Identification of furin pro-region determinants involved in folding and activation.
The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the trans -Golgi network. To identify residues that are important for pro-region function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's pro-region.
Bissonnette
Lyne
L
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, 3001, 12th Ave. North, Sherbrooke, Quebec, Canada J1H 5N4.
Charest
Gabriel
G
Longpré
Jean-Michel
JM
Lavigne
Pierre
P
Leduc
Richard
R
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002460:Cell Line; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045683:Furin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism
D004792:Enzyme Precursors; D011498:Protein Precursors; D014841:von Willebrand Factor; C067003:von Willebrand factor propolypeptide; D045683:Furin