The isolation of a proteinaceous putative inhibitor of cyste
The isolation of a proteinaceous putative inhibitor of cysteine proteases from the extracts of Y. enterocolitica and E. coli was performed by affinity chromatography on immobilized and inactivated papain (Fig. 7A). Instead of the column variant of this technique (Tsushima et al., 1992), we used a batch variant of affinity chromatography, which had not been previously employed to isolate the inhibitors. This method permitted the very efficient single-step isolation of the protein with a molecular weight of less than 20 kDa from both tested bacterial species. Only the eluates containing this protein inhibited the activity of papain, cathepsin L and cysteine cathepsins produced by human dermal fibroblasts (Figs. B and), hence allowing us to suggest that the isolated protein was likely an inhibitor of cysteine proteases. Since the protein could only be isolated with inactivated papain, we also speculated whether it may be a substrate rather than an inhibitor of this enzyme. Our results demonstrated that the protein was not cleaved by papain and cathepsin L used at the same concentrations as in the fluorimetric inhibition assays, but it was degraded by these peptidases used at much higher concentrations (Fig. 8). However, it has already been proven that papain and cathepsin L may degrade effectively their potent proteinaceous inhibitors (i.e., cystatins) under certain conditions (e.g., when altering the pH value or cystatin/peptidase molar ratio in the reaction mixture) (Popovič et al., 1999). The protein isolated from both Y. enterocolitica and E. coli was then identified as the periplasmic chaperone Skp. It is a homotrimeric protein with a jellyfish-like architecture, formed by three α-helical coiled-coils extending from a central β-barrel (Korndörfer et al., 2004). The SR 1555 hydrochloride of Skp are synthesized by many bacterial species of the family Enterobacteriaceae. Skp is involved in the biogenesis of diverse outer membrane proteins, many of which constitute important virulence factors; the chaperone is required for the proper folding of these proteins and protects them from aggregation during their transport across the periplasm (Schiffrin et al., 2016). Skp localizes mostly to the cytosol, periplasmic space and cell surface (Thome et al., 1990), and in the present work, the bacterial cytoplasmic extracts and cell surface were shown to exhibit inhibitory potential on cysteine proteases (Figs. 2A and C). The molecules of Skp bind to the outer membrane proteins through electrostatic and hydrophobic interactions, hence being peripherally associated with the cell surface (Qu et al., 2007), and in our study, the inhibitors of papain and cathepsin L also proved to bind peripherally to the surface of Y. enterocolitica cells (Fig. 3). Qu et al. (2007) demonstrated that Skp is structurally stable within a range of pH values from 3.0 to 11.0, and we discovered at least over 60% stability of the inhibitory activity of Y. enterocolitica extracts against cysteine proteases at similar pH values ranging from 3.0 to 12.0 (Fig. 5B). Additionally, the amino acid sequence of Skp from both Y. enterocolitica and E. coli shares minor common motifs with cystatins. The above observations support our suggestion that the chaperone Skp may function as a cysteine protease inhibitor. So far, however, such a role for this protein has not been demonstrated. Simultaneous chaperonic and inhibitory properties were shown for the propeptide domains of zymogenic cysteine cathepsins, which assist in the proper folding of the proenzymes and prevent their premature activation (Wiederanders et al., 2003). We isolated Skp together with much lower amount of several ribosomal proteins. Since Skp is a cationic protein, which may interact strongly with other cellular components, such as the negatively charged DNA molecules, lipopolysaccharides and ribosomes (Thome et al., 1990), the ribosomal proteins could be co-isolated in our chromatographic approach through their affinity towards Skp rather than papain. Besides, since Skp is exposed on the bacterial cell surface, we suggest that it may interact directly with the lysosomal cysteine cathepsins once the bacteria are internalized by phagocytes and enter the phagosomes. Through the inhibition of the cathepsins involved in immune responses (e.g., cathepsin L), Skp may lead to the limitation of these responses and, hence, survival of the bacteria within their host.