The opioid peptide precursors proopiomelanocortin, proenkephalin, and prodynorphin are first synthesized as large inactive proteins which must be activated by proteolytic maturation. The work in this grant focuses on the enzymes that accomplish this proteolytic step, prohormone convertases 1 and 2 (PC1 and PC2). During past funding cycles we have expressed and purified recombinant enzymes in an effort to characterize their biochemical properties and specificity. We later established their cleavage preferences for proenkephalin, both within cells and in vitro. In collaboration with Wolfram Bode and Manuel Than, we obtained the first crystal structure of a related family member, furin. Our long-standing collaboration with the Torrey Pines Institute for Molecular Studies has identified potent furin and PC-inhibitory short peptides;and in the last funding cycle, we successfully obtained stable small molecule PC2 inhibitors with micromolar potency. In the next funding cycle, we propose to continue our successful collaborations with these two groups. We will continue our crystallization of different forms of PC1, and expand our inhibition work both by chemical modification of current hits, as well as by screening further compounds and libraries. Lastly, since our recent work shows that both PC2 as well as the small form of PC1 are extremely susceptible to mixed-disulfide aggregation, we will investigate the role of this phenomenon in the regulation of PC1 activity. We will perform new studies on labile, aggregation-prone forms of PC1 to ask whether either chemical chaperones or interaction with the natural PC1 binding protein proSAAS can prevent PC1 aggregation and preserve enzymatic activity. We will attempt to derive structure-function information for the proSAAS-PC1 interaction, which may represent a regulatory mechanism in vivo. Taken together, these studies are expected provide additional information on the biochemical mechanisms involved in neuropeptide formation, including the opioid peptides met- and leu-enkephalin. Additionally this work should lead to structures and reagents useful for the therapeutic inhibition of convertases.
The work in this proposal will give us a more complete understanding of biosynthetic mechanisms involved in the production of neuropeptides, using opioid peptide synthesis as a model system. We are also interested in describing the molecular forms of a new peptide precursor we have observed in brain tissue.
|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|
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