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.
|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|
|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|
|Wilschanski, Michael; Abbasi, Montaser; Blanco, Elias et al. (2014) A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity. PLoS One 9:e108878|
|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|
|Pickett, Lindsay A; Yourshaw, Michael; Albornoz, Valeria et al. (2013) Functional consequences of a novel variant of PCSK1. PLoS One 8:e55065|
|Martín, Martín G; Lindberg, Iris; Solorzano-Vargas, R Sergio et al. (2013) Congenital proprotein convertase 1/3 deficiency causes malabsorptive diarrhea and other endocrinopathies in a pediatric cohort. Gastroenterology 145:138-48|
|Yongye, Austin B; Vivoli, Mirella; Lindberg, Iris et al. (2013) Identification of a small molecule that selectively inhibits mouse PC2 over mouse PC1/3: a computational and experimental study. PLoS One 8:e56957|
|Becker, Gero L; Lu, Yinghui; Hardes, Kornelia et al. (2012) Highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases. J Biol Chem 287:21992-2003|
|Lindberg, Iris; Appel, Jon R (2011) Inhibitor screening of proprotein convertases using positional scanning libraries. Methods Mol Biol 768:155-66|
|Kuester, Miriam; Becker, Gero L; Hardes, Kornelia et al. (2011) Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors. Biol Chem 392:973-81|
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