10 research outputs found
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Proteolytic activation of human procathepsin D
Procathepsin D is a short-lived inactive precursor of the lysosomal aspartyl protease, cathepsin D. Pulse-chase analysis using radiolabeled amino acids demonstrated the existence of several biosynthetic intermediates during formation of mature cathepsin D (summarized in Figure 1). Procathepsin D is capable of autocatalytic cleavage to pseudocathepsin D. This was demonstrated using small quantities of procathepsin D isolated from cell culture media as well as using a non-glycosylated form of procathepsin D synthesized in a bacterial expression system. Complete conversion to the single-chain cathepsin D appears to require a second enzyme which is inhibited by leupeptin. This conclusion was drawn from the inability to produce single-chain enzyme from either procathepsin D or pseudocathepsin D in vitro as well as observations from addition of protease inhibitors to cell cultures. It appears that the conversion of procathepsin D to active single-chain enzyme falls between the paradigms of pepsinogen autoactivation and prorenin conversion by a separate enzyme
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Structural requirements of procathepsin D activation and maturation
Cathepsin D biosynthesis involves several proteolytic events; however, the enzymology and sequence of these events are not known. Procathepsin D undergoes a pH-dependent, intramolecular proteolysis in vitro which removes 26 residues yielding an active form that is intermediate in size between procathepsin D and single-chain cathepsin D. This form, designated pseudocathepsin D, has not been shown to be an in vivo intermediate. The N-terminal sequence of the light chain of cathepsin D, isolated from human placenta, showed that 42 residues were removed as compared with 44 residues predicted by comparison with porcine cathepsin D. Site-directed mutations were generated at both processing sites within the propeptide of procathepsin D. Mutation at the autocatalytic site prevented in vitro autoactivation, but, after transfection of mouse Ltk- cells, the mutant procathepsin D was transported to the lysosome and processed normally to the mature enzyme despite its inability to autoactivate in vitro. Mutation at the mature N terminus of cathepsin D prevented in vivo formation of the single-chain form of the enzyme; however, the protein was still processed to the two-chain form of human cathepsin D. This change at the mature N terminus did not prevent in vitro autoactivation. Procathepsin D with mutations at both cleavage sites was processed to the two-chain form despite the inability to undergo removal of the propeptide. These results indicated that stepwise autoactivation and propeptide removal were not necessary for later processing or delivery of human cathepsin D to the lysosome. The results also suggested that pseudocathepsin D was not a normal intermediate of procathepsin D processing in vivo
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Comparison of kinetic properties of native and recombinant human cathepsin D
Phytepsin, a barley vacuolar aspartic proteinase, is highly expressed during autolysis of developing tracheary elements and sieve cells
HaCaT human keratinocytes express IGF-II, IGFBP-6, and an acid-activated protease with activity against IGFBP-6
Cathepsin D in the Tumor Microenvironment of Breast and Ovarian Cancers
Cancer remains a major and leading health problem worldwide. Lack of early diagnosis, chemoresistance, and recurrence of cancer means vast research and development are required in this area. The complexity of the tumor microenvironment in the biological milieu poses greater challenges in having safer, selective, and targeted therapies. Existing strategies such as chemotherapy, radiotherapy, and antiangiogenic therapies moderately improve progression-free survival; however, they come with side effects that reduce quality of life. Thus, targeting potential candidates in the microenvironment, such as extracellular cathepsin D (CathD) which has been known to play major pro-tumorigenic roles in breast and ovarian cancers, could be a breakthrough in cancer treatment, specially using novel treatment modalities such as immunotherapy and nanotechnology-based therapy. This chapter discusses CathD as a pro-cancerous, more specifically a proangiogenic factor, that acts bi-functionally in the tumor microenvironment, and possible ways of targeting the protein therapeutically.published version, accepted version (12 month embargo