Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.     

Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.     

Dual role of cathepsin D: ligand and protease

Cathepsin D is peptidase belonging to the family of aspartic peptidases. Its mostly described function is intracellular catabolism in lysosomal compartments, other physiological effect include hormone and antigen processing. For almost two decades, there have been an increasing number of data describing additional roles imparted by cathepsin D and its pro-enzyme, resulting in cathepsin D being a specific biomarker of some diseases. These roles in pathological conditions, namely elevated levels in certain tumor tissues, seem to be connected to another, yet not fully understood functionality. However, despite numerous studies, the mechanisms of cathepsin D and its precursor's actions are still not completely understood. From results discussed in this article it might be concluded that cathepsin D in its zymogen status has additional function, which is rather dependent on a "ligand-like" function then on proteolytic activity.     

Regulatory role of cathepsin L in induction of nuclear laminopathy in Alzheimer’s disease

Experimental and clinical therapies in the field of Alzheimer''s disease (AD) have focused on elimination of extracellular amyloid beta aggregates or prevention of cytoplasmic neuronal fibrillary tangles formation, yet these approaches have been generally ineffective. Interruption of nuclear lamina integrity, or laminopathy, is a newly identified concept in AD pathophysiology. Unraveling the molecular players in the induction of nuclear lamina damage may lead to identification of new therapies. Here, using 3xTg and APP/PS1 mouse models of AD, and in vitro model of amyloid beta42 (Aβ42) toxicity in primary neuronal cultures and SH‐SY5Y neuroblastoma cells, we have uncovered a key role for cathepsin L in the induction of nuclear lamina damage. The applicability of our findings to AD pathophysiology was validated in brain autopsy samples from patients. We report that upregulation of cathepsin L is an important process in the induction of nuclear lamina damage, shown by lamin B1 cleavage, and is associated with epigenetic modifications in AD pathophysiology. More importantly, pharmacological targeting and genetic knock out of cathepsin L mitigated Aβ42 induced lamin B1 degradation and downstream structural and molecular changes. Affirming these findings, overexpression of cathepsin L alone was sufficient to induce lamin B1 cleavage. The proteolytic activity of cathepsin L on lamin B1 was confirmed using mass spectrometry. Our research identifies cathepsin L as a newly identified lamin B1 protease and mediator of laminopathy observed in AD. These results uncover a new aspect in the pathophysiology of AD that can be pharmacologically prevented, raising hope for potential therapeutic interventions.     

Human cathepsin D

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.     

The role of the cathepsin D propeptide in sorting to the lysosome.

The propeptides of lysosomal enzymes have been implicated in membrane association and mannose 6-phosphate-independent sorting to the lysosome (Rijnboutt, S., Aerts, H., Geuze, H. J., Tager, J. M., and Strous, G. J. (1991) J. Biol. Chem. 266, 4862-4868; McIntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445). In this report, the function of the propeptide of procathepsin D in sorting to the lysosome was directly assessed using a cathepsin D deletion mutant lacking the propeptide, and using a chimeric cDNA encoding the cathepsin D propeptide fused to the secretory protein alpha-lactalbumin. Proteins encoded by these cDNAs were expressed in mouse Ltk- cells and in human hepatoma Hep G2 cells, and then immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The deletion mutant was glycosylated but was rapidly degraded in a chloroquine-independent fashion and did not assume an active conformation. Thus the propeptide appeared to be necessary for correct folding. The chimeric protein was glycosylated and secreted. The coincidence of complex oligosaccharide modification and secretion of the chimeric protein suggested that it was slowly released from the endoplasmic reticulum and rapidly passed through the cell to the extracellular compartment. This was confirmed by immunofluorescent localization of the proteins. The data indicated that the propeptide appeared to be necessary for folding of cathepsin D but, unlike the yeast vacuolar propeptides, was not sufficient to direct a secretory protein to the lysosome in fibroblasts or in epithelial cells.     

Acetate-induced apoptosis in colorectal carcinoma cells involves lysosomal membrane permeabilization and cathepsin D release

Colorectal carcinoma (CRC) is one of the most common causes of cancer-related mortality. Short-chain fatty acids secreted by dietary propionibacteria from the intestine, such as acetate, induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. We previously reported that acetic acid-induced apoptosis in Saccharomyces cerevisiae cells involves partial vacuole permeabilization and release of Pep4p, the yeast cathepsin D (CatD), which has a protective role in this process. In cancer cells, lysosomes have emerged as key players in apoptosis through selective lysosomal membrane permeabilization (LMP) and release of cathepsins. However, the role of CatD in CRC survival is controversial and has not been assessed in response to acetate. We aimed to ascertain whether LMP and CatD are involved in acetate-induced apoptosis in CRC cells. We showed that acetate per se inhibits proliferation and induces apoptosis. More importantly, we uncovered that acetate triggers LMP and CatD release to the cytosol. Pepstatin A (a CatD inhibitor) but not E64d (a cathepsin B and L inhibitor) increased acetate-induced apoptosis of CRC cells, suggesting that CatD has a protective role in this process. Our data indicate that acetate induces LMP and subsequent release of CatD in CRC cells undergoing apoptosis, and suggest exploiting novel strategies using acetate as a prevention/therapeutic agent in CRC, through simultaneous treatment with CatD inhibitors.     

