The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity.
All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.
Zhong
M
M
Laboratory of Biochemical, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7.
Munzer
J S
JS
Basak
A
A
Benjannet
S
S
Mowla
S J
SJ
Decroly
E
E
Chrétien
M
M
Seidah
N G
NG
D000595:Amino Acid Sequence; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D045683:Furin; D015870:Gene Expression; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D020932:Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019208:Brain-Derived Neurotrophic Factor; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D020932:Nerve Growth Factor; D013381:Subtilisins; C107583:proprotein convertase PC7; D045683:Furin