Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P5-P1 part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'1-P'2 dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'1-S'2 sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P4 is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P2 causes an increase of the substrate preference towards this activity.
Periodontal diseases are the most prevalent bacterial ailments, affecting 10–15% of the global population, and eventually leading to tooth loss if left untreated. Shogaols obtained from ginger (Zingiber officinale) exhibit significant anti-inflammatory activity. However, the antibacterial potential of shogaols against periodontitis remains unexplored. Therefore, we investigated the effects of 6-shogaol (6-SH) on various factors responsible for periodontitis such as inflammation. Escherichia coli endotoxin was used to induce experimental periodontitis, and the effects of 6-SH on hydrogen peroxide, superoxide anion, myeloperoxidase activity, and lipid peroxides were estimated together with cathepsin B, cathepsin D, β-glucuronidase, acid phosphatase, and C-reactive protein activity in serum. In addition, the levels of ascorbic acid, α-tocopherol, ceruloplasmin, reduced glutathione, superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase were estimated in serum after 6-SH treatment. Reactive oxygen species and lipid peroxide levels were significantly reduced in the 6-SH-treated group. Moreover, lysosomal enzyme (cathepsin B, cathepsin D, β-glucuronidase and acid phosphatase) and acute-phase protein (C-reactive protein and fibrinogen) levels significantly declined after administration of 6-SH. Meanwhile, non-enzymatic antioxidant systems (e.g., ascorbic acid, α-tocopherol, ceruloplasmin, and reduced glutathione) and antioxidant enzymes (e.g., catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferase) were significantly increased in the 6-SH-treated group. These results suggest a protective effect of 6-SH against experimental periodontitis via the regulation of key disease markers.
The extracellular matrix components are differentially distributed among various structures of the umbilical cord. Wharton's jelly is especially rich in collagens and growth factors. Cathepsin B is a major cysteine protease involved in collagen degradation, as well as in the activation of precursor forms of other collagenolytic enzymes and growth factors. We assessed the activity and expression of cathepsin B in the umbilical cord arteries, veins and Wharton's jelly. Extracts of separated umbilical cord components were subjected to an activity assay with the use of specific fluorogenic substrate. The expression of cathepsin B protein was qualitatively evaluated by Western immunoblotting and quantitatively determined with an immunoenzymatic method. The total cathepsin B activity and content calculated per gram of DNA were higher in Wharton's jelly than in the umbilical cord vessels, and the latter parameter was the lowest in the umbilical cord arteries. Moreover, the expression and the activity of latent cathepsin B (following activation by pepsin digestion) calculated per gram of DNA were the highest in Wharton's jelly and the lowest in the umbilical cord arteries. High expression and activity of latent, pepsin-activatable cathepsin B related to DNA content in Wharton's jelly seem to reflect the stimulation of its cells by high amounts of collagen I and growth factors.
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