The sorting signal motif for targeting pro-opiomelanocortin (POMC, pro-ACTH/endorphin) pro-enkephalin (pro-ENK) and pro- insulin to the regulated secretory pathway (RSP) was identified. Site directed mutagenesis studies identified a concensus sorting motif containing two pairs of acidic/hydrophobic residues exposed on the surface of these three molecules. For POMC, the residues are D10, L11; E14, L18 located at the N-terminus. A similar sorting motif consisting of residues D18, I19; E29,L32 was found in the N-terminus of pro-enkephalin. In monomeric proinsulin, the sorting signal motif consists of residues E13 and L17 located on the B chain and L16 and E17 located on the A chain. In hexameric proinsulin, residue E13 on the B chain is burried and the motif is contributed by the two residues in the A chain from two adjacent proinsulin dimers in the hexamer. A RSP sorting receptor that is specific for the sorting signal of POMC, pro- insulin and pro-enkephalin was identified as membrane carboxypeptidase E (CPE). CPE was shown to be a transmembrane protein which is anchored in cholesterol-glycosphingolipid rich microdomains in the trans-golgi network (TGN). The acidic residues in the prohormone sorting signal motif specifically bind the two basic residues, R255 and K260, on CPE, to effect sorting at the TGN. Depletion of CPE by antisense RNA in Neuro2a cells resulted in the missorting of POMC, pro- enkephalin and pro-insulin to the constitutive pathway, indicating that CPE functions as a sorting receptor in vivo. Using a mouse model which synthesizes a mutant CPE that is differentially degraded in pituitary and pancreas, we were able to show a correlation between lowered CPE levels and the degree of missorting of endogenous prohormones in the cells of these tissues. These studies provide evidence for a sorting signal/receptor mediated mechanism for sorting prohormones to the regulated secretory pathway in neuro-endocrine cells.The biosynthesis, processing and molecular basis for the specificity of yapsin 1, a member of the novel class of yapsin aspartic proteases was studied. Yapsin 1, a yeast prohormone processing enzyme is synthesized as an inactive proenzyme and is activated by removal of the pro-region. The enzyme is first cleaved internally in the proregion autocatalytically to form pseudoyapsin 1. Pseudoyapsin 1 then undergoes autocatalytic cleavage 10-20 residues upstream of Asp45 to yield two subunits, a and b, which are linked by a disulfide bridge. Pulse-chase studies using wild type and the Sec 18 yeast mutant showed that these processing steps occur in the endoplasmic reticulum. Prior to secretion, the remaining pro- region is cleaved to form mature yapsin 1. Molecular modeling of yapsin1 revealed that it has an open, highly electronegative active site pocket in the S1 subsite, favoring substrates with a basic residue in the P1 position. The model also showed that the S6, S2, and S3 subsites were electronegative, accounting for our observations that substrates with basic residues in the P6, P2 and P3 positions are cleaved with higher catalytic efficiency due to enhanced binding to these active site pockets. Another member, yapsin 3 was cloned and shown to preferentially cleave single Lys of peptide precursors. This differs from yapsin 1 which prefers paired basic residues. Analysis of yapsin 2 specificity indicated that it prefers to cleave after a pair of Arg or Lys, differing from yapsin 1 which cleaves in between the Lys. Thus the various members of the yapsin family show subtle differences in specificity and are likely tailored to cleave different substrates in vivo.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Intramural Research (Z01)
Project #
1Z01HD000056-24
Application #
6290145
Study Section
Special Emphasis Panel (LDN)
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
1999
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Cawley, Niamh X; Li, Zhaojin; Loh, Y Peng (2016) 60 YEARS OF POMC: Biosynthesis, trafficking, and secretion of pro-opiomelanocortin-derived peptides. J Mol Endocrinol 56:T77-97
Cawley, Niamh X; Portela-Gomes, Guida; Lou, Hong et al. (2011) Yapsin 1 immunoreactivity in {alpha}-cells of human pancreatic islets: implications for the processing of human proglucagon by mammalian aspartic proteases. J Endocrinol 210:181-7
Koshimizu, Hisatsugu; Senatorov, Vladimir; Loh, Y Peng et al. (2009) Neuroprotective protein and carboxypeptidase E. J Mol Neurosci 39:1-8
Park, Joshua J; Cawley, Niamh X; Loh, Y Peng (2008) A bi-directional carboxypeptidase E-driven transport mechanism controls BDNF vesicle homeostasis in hippocampal neurons. Mol Cell Neurosci 39:63-73
Park, Joshua J; Loh, Y Peng (2008) How Peptide Hormone Vesicles are Transported to the Secretion Site for Exocytosis. Mol Endocrinol :
Samuni, Yuval; Zheng, Changyu; Cawley, Niamh X et al. (2008) Sorting of growth hormone-erythropoietin fusion proteins in rat salivary glands. Biochem Biophys Res Commun 373:136-9
Arnaoutova, Irina; Cawley, Niamh X; Patel, Nimesh et al. (2008) Aquaporin 1 is important for maintaining secretory granule biogenesis in endocrine cells. Mol Endocrinol 22:1924-34
Samuni, Yuval; Cawley, Niamh X; Zheng, Changyu et al. (2008) Sorting behavior of a transgenic erythropoietin-growth hormone fusion protein in murine salivary glands. Hum Gene Ther 19:279-86
Park, Joshua J; Cawley, Niamh X; Loh, Y Peng (2008) Carboxypeptidase E cytoplasmic tail-driven vesicle transport is key for activity-dependent secretion of peptide hormones. Mol Endocrinol 22:989-1005
Woronowicz, Alicja; Koshimizu, Hisatsugu; Chang, Su-Youne et al. (2008) Absence of carboxypeptidase E leads to adult hippocampal neuronal degeneration and memory deficits. Hippocampus 18:1051-63

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