The noncanonical role of the protease cathepsin D as a cofilin phosphatase

Cathepsin D (cathD) is traditionally regarded as a lysosomal protease that degrades substrates in acidic compartments. Here we report cathD plays an unconventional role as a cofilin phosphatase orchestrating actin remodeling. In neutral pH environments, the cathD precursor directly dephosphorylates and activates the actin-severing protein cofilin independent of its proteolytic activity, whereas mature cathD degrades cofilin in acidic pH conditions. During development, cathD complements the canonical cofilin phosphatase slingshot and regulates the morphogenesis of actin-based structures. Moreover, suppression of cathD phosphatase activity leads to defective actin organization and cytokinesis failure. Our findings identify cathD as a dual-function molecule, whose functional switch is regulated by environmental pH and its maturation state, and reveal a novel regulatory role of cathD in actin-based cellular processes.Subject terms: Actin, Cytokinesis     

The catalytically inactive precursor of cathepsin D induces apoptosis in human fibroblasts and HeLa cells

In several reports cathepsin D has been implicated in apoptosis. In some systems the effects of agents considered to be mediated by cathepsin D were inhibited in the presence of pepstatin A, an inhibitor of the enzyme. In other studies the effect of a mutant cathepsin D deprived of activity was indistinguishable from that of the normal enzyme. Here we show that in human fibroblasts and in HeLa cells apoptosis can be induced by microinjecting into cytosol either mature cathepsin D or its inactive precursor procathepsin D. The microinjected precursor remains in the uncleaved form. These results confirm that the proapoptotic effect of cathepsin D in the cytosol is independent of its catalytic activity and suggest that the interaction of cathepsin D with the downstream effector does not involve the active site of the enzyme, since in the proenzyme the active site is masked by the prosequence.     

Isolation of rabbit testicular cathepsin D and its role in the activation of proacrosin

Cathepsin D was purified 900-fold with 30% recovery from rabbit testes using pepstatin bound Sepharose affinity chromatography. The enzyme is homogeneous as observed by acrylamide gel electrophoresis. The heat stable enzyme exhibits an apparent molecular weight of 42,000 with identical subunits of 20,000. Purified cathepsin D catalyses the conversion of proacrosin to acrosin.     

Phospholipids activate cathepsin D

Total lipids as well as phospholipids extracted from the mitochondrial-lysosomal fraction of porcine adrenal cortex activated the lysosomal cathepsin D of this tissue 30- and 40-fold, respectively, with bovine serum albumin as the substrate. Phosphatidic acid, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol and cardiolipin were found to activate greatly the cathepsin D. The degree of activation ranged from 6-fold by phosphatidyl ethanolamine to 40-fold by cardiolipin at 1 mM, respectively. These results strongly point to the importance of phospholipids in intracellular protein degradation by lysosomal cathepsin D.     

Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy

The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.     

Role of cathepsin B in dengue virus-mediated apoptosis

Dengue virus (DENV) infection is one of the most important mosquito-borne viral diseases, which is endemic in the tropical and sub-tropical regions. Patients with dengue hemorrhagic fever (DHF) generally present hemorrhagic tendencies, plasma leakage, thrombocytopenia, and hemoconcentration. Hepatic dysfunction is also a crucial feature of DENV infection. Hepatic biopsy specimens obtained from fatal cases of DENV infection show cellular apoptosis, which apparently relate to the pathogenesis. Cathepsins, which are cysteine proteases inside the lysosome, were previously reported to be up-regulated in patients with DHF. However, their functions during DENV infection have not been thoroughly investigated. We show for the first time that DENV induces lysosomal membrane permeabilization. The resulting cytosolic cathepsin B and S contributed to apoptosis via caspase activation. The activity of caspase 3 was significantly reduced in DENV-infected HepG2 cells treatedwith cathepsin B or S inhibitors. Treatment with cathepsin B inhibitor also reduced the activity of caspase 9, suggesting that cathepsin B activates both caspase-9 and caspase-3. Reduced cathepsin B expression, effected by RNA interference, mimicked pharmacological inhibition of the enzyme and confirmed the contribution of cathepsin B to apoptotic events induced by DENV in HepG2 cells.     

Structural differences between rabbit cathepsin E and cathepsin D

Rabbit cathepsins D and E were isolated from bone marrow. Both enzymes were purified by affinity chromatography on pepstatin-Sepharose 4B and Con A-Sepharose 4B. Purity of the enzymes was ascertained by two-dimensional gel electrophoresis after iodination. The isoelectric point of cathepsin D was found to be 6.95. Cathepsin E was shown to consist of two subunits having molecular masses each of 40 kDa and isoelectric points of 4.60 and 4.65, respectively. The amino-acid composition of cathepsin E was found to be different from that of cathepsin D.     

Localization of cathepsin D in rat liver

Summary Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.