Two Faces of Cathepsin D: Physiological Guardian Angel and Pathological Demon

Abstract

Since its discovery as a lysosomal hydrolase, Cathepsin D (CatD) has been the subject of intensive scrutiny by numerous scientists. Those accumulated efforts have defined its biosynthetic pathway, structure, and companion proteins in the context of its perceived "house keeping" function. However, in the past two decades CatD has emerged as a multifunctional enzyme, involved in myriad biological processes beyond its original "housekeeping" role. CatD is responsible for selective and limited cleavage (quite distinct from non-specific protein degradation) of particular substrates vital to proper cellular function. These proteolytic events are critical in the control of biological processes, including cell cycle progression, differentiation and migration, morphogenesis and tissue remodeling, immunological processes, ovulation, fertilization, neuronal outgrowth, angiogenesis, and apoptosis. Consistent with the biological relevance of CatD, its deficiency, altered regulation or post-translational modification underlie important pathological conditions such as cancer, atherosclerosis, neurological and skin disorders. Specifically, deregulated synthesis, post-translational modifications and hyper-secretion of CatD, along with its mitogenic effects, are established hallmarks of cancer. More importantly, but less studied, is its significance in regulating the sensitivity to anticancer drugs. This review outlines CatD's post-translational modifications, cellular trafficking, secretion and protein binding partners in normal mammary gland, and restates the "site-specific" function of CatD which is most probably dictated by its post-translational modifications and binding partners. Noteworthy, CatD's association with one of its binding partners in the context of drug sensitivity is highlighted, with the optimism that it could contribute to the development of more effective chemotherapeutic agent(s) tailored for individual patients.

Keywords: Binding partners; Cancer; Cathepsin D; Mammary gland; Post-translational modification.

Figure 1

(A) Schematic presentation of CatD promoter region. The TATA and GC sequences are represented by square boxes, five transcription start sites are indicated by arrows and their distance from the +1 nucleotide are indicated. (B) Pictorial presentation of proteolytic processing of pre-pro-CatD. CatD is synthesized in the ER as a pre-pro enzyme containing a signal peptide at its amino terminus. As the enzyme traverses the ER, it loses its signal peptide and is glycosylated at two N-glycosylation sites. The pro-enzyme is transported to the Golgi, tagged with Man-6-P for binding to Man-6-PR. The complex is transported across the Golgi and reaches the endosomal compartment. The acidic environment causes the release of the receptor and the pro-peptide is cleaved generating the single chain active enzyme. Further removal of seven amino acids generates the light chain and the heavy chain mature enzyme (please see the text). (C) Developmental regulation of CatD level and proteolytic processing in mouse mammary tissue. Cytosolic extracts prepared from the mammary gland at different stages of development were subjected to SDS-PAGE and Western blot analysis for the presence of CatD cleavage products. V: virgin, P: pregnant, LD: lactation days 1, 3 and 7, IND: involution days 1–15. Molecular mass of CatD proteolytic products are indicated on the right.

Figure 2

CatD cleavage products and N-glycan structures in normal mammary epithelial cells (HMEpCs) compared to breast cancer cell lines MCF-7 and MDA-MB231. Cytosolic fractions (25 μg total protein), and conditioned media (CM) from HMEpCs and breast cancer cell lines MCF-7 and MDA-MB231 were treated with or without endoglycosidase H (Endo-H, to remove the chitobiose core of high mannose and some hybrid oligosaccharides), and peptide-N-glycosidase F (PGNase, to remove high mannose, hybrid and complex glycans), then subjected to SDS-PAGE (10% acrylamide gel) and Western blot analysis. CM from the three cell lines was concentrated prior to treatment (HMEpC: 35×, MCF-7 and MDA-MB231: 2×). Differences in the abundance of the processed forms (A), and the N-glycan moieties of CatD (B&C), between normal mammary epithelial cells and breast cancer cell lines are evident in these Western blots. Note the preferential presence of multiple high mannose N-glycan structures (indicated by the appearance of multiple bands following Endo-H treatment) of the CM from the cancer cell lines.

Figure 3

Model depicting different routes of CatD trafficking in association with its binding partners in polarized normal mammary epithelial cells, and their relevance to cancer: 1). When in the Golgi, the Man-6-P tagged CatD binds Man-6-PR (and/or sortilin) and is transported to the endosomal compartment. In the acidic environment, the complex is dissociated and Man-6-PR returns to the membrane with the retromer complex, while CatD is cleaved and processed in the late endosome and lysosomes. 2). Under normal conditions ≤5% of pro-CatD (either alone or in a complex with Pro-Sap) is secreted from the ER. 3). In polarized epithelial cells, the basolateral release of CatD is also observed, the binding partner in this case is unknown. However, in Caco-2 colon epithelial cell line, Man-6PR binds CatD and transports it basolateraly. 4). Generation of ceramide by acid sphingomyelinase results in the limited permeability of lysosomal membrane and release of CatD, leading to the induction of apoptosis. The majority of these pathways are altered in breast cancer. In pathway 1, the reduced acidification of endosomal/lysosomal compartment noted in cancer cells affects proper processing of CatD, resulting in increased secretion of pro-CatD. Routes 2 and 3 are greatly elevated and could lead to excessive ECM degradation. Route 4 could be equally affected by changes in vacuolar acidification, which alters CatD processing, its lysosomal release and participation in apoptosis.