Precursor Protein Processing Inhibitors

Many biological proteins are synthesized as precursor which are activated via the limited proteolysis at the peptide bond composed of paired basic residues with proprotein convertases(PCs). Thus, these PCs play a very important role in the generation of many physiological and pathological active peptides or proteins.The progress in PCs and their inhibitors in recent yearswas reviewed, including its tissue localization, maturation and modification after translation, its relationship with diseases and pathogens as well as the studies. PCs are involved in many diseases and pathogens, such as the infection of HIV and the intrusion of anthrax. Base on the previous work, we chosed a mutantof eglin c (P1, P4 are Arg) as the starting material to be further bioengineered at its P' positions. The results showed that 1) the residue Leu47 at P2' substituted with either a positively or negatively charged residue resulted in a decrease in inhibitory activities to both enzymes. 2) The substitution of Arg with Asp at P3' was favorable for inhibiting furin with a Ki of 7.8X10-9 M, but not for inhibiting kexin 3) The substitution of Tyr with Glu at P4' increased the inhibitory activity to kexin, with a Ki of 3X10-11M, but almost without any influence on furin inhibition. It was indicated that the inhibitory specificity of eglin c could be changed from inhibiting elastase to inhibiting PCs by site-directed mutation at the P positions, while the inhibitory selectivity to furin or kexin could be optimized by mutation at the P' positions.The natural inhibitor has strong inhibition to kexin. The IC50 is about 10-8M. Its gene was then cloned and sequenced. ORF (open-reading frame) encodes 106 residues including a signal peptide (20 residues), a pro-peptide (12 residues) and a mature peptide (74 residues). The genomic sequence analysis of the inhibitor reveals that it contains no intron. In order to obtain a mutant of this inhibitor with more strong inhibitory activity to furin and kexin, the mutagenesis and gene expression of this inhibitor is being carried out.