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The bacterium Vibrio alginolyticus, an opportunistic pathogen in humans, has a type III secretion system (T3SS) that is responsible for its cytotoxicity toward eukaryotic cells. The effector of T3SS that is responsible for the cytotoxicity had not been identified. Here we demonstrate that VepA, a homolog of the T3SS effector in V. parahaemolyticus, is required for cytotoxicity in V. alginolyticus. VepA induces lysosomal membrane permeabilization, and it allows the leakage of only small molecules into the cytosol. Our findings revealed that VepA induces cathepsin-independent cell death in mammalian cells. The ferrous ion, one of the small molecules in the lysosome contents, appears to be involved in the cell death caused by V. alginolyticus VepA., Competing Interests: No potential conflict of interest relevant to this article was reported.

Lysosomes are the key degradative compartments of the cell. Lysosomal cathepsins, which are enclosed in the lysosomes, help to maintain the homeostasis of the cell's metabolism by participating in the degradation of heterophagic and autophagic material. Following the targeted lysosomal membrane's destabilization, the cathepsins can be released into the cytosol and initiate the lysosomal pathway of apoptosis through the cleavage of Bid and the degradation of the anti-apoptotic Bcl-2 homologues. Cathepsins can also amplify the apoptotic signaling, when the lysosomal membranes are destabilized at a later stage of apoptosis, initiated by other stimuli. However, the functional integrity of the lysosomal compartment during apoptosis enables efficient autophagy, which can counteract apoptosis by providing the energy source and by disposing the damaged mitochondria, which generate the ROS. Impairing autophagy by disabling the lysosome function is being investigated as an adjuvant therapeutic approach to sensitize cells to apoptosis-inducing agents. Destabilization of the lysosomal membranes by the lysosomotropic detergents seems to be a promising strategy in this context as it would not only disable autophagy, but also promote apoptosis through the initiation of the lysosomal pathway. In contrast, the impaired autophagy and lysosomal degradation linked with the increased oxidative stress underlie degenerative changes in the aging neurons. This further suggests that lysosomes and lysosomal cathepsins have a dual role in cell death. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Cathepsins B and L are lysosomal cysteine proteases which have been implicated in a variety of pathological processes such as cancer, tumor angiogenesis, and neurodegeneration. However, only a few protein substrates have thus far been described and the mechanisms by which cathepsins B and L regulate cell proliferation, invasion, and apoptosis are poorly understood. Combined deficiency of both cathepsins results in early-onset neurodegeneration in mice reminiscent of neuronal ceroid lipofuscinoses in humans. Therefore, we intended to quantify accumulated proteins in brain lysosomes of double deficient mice. A combination of subcellular fractionation and LC-MS/MS using isobaric tagging for relative and absolute quantitation (iTRAQ) allowed us to simultaneously assess wildtype and cathepsin B(-/-)L(-/-) cerebral lysosomes. Altogether, 19 different proteins were significantly increased in cathepsin B(-/-)L(-/-) lysosomes. Most elevated proteins had previously been localized to neuronal biosynthetic, recycling/endocytic or lysosomal compartments. A more than 10-fold increase was observed for Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E. Intriguingly, immunohistochemistry demonstrated that Rab14 and DNER specifically stain swollen axons in double deficient brains. Since dense accumulations of expanded axons are the earliest phenotypic and pathognomonic feature of cathepsin B(-/-)L(-/-) brains, our data suggest a role for cathepsins B and L in recycling processes during axon outgrowth and synapse formation in the developing postnatal central nervous system.

