Prohormone-Converting Enzymes: Regulation and Evaluation of Function Using Antisense RNA

Abstract

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).

Sorting and processing of secretory proteins

“…PCI and POMC have been co-localized in the anterior corticotrophs, while PC2 and POMC mRNAs were rarely observed together. These results are in agreement with the actions of PCI and PC2 on POMC [224], and with the co-ordinate regulation of POMC, PCI and PC2 mRNAs that has been demonstrated in the pituitary [253].…”

PACE4: a subtilisin-like endoprotease with unique properties

“…This is in marked contrast with the striking changes in POMC processing seen when PC1 or PC2 are over-or under-expressed in AtT-20 cells [20,21,35,36,74]. This raised the possibility that PACE4 might need activation by another factor such as 7B2 [75,76], that PACE4 is secreted as a zymogen (like the α-subunit of endopeptidase 24.18) [77,78] or that PACE4 may not have access to the POMC in the cell (as von Willebrand Factor is not a good intracellular substrate for PC1 and PC2, although furin activates von Willebrand Factor well) [79].…”

“…The expression vector for rat PACE4s (truncated PACE4) was created in an identical manner but with a stop codon inserted to make the protein terminate after amino acid residue 650, producing a molecule the same size as PC2 [20]. The PACE4s protein lacks the entire Cysrich region (residues 663-937) [13].…”

Antisense Oligonucleotides in the Study of Neuroendocrine Systems

“…A generalized effect on neurosecretion could be ruled out since there were no changes in the level of chromogranin, another precursor of bioactive peptides. Chronic changes in peptide secretion were observed after the genomic introduction of an antisense oligomer for a processing enzyme in cultured AtT20 endocrine cells (60).…”