Propeptides of papain-like cysteine proteinases such as papain, cathepsins B, L and S are potent inhibitors of their cognate cysteine proteinases with Ki values in the nanomolar range, and they exhibit highest inhibition selectivity for enzymes from which they originate. Recent studies have identified novel inhibitor proteins that are homologous to the proregions of papain-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. CTLA-2 exhibits inhibitory activities to several cysteine proteinases. We have also identified a similar propeptide-like cysteine proteinase inhibitor, Bombyx cysteine proteinase inhibitor (BCPI), in the silkmoth Bombyx mori. BCPI is a slow and tight binding inhibitor of cathepsin L-like cysteine proteinases with Ki values in picomolar range, and the inhibition is highly selective towards these proteinases just like the propeptides. Recent genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.
Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.
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