Although lysosomal proteases are expressed in the heart at considerable levels, their specific functions in this organ remain elusive. Mice deficient for the lysosomal cysteine protease cathepsin L (CTSL) develop a late onset dilated cardiomyopathy (DCM) that is characterized by cardiac chamber dilation, fibrosis, and impaired cardiac contraction at 12 month of age. Investigation of the pathogenic sequence of DCM in ctsl-/- mice revealed numerous dysmorphic lysosome-like structures in heart muscle as early as 3 days after birth, whereas skeletal muscle was not affected. Labeling of the acidic cell compartment of neonatal cardiomyocytes and detection of lysosomal markers after subcellular fractionation confirmed increased lysosome content in CTSL deficient myocardium; however, specific storage materials were not detected. The myocardium of ctsl+/+ and ctsl-/- mice revealed no differences in incidence of cell death, proliferation, and capillary density during DCM progression. However, an observed increase in mRNA expression of natriuretic peptides in young adult mice indicates the activation of the adaptive "fetal" gene program, while proteome analysis revealed decreased levels of the sarcomere-associated proteins alpha-tropomyosin, desmin, and calsarcin 1, as well as considerable changes of metabolic enzymes. Bioinformatic pathway analysis suggested a switch to anaerobic catabolism and impairment of mitochondrial respiration. This interpretation was supported by a 50% reduction in resting state oxygen consumption and impaired respiration capacity in ctsl-/- myocardial homogenates. In summary, the data indicate an essential role of CTSL in maintaining the structure of the endosomal/lysosomal compartment in cardiomyocytes. Lysosomal impairment in ctsl-/- hearts results in metabolic and sarcomeric alterations that promote DCM development.

Dendritic cells (DC) initiate immunity and maintain tolerance. Although in vitro-generated DC, usually derived from peripheral blood monocytes (MO-DC), serve as prototype DC to analyze the biology and biochemistry of DC, phenotypically distinct primary types of DC, including CD1c-DC, are present in peripheral blood (PB-DC). The composition of lysosomal proteases in PB-DC and the way their MHC class II-associated Ag-processing machinery handles a clinically relevant Ag are unknown. We show that CD1c-DC lack significant amounts of active cathepsins (Cat) S, L, and B as well as the asparagine-specific endopeptidase, the major enzymes believed to mediate MHC class II-associated Ag processing. However, at a functional level, lysosomal extracts from CD1c-DC processed the multiple sclerosis-associated autoantigens myelin basic protein and myelin oligodendrocyte glycoprotein in vitro more effectively than MO-DC. Although processing was dominated by CatS, CatD, and asparagine-specific endopeptidase in MO-DC, it was dominated by CatG in CD1c-DC. Thus, human MO-DC and PB-DC significantly differ with respect to their repertoire of active endocytic proteases, so that both proteolytic machineries process a given autoantigen via different proteolytic pathways.

Tumorigenesis is associated with several changes that alter the cellular susceptibility to programmed cell death. Here, we show that immortalization and transformation sensitize cells in particular to the cysteine cathepsin-mediated lysosomal death pathway. Spontaneous immortalization increased the susceptibility of wild-type murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF)-mediated cytotoxicity >1000-fold, whereas immortalized MEFs deficient for lysosomal cysteine protease cathepsin B (CathB) retained the resistant phenotype of primary cells. This effect was specific for cysteine cathepsins, because also lack of cathepsin L (a lysosomal cysteine protease), but not that of cathepsin D (a lysosomal aspartyl protease) or caspase-3 (the major executioner protease in classic apoptosis) inhibited the immortalization-associated sensitization of MEFs to TNF. Oncogene-driven transformation of immortalized MEFs was associated with a dramatic increase in cathepsin expression and additional sensitization to the cysteine cathepsin-mediated death pathway. Importantly, exogenous expression of CathB partially reversed the resistant phenotype of immortalized CathB-deficient MEFs, and the inhibition of CathB activity by pharmacological inhibitors or RNA interference attenuated TNF-induced cytotoxicity in immortalized and transformed wild-type cells. Thus, tumorigenesis-associated changes in lysosomes may counteract cancer progression and enhance therapeutic responses by sensitizing cells to programmed cell death.

