A novel antimicrobial peptide M1-8 targets the lysosomal pathway to inhibit autolysosome formation and promote apoptosis in liver cancer cells

Lysosomes, a central regulator of autophagy, play a critical role in tumour growth. Lysosomal protease cathepsin D can initiate apoptosis when released from lysosomes into the cytosol. In this study, we observed that Musca domestica cecropin (Mdc) 1–8 (M1-8), a small anti-tumour peptide derived from Mdc, inhibits hepatoma cell growth by blocking autophagy–lysosome fusion. This effect is likely achieved by targeting lysosomes to activate lysosomal protease D. Additionally, we examined whether lysosomal content and cathepsin D release were involved in M1-8-induced apoptosis. After exposure to M1-8, human hepatoma HepG2 cells rapidly co-localized with lysosomes, disrupted lysosomal integrity, caused leakage of lysosomal protease cathepsin D, caspase activation and mitochondrial membrane potential changes; and promoted cell apoptosis. Interestingly, in M1-8-treated HepG2 cells, autophagic protein content increased and the lysosome–autophagosome fusion was inhibited, suggesting that M1-8 can cause apoptosis through autophagy and lysosomes. This result indicates that a small accumulation of autophagy and autolysosome inhibition in cells can cause cell death. Taken together, these data suggest a novel insight into the regulatory mechanisms of M1-8 in autophagy and lysosomes, which may facilitate the development of M1-8 as a potential cancer therapeutic agent.     

Lysosomal–mitochondrial cross-talk during cell death

Lysosomes are membrane-bound organelles, which contain an arsenal of different hydrolases, enabling them to act as the terminal degradative compartment of the endocytotic, phagocytic and autophagic pathways. During the last decade, it was convincingly shown that destabilization of lysosomal membrane and release of lysosomal content into the cytosol can initiate the lysosomal apoptotic pathway, which is dependent on mitochondria destabilization. The cleavage of BID to t-BID and degradation of anti-apoptotic BCL-2 proteins by lysosomal cysteine cathepsins were identified as links to the mitochondrial cytochrome c release, which eventually leads to caspase activation. There have also been reports about the involvement of lysosome destabilization and lysosomal proteases in the extrinsic apoptotic pathway, although the molecular mechanism is still under debate. In the present article, we discuss the cross-talk between lysosomes and mitochondria during apoptosis and its consequences for the fate of the cell.     

Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose

Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.     

Clathrin and phosphatidylinositol-4,5-bisphosphate regulate autophagic lysosome reformation

Autophagy is a lysosome-based degradation pathway. During autophagy, lysosomes fuse with autophagosomes to form autolysosomes. Following starvation-induced autophagy, nascent lysosomes are formed from autolysosomal membranes through an evolutionarily conserved cellular process, autophagic lysosome reformation (ALR), which is critical for maintaining lysosome homeostasis. Here we report that clathrin and phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulate ALR. Combining a screen of candidates identified through proteomic analysis of purified ALR tubules, and large-scale RNAi knockdown, we unveiled a tightly regulated molecular pathway that controls lysosome homeostasis, in which clathrin and PtdIns(4,5)P(2) are the central components. Our functional study demonstrates the central role of clathrin and its associated proteins in cargo sorting, phospholipid conversion, initiation of autolysosome tubulation, and proto