Abstract
BackgroundBefore furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.Methodology/Principal FindingsWe constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg107↓Asp108, but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg75↓Ser76) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S′ sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg89↓Glu90 site, is required for the efficient activation of furin.Conclusions/SignificanceCollectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases.
Highlights
A variety of proteins, including serine proteinases and metalloproteinases, growth factors, and adhesion molecules as well as bacterial and viral pathogens, is initially synthesized as inactive precursors
Modeling of the activation mechanism In our modeled profurin structure, the relative position of the prodomain and the catalytic domain was similar to that reported for the subtilisin-propeptide complex (Protein Data Bank code 1SCJ)
To prevent unwanted protein degradation and to enable spatial and temporal regulation of proteolytic activity, proteolytic enzymes are synthesized as latent precursors [9,33]
Summary
A variety of proteins, including serine proteinases and metalloproteinases, growth factors, and adhesion molecules as well as bacterial and viral pathogens, is initially synthesized as inactive precursors. Specific processing is required to transform these proproteins into biologically active proteins [1]. Furin and related proprotein convertases (PCs) are specialized serine endoproteinases, which cleave the multibasic motifs R-X-R/K/ X-R to transform proproteins into biologically active proteins and peptides [2]. Furin is synthesized as a preproprotein which contains a signal peptide, a prodomain, a subtilisin-like catalytic domain, a middle P domain, a cysteine-rich region, a transmembrane anchor and a cytoplasmic tail. Furin and other PCs require proteolytic removal of the inhibitory prodomain to become proteolytically potent enzymes [2,9]. Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme’s N-terminus had to be ejected from the catalytic cleft
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