The asparagine-specific endoprotease (AEP) controls lysosomal processing of the potential autoantigen myelin basic protein (MBP) by human B lymphoblastoid cells, a feature implicated in the immunopathogenesis of multiple sclerosis. In this study, we demonstrate that freshly isolated human B lymphocytes lack significant AEP activity and that cleavage by AEP is dispensable for proteolytic processing of MBP in this type of cell. Instead, cathepsin (Cat) G, a serine protease that is not endogenously synthesized by B lymphocytes, is internalized from the plasma membrane and present in lysosomes from human B cells where it represents a major functional constituent of the proteolytic machinery. CatG initialized and dominated the destruction of intact MBP by B cell-derived lysosomal extracts, degrading the immunodominant MBP epitope and eliminating both its binding to MHC class II and a MBP-specific T cell response. Degradation of intact MBP by CatG was not restricted to a lysosomal environment, but was also performed by soluble CatG. Thus, the abundant protease CatG might participate in eliminating the immunodominant determinant of MBP. Internalization of exogenous CatG represents a novel mechanism of professional APC to acquire functionally dominant proteolytic activity that complements the panel of endogenous lysosomal enzymes.

Dilated cardiomyopathy is a frequent cause of heart failure and is associated with high mortality. Progressive remodeling of the myocardium leads to increased dimensions of heart chambers. The role of intracellular proteolysis in the progressive remodeling that underlies dilated cardiomyopathy has not received much attention yet. Here, we report that the lysosomal cysteine peptidase cathepsin L (CTSL) is critical for cardiac morphology and function. One-year-old CTSL-deficient mice show significant ventricular and atrial enlargement that is associated with a comparatively small increase in relative heart weight. Interstitial fibrosis and pleomorphic nuclei were found in the myocardium of the knockout mice. By electron microscopy, CTSL-deficient cardiomyocytes contained multiple large and apparently fused lysosomes characterized by storage of electron-dense heterogeneous material. Accordingly, the assessment of left ventricular function by echocardiography revealed severely impaired myocardial contraction in the CTSL-deficient mice. In addition, echocardiographic and electrocardiographic findings to some degree point to left ventricular hypertrophy that most likely represents an adaptive response to cardiac impairment. The histomorphological and functional alterations of CTSL-deficient hearts result in valve insufficiencies. Furthermore, abnormal heart rhythms, like supraventricular tachycardia, ventricular extrasystoles, and first-degree atrioventricular block, were detected in the CTSL-deficient mice.

The lysosomal cysteine peptidases cathepsin B and cathepsin L are abundant and ubiquitously expressed members of the papain family, and both enzymes contribute to the terminal degradation of proteins in the lysosome. However, there is accumulating evidence for specific functions of lysosomal proteases in health and disease. The generation of 'knock out' mouse strains that are deficient in lysosomal proteases provides a valuable tool for evaluation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by analysis of mice that are devoid of cathepsin B or cathepsin L. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, regulation of the hair cycle, and MHC class II-mediated antigen presentation in cortical epithelial cells of the thymus. Cathepsin B plays a major role in pathological trypsinogen activation in the early course of experimental pancreatitis and contributes significantly to TNF-alpha induced hepatocyte apoptosis.

The contribution of mannose 6-phosphate (Man 6-P)-dependent and -independent systems to lysosomal targeting of cathepsin H, a lysosomal cysteine protease, was investigated by metabolic labeling with [32P]phosphate and [35S]methionine/cysteine in primary cultures of rat hepatocytes. Metabolic labeling experiments with [32]phosphate revealed that only the proform of cathepsin H acquired Man 6-P residues on its high mannose type oligosaccharide, and that most of the phosphorylated procathepsin H was secreted into the medium without having undergone significant intracellular dephosphorylation. Thus, acquisition of Man 6-P residues did not correlate with targeting of cathepsin H to lysosomes. Pulse-chase experiments with [35S]methionine/cysteine showed that only about 10% of the newly synthesized cathepsin H was secreted as a proform while the remainder was retained intracellularly in processed mature form. These results indicate that the majority of newly synthesized cathepsin H is targeted to lysosomes by a Man 6-P-independent mechanism, at least in rat hepatocytes.

