The proprotein convertases (PCs) are involved in the activation of a wide variety of precursors via limited proteolysis at either single or paired basic residues, a crucial regulatory process in both normal and disease states. Seven members of this family were identified including furin, PC I, PC2, PC4, PACE4, PC5 and PC7. All of which exhibit a signal peptide, a prosegment, a catalytic domain, a P-domain and a specific C-terminal segment. The present work concentrated on the characterization of two structural elements, namely the prosegment and the P-domain, which are critical for enzymatic function and cellular trafficking. We examined the biosynthesis, functional activity and cellular localization of two PACE4 isoforms generated by differential splicing, the full length PACE4-A and the C-terminally truncated PACE4-CS that lacks 11 amino acids at the end of its chaperone-like P-domain. Cellular expression demonstrated that PACE4-A codes for a functional secretable enzyme capable of cleaving pro7B2 into 7132. However, PACE4-CS is not secreted and remains in the endoplasmic reticulum as an inactive zymogen form, therefore emphasizing the importance of the integrity of the P-domain. The prosegment is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. We purified recombinant prosegments of furin (pFurin) and PC7 (pPC7) from bacteria. The inhibitory potencies and selectivities of the prosegments were tested in vitro and ex vivo. The pFurin and pPC7 are potent inhibitors (IC50 low nM) of their parent enzymes. Whereas pPC7 is more selective than pFurin. Most of the inhibitory potency seems to reside at the C-terminal region immediately preceding the primary cleavage site of the prosegment with the P1 Arg playing a critical role. Furthermore, overexpression of prosegments in cell lines efficiently blocked precursor processing of the neurotrophins NGF and BDNF. For the first time our results showed that PC prosegments expressed ex
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37603 |
| Date | January 1999 |
| Creators | Zhong, Mei, 1969- |
| Contributors | Seidah, Nabil G. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001740492, proquestno: NQ64707, Theses scanned by UMI/ProQuest. |
Page generated in 0.1103 seconds