Exocytosis of active cathepsin B

Abstract

Lysosomal cathepsin B has been implicated in parasitic, inflammatory and neoplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is not well defined. The activity of extracellular cathepsin B is difficult to assess because of the presence of inhibitors and inactivation of the enzyme by oxidizing agents. Therefore, we have developed a continuous assay for measurement of cathepsin B activity produced pericellularly by living cells. The kinetic rate of Z-Arg-Arg-NHMec conversion was monitored and the assay optimized for enzyme stability, cell viability and sensitivity. To validate the assay, we determined that human liver cathepsin B was stable and active under the conditions of the assay and its activity could be inhibited by the selective epoxide derivative CA-074. Via this assay, we were able to demonstrate that active cathepsin B was secreted pericellularly by viable cells. Both preneoplastic and malignant cells secreted active cathepsin B. Pretreatment of cells with the membranepermeant proinhibitor CA-074Me completely abolished pericellular and total cathepsin B activity whereas pretreatment with the active drug CA-074 had no effect. Immunoprecipitation and immunoblotting experiments suggested that the active enzyme species was 31-kDa single-chain cathepsin B. Exocytosis of cathepsin B was not related to secretion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.

Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice

“…Ctsb, one of the most potent tumorpromoting cysteine cathepsins, is overexpressed in various human cancers and is secreted by malignant cells and cells of the tumor microenvironment (20,33). Although Ctsb is most stable and active at pH 5.5, the enzyme also exerts proteolytic activity at neutral or slightly acidic pH (34) and is capable of degrading the ECM proteins laminin, fibronectin, and collagen (35). Ctsb also is known to initiate proteolytic cascades through activation of other tumor-promoting proteases, including matrix metalloproteinases and urokinase-type plasminogen activator (35).…”

Diverse regulatory roles for lysosomal proteases in the immune response

“…Nonetheless, there is clear evidence that some activity persists most likely against a limited range of substrates [87][88][89]. Identification of the relevant physiological targets of cytosolic cathepsins has been complicated by the fact that many apoptotic signalling cascades occur in parallel.…”

Caveolin-1 mediates the expression and localization of cathepsin B, pro-urokinase plasminogen activator and their cell-surface receptors in human colorectal carcinoma cells

“…Increases in expression of cathepsin B and translocation of active cathepsin B to the basal membrane are also observed during the progression of adenomas in Min mice (Marten et al, 2002), a model for familial adenomatous polyposis in humans (for a review, see Dove et al, 1995). Membrane-associated cathepsin B can be isolated from a range of cancer cells (Rozhin et al, 1987;Rozhin et al, 1994), and activities can be assayed on the surface of living cells in culture (Linebaugh et al, 1999), suggesting a functional role for this enzyme on tumor-cell surfaces.…”