Brain-derived neurotrophic factor (BDNF) expression in hippocampus and cortex is considerably reduced in Alzheimer's disease. The present study tested if lysosomal disturbances, a concomitant of brain aging, impair basal and/or induced expression of BDNF. Cultured hippocampal slices were incubated with N- CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), an inhibitor of cathepsins B and L, for 6 days and processed for in situ hybridization using radiolabeled cRNA probes against BDNF mRNA. Multiple densitometric readings were collected from each of the three principal hippocampal subdivisions. Within-slice averages were substantially lower in the ZPAD-treated group compared to controls. Treatment with the inhibitor did not change average neuron diameter or packing density. Intense stimulation of glutamate receptors with kainate for 30 min (followed by a 90-min recovery period) caused a nearly threefold increase in BDNF mRNA concentrations in the dentate gyrus while having only marginal effects in the other subdivisions. Slice averages of ZPAD-exposed cultures treated with kainate were lower than those of controls exposed to the excitotoxin; however, on a percentage basis, the kainate-induced increase in the dentate gyrus was comparable for the two groups (175 +/- 31 vs 179 +/- 39%). Kainate for 1 h (with a 5-h recovery) affected BDNF mRNA in a manner similar to that found with shorter infusions, i.e., induction in stratum granulosum but not elsewhere, lower overall slice averages with ZPAD treatment, and no evidence that ZPAD blocked the percentage increase in the dentate gyrus. These results provide evidence that lysosomal dysfunction occurring during brain aging could disrupt ongoing BDNF production without substantially impairing the neurotrophin response to intense physiological activity. The first observation suggests a plausible aging sequence leading to pathology while the second may be of interest with regard to possible therapeutics.

Serum IL-6 level increased after the injection of turpentine oil into the right gastrocnemius muscle in mice. The mRNA level of IL-6 was highest in the injected muscle at 12 h after injection, but was not identified in the opposite muscle. The activities of cathepsins B and B+L started to elevate after 12 h in the injected muscle and markedly increased after day 3. Likewise, the mRNA levels of cathepsins B and L markedly increased from day 1 to day 5 in the injected muscle. However, a very mild increase was also observed in the opposite muscle. Immunohistochemical staining of cathepsins B and L exhibited positive reactions as fine granules in myofibers at 12 h and strong positive reactions in the infiltrating macrophages at 3 days. Atrophy of myofibers type 1 and 2 was evident in a time-dependent manner in the injected muscle. Treatment with rat anti-mouse IL-6 receptor monoclonal antibody inhibited the increase in cathepsin activities in the injected muscle. We conclude that IL-6 produced in the inflamed muscle is involved in the process of muscle degeneration, especially through the activation of lysosomal cathepsins.

The lysosomal cysteine proteinases are synthesized in a form of pre-proenzymes. They are submitted to posttranslational glycosylation and phosphorylation. These modifications make possible transport the modified proteins into Golgi apparatus and into lysosomes. Some disturbances of transport which occur mainly in tumour cells result in an increase of these enzymes activities in cytosol and in intercellular compartment. The activity of cysteine proteinases is regulated by specific inhibitors (cystatin, kininogen) which exist in some tissues and body fluids. The evaluation of activity and concentration of proteinases and inhibitors is important in clinical diagnostics.

We review the role played by the lysosomal proteases on cellular protein breakdown and their relation to nutrition research. Several recent works have shown that an important group of lysosomal proteases (cathepsins) are directly responsible for the maintenance of protein breakdown. The determination of their enzyme activity or measurement of mRNAs as an index of gene expression have provided new views about the mechanisms of protein-breakdown regulation. The introduction of molecular biology could provide new ways with which to manipulate the catabolic response of injured patients.

Both lysosomal cysteine-proteinases and collagenase appear to be necessary for the resorption of actively growing, immature woven bone, but their relative roles are not yet clearly elucidated. The present evidence indicates that, during bone resorption, the osteoclast first solubilizes the mineral by a secretion of acid and then removes the exposed demineralized collagen by the action of secreted lysosomal collagenolytic cysteine-proteinases. Collagenase in bone seems to be mainly a product of osteoblasts and related cells, not osteoclasts. Its role could be limited in the removal of any non-mineralized collagen layers which could be covering mineralized bone surfaces and which seem to prevent the activation of osteoclasts and thus their action; such a "shield" of unmineralized osteoid is well-established at the surface of actively growing woven bone, although not on the resorbing surfaces of mature lamellar bone. Moreover, some osteoblast-derived procollagenase is stored in the mineralized bone matrix from which it can be released by demineralization. It is therefore possible that it may also contribute to the degradation of demineralized bone collagen once it has been released and activated by lysosomal cysteine-proteinases under the osteoclast.

