Neurosciences
Type
Schools of Medicine
DUNS #
114400633
City
New York
State
NY
Country
United States
Zip Code
10029

  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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