Substantial progress has been made in deciphering the enzymatic mechanisms responsible for opioid 3eptide synthesis within the past decade. Opioid precursors are now known to be cleaved by prohormone convertases 1 and 2 (PC1 and PC2), subtilisin-like proteases restricted to neuroendocrine tissues, to produce small bioactive opioid peptides. Our work during the past two cycles of this grant has focused on the expression and biochemical characterization of PC1 and PC2. We have produced both enzymes in recombinant form, and have defined their specificity on the model opioid peptide precursor proenkephalin, both in vitro and in vivo, and have identified potent inhibitors of both enzymes through the use of combinatorial peptide library screening. Most recently, in collaboration with the crystallography group of Wolfram Bode, we have obtained the first crystal structure of this family of enzymes, the related enzyme furin. In this renewal application, we propose to apply the structural information on convertases gained thus far to perform site-directed mutagenesis of PC1 and PC2; this will help us to understand the molecular basis for the specific biochemical properties of each enzymes. We will continue our collaboration with the Bode group to obtain the crystal structures of PC1, PC2 and proPC2. We will also continue our successful collaboration with the Houghten group in identifying specific and potent non-peptide convertase inhibitors. Lastly, in collaboration with Melvin Simon, we will combine the reagents generated through previous funding of this grant, recombinant proenkephalin and recombinant convertases, with other recombinant substrates and processing enzymes to produce a robust in vitro system for the synthesis of natural peptides from recombinant precursors for functional studies. Taken together these studies will provide us with novel information on the biochemical mechanisms of opioid peptide formation and may provide reagents useful for he therapeutic inhibition of convertases. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA005084-22
Application #
7473173
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Purohit, Vishnudutt
Project Start
1988-04-01
Project End
2010-02-27
Budget Start
2008-08-01
Budget End
2010-02-27
Support Year
22
Fiscal Year
2008
Total Cost
$313,610
Indirect Cost
Name
University of Maryland Baltimore
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Hardes, Kornelia; Ivanova, Teodora; Thaa, Bastian et al. (2017) Elongated and Shortened Peptidomimetic Inhibitors of the Proprotein Convertase Furin. ChemMedChem 12:613-620
Winters, Alexandra; Ramos-Molina, Bruno; Jarvela, Timothy S et al. (2017) Functional analysis of PCSK2 coding variants: A founder effect in the Old Order Amish population. Diabetes Res Clin Pract 131:82-90
Ramos-Molina, B; Martin, M G; Lindberg, I (2016) PCSK1 Variants and Human Obesity. Prog Mol Biol Transl Sci 140:47-74
Ramos-Molina, Bruno; Lick, Adam N; Blanco, Elias H et al. (2015) Identification of potent and compartment-selective small molecule furin inhibitors using cell-based assays. Biochem Pharmacol 96:107-18
Lindberg, Iris; Shorter, James; Wiseman, R Luke et al. (2015) Chaperones in Neurodegeneration. J Neurosci 35:13853-9
Ramos-Molina, Bruno; Lick, Adam N; Nasrolahi Shirazi, Amir et al. (2015) Cationic Cell-Penetrating Peptides Are Potent Furin Inhibitors. PLoS One 10:e0130417
Blanco, Elias H; Ramos-Molina, Bruno; Lindberg, Iris (2015) Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants. Endocrinology 156:3625-37
Blanco, Elias H; Peinado, Juan R; Martín, Martín G et al. (2014) Biochemical and cell biological properties of the human prohormone convertase 1/3 Ser357Gly mutation: a PC1/3 hypermorph. Endocrinology 155:3434-47
Liew, Chong Wee; Assmann, Anke; Templin, Andrew T et al. (2014) Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic ? cells. Proc Natl Acad Sci U S A 111:E2319-28
Prabhu, Yogikala; Blanco, Elias H; Liu, Ming et al. (2014) Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function. Endocrinology 155:2391-401

Showing the most recent 10 out of 50 publications