Catalytic Activity Of Cathepsin Research Articles

Cellular proliferation in mouse and human pancreatic islets is regulated by serpin B13 inhibition and downstream targeting of E-cadherin by cathepsin L.

We previously reported that exposure to antibodies neutralising serpin B13, a protease inhibitor expressed in exocrine pancreatic ducts, promotes beta cell proliferation, underscoring the importance of a functional relationship between exocrine and endocrine pancreas. The aim of the present study was to identify the molecular events that link inhibition of serpin B13 to islet cell proliferation. We used an in vitro culture system consisting of isolated pancreatic islets, an extract of pancreatic ductal epithelium and a monoclonal antibody (mAb) to serpin B13 or IgG isotype control. In vivo studies involved treatment of mice with these mAbs. The catalytic activity of cathepsin L (CatL), a cysteine protease target of serpin B13, was augmented in the pancreas of mice injected with serpin B13 mAb. Furthermore, the addition of serpin B13 mAb to the islets, together with the pancreatic ductal epithelium lysate, caused CatL-dependent cleavage of E-cadherin and concomitant upregulation of REG genes, ultimately leading to beta cell proliferation. Direct blockade of E-cadherin with mAb also markedly enhanced REG gene induction, while chemical inhibition of β-catenin, a binding target of E-cadherin, prevented the serpin B13 mAb-induced upregulation of REG genes. Our work implicates the CatL-E-cadherin-REG pathway in the regulation of islet cell proliferation in response to signals generated in exocrine pancreatic tissue and demonstrates that protease activity may promote adaptive changes in the islets. Microarray data that support the findings of this study have been deposited in Gene Expression Omnibus (GEO) with the accession no. GSE125151.

Cathepsin G and thrombin: evidence for two different platelet receptors

Neutrophil cathepsin G and thrombin, the only platelet agonists that are proteases, exhibit a mandatory requirement for catalytic activity to induce platelet aggregation and signal transduction. The thrombin receptor is a G-protein-coupled receptor which undergoes proteolysis to generate a tethered ligand that causes self-activation. Since cathepsin G strongly resembles thrombin in its ability to activate platelets, we have attempted to determine whether cathepsin G and thrombin function through the same or different receptors. Evidence that thrombin and cathepsin G act at different receptors was as follows: (a) an antibody directed against the thrombin receptor blocked thrombin-induced but not cathepsin G-induced platelet responses; (b) human fibroblasts responded to thrombin and to a synthetic thrombin receptor peptide (comprising residues 42-55 of the thrombin receptor) by exhibiting an elevation in cytosolic Ca2+ concentration but did not respond to cathepsin G; and (c) platelets pretreated with neutrophil elastase failed to respond to thrombin but responded when rechallenged by cathepsin G. Thrombin and cathepsin G exhibit heterologous desensitization that is potentiated by okadaic acid and is attenuated by staurosporine, indicating that phosphorylation of serine/threonine residues is important for desensitization and that protein kinase C may be involved. Since catalytic activity of cathepsin G is required for platelet stimulation, it is probable that platelet activation by cathepsin G requires receptor proteolysis and that a tethered ligand mechanism is involved, suggesting that platelets may possess a family of protease receptors.

Interaction of human cathepsin D with the inhibitor pepstatin

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.