We have determined the sequence of cDNAs encoding the precursors of rat cathepsins H and L. In order to understand the mechanism and regulation of expression and processing of these genes we have also determined the genomic restriction maps and the genomic structures in the extension sequences of the 5'-regions. We have found that the intracellular locations of cathepsins B, H and L in various cells and organs are different. Lysosomes are shown to have different amounts of cathepsins B, H and L and this heterogeneity may reflect their different functions in autophagy and heterophagy. The intracellular role of cystatins, endogenous inhibitors of cathepsins, in suppressing intralysosomal proteolysis has been clarified. Electron microscopic studies of islet cells of the pancreas using double gold particle immunostaining for insulin and cystatin beta, indicate that cystatin beta is located in the insulin secretory granules. We propose the following hypothesis for the mode of action of cystatin beta: cystatin beta is secreted from its primary location in the secretory granules and is then taken up by target cells which direct it into lysosomes for the regulation of intralysosomal cathepsin activities. The inhibitory activity of cystatin beta is controlled by formation of a mixed-disulfate with glutathione and cysteine at position 3: the free form is active and the "glutathionated" form is inactive. The changes in glutathione balance (oxidized/reduced) in cells may regulate the inhibitory activities of cystatin beta. Further, the regulation of cathepsins under various nutritional and hormonal conditions, as well as the abnormal expressions of cathepsins in various pathological conditions, are discussed.

A review of continuing studies on the physiological and pathological roles of the lysosomal proteinases, cathepsin D and cathepsin B is presented. Intracellular release of cathepsin D from lysosomes has been demonstrated during myocardial ischemia. Both this proteinase and cathepsin B have been found, by organ culture in the presence of the appropriate antiserum, to be released into the extracellular space in rheumatoid synovium, where they may play a part in cartilage destruction. Also reviewed is the finding of a cathepsin B-like proteinase that, in organ culture, is secreted in markedly increased amounts from human malignant breast carcinomas in comparison to amounts secreted from benign tumors or normal tissue.

The cycloheximide-related loss of transport activity (manifested as a decrease in Vmax for transport) in cultured Nil hamster fibroblasts was blocked by the addition of carbamoyl phosphate, cyanate (a product of spontaneous phosphate elimination from carbamoyl phosphate), or ammonium salts to the culture medium. Acid proteases capable of hydrolyzing alpha-N-benzoyl-D,L-arginine-beta-naphthylamine (cathepsins B1, H, and L) were also inhibited in situ by ammonia and cyanate. The inactivation of these cathepsins by ammonia was irreversible and probably was related to the increase in the intralysosomal pH known to be caused by an accumulation of ammonia in the lysosomes. The inhibition of the cathepsin activity by cyanate in situ (and in cell-free extracts) was completely reversible and blocked irreversible inhibition of the cathepsin(s) by N-ethylmaleimide. The inactivation of the cathepsins caused by cyanate was deduced to be the result of reversible blocking of sulfhydryl groups essential to the thiol cathepsin activity. The concomitant inhibition of thiol cathepsins and hexose carrier inactivation provided further evidence for the involvement of lysosomal proteases in at least part of the mechanism that regulates the rate of hexose transport in animal cells.

Stress dependent variations in th properties of the rat muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) have been linked to the corresponding changes in the levels of proteolytic activities in rat muscle. Whole-body X-irradiation of rat was shown to result in loss of muscle aldolase activity towards fructose 1,6-bisphosphate by 50% while fructose 1-phosphate activity remained unchanged (Pote, M.S. and Altekar, W. (1980) Ind. J. Biochem, Biophys. 17, 255-262). Incubation of muscle extract of irradiated rat with that from control rat or rabbit muscle aldolase caused similar changes in aldolase activity. The changes are attributed to the action of catheptic enzymes possessing latency characteristics and capable of using aldolase as a substrate; the time course of their increase after irradiation corresponds to that of loss in muscle aldolase activities. Exposure of rats to stress resulted in an increase in the 'free' proteolytic activity, and the concomitant loss of 'bound' activity in muscle lysosomes indicates labilization of lysosomal membrane. The observed degradation of aldolase in vivo by muscle lysosomes is shown to be due to the action of cathepsin B (EC 3.4.22.1) present in the proteolytic enzymes released into cytosol under stress. Inactivation of rabbit muscle aldolase and rat muscle aldolase by rat muscle cathepsin B inhibited by leupeptin, antipain an iodoacetamide, but not be pepstatin. Inactivation is shown to be due to the release of C-terminal tyrosine if aldolase, required for its catalytic activity. Cathepsin B who acts as a rate-limiting enzyme in the degradation of aldolase. Such a proteolytic modification of aldolase in vivo could be relevant not only to the regulation of aldolase activity of glycolysis in muscle but also to the degradation of aldolase during stress conditions related to tissue damage and the maintenance of normal aldolase levels in the blood.

Institute of Nutrition, Academy of Medical Sciences of the USSR, Moscow Directed change of the genotype in cell cultures (Hela, fibroblasts, L929), which leads to the resistance to challenge with some of the viruses is accompanied by pronounced modulation of the activity of lysosomal proteinases, cathepsins A, B1, D and, in the first turn, by a considerable reduction (by 2--12 times) of cathepsin C activity. The data obtained indicate an important role of the proteolytic system of the cell lysosomal apparatus in providing defence reactions.

The electrophoretic behaviour of rat-liver catalase in polyacrylamide gel depends on the subcellular fraction the enzyme was isolated from. Catalase extracted from the mitochondrial fraction is more anodic than catalase recovered from the unsedimentable fraction of the homogenate. The difference disappears if the sedimentable enzyme is extracted from purified peroxisomes. On the other hand, when treated with a lysosomal extract, catalase present in the unsedimentable fraction or in purified peroxisomes, behaves like the enzyme isolated from the mitochondrial fraction. Factors that influence that effect of lysosomes on catalase indicate that it is due to a proteolysis by an enzyme like cathepsin B. These results suggest that catalase isolated from the mitochondrial fraction differs from catalase isolated from peroxisomes or from the unsedimentable fraction, because it has been subjected to a proteolysis caused by lysosomes present in the mitochondrial fraction, during the extraction procedure. As a matter of fact, catalase isolated from the mitochondrial fraction is endowed with a lower molecular weight than catalase extracted from purified peroxisomes or from the unsedimentable fraction. This structural modification does not apparently affect the stability and the catalytic power of the enzyme.

Lysosomal enzymes acid phosphatase, acid DNAase, acid RNAase cathepsin D,beta-galactosidase beta-glucuronidase were studied in mice and rat liver tissue within 3 days beginning from 4 o'clock a.m. at 4 hrs intervals. Activities of acid phosphatase, acid RNA ase in mice liver tissue and of cathepsin D in rat and mice liver tissues was higher at morning and day time than at evening and night. Alterations in enzymatic activity of non-sedimenting fraction of mice liver tissue correlated with that of total activity. In non-sedimenting fraction of rat liver tissue the enzymatic activity was altered only slightly. Within the second half of a day the enzymatic activity redistributed in the cells with an increase of the activity in non-sedimenting fraction, caused by labilization of lysosomal membranes.

Gastric ulcers were induced in rats by i.m. injections of vitamin A, and i.p. injection of histamine, or injections of both vitamin A and histamine. The incidence of ulcer formation was highest in the vitamin A-histamine group. However, ulcer formation also occurred after the administration of vitamin A alone as a lysosomal labilizer. Furthermore, the vitamin A-histamine group showed a remarkably elevated cathepsin activity in the tissue of gastric wall. In the experiments of ulcer formation by vitamin A injections, the specific activity and the enzyme release of cathepsin were elevated already in the early stage after vitamin A administration. Consequently, cathepsin activity in stomach juice was remarkably elevated in the stage of the ulcer formation. In the clinic, the cathepsin activity in the mucous membrane of human gastric wall was remarkably high in the antrum and the angulus of the stomach, in which ulcer formation tends to occur most frequently. In view of these results, cathepsin in the stomach seems to play an important role in the formation of gastric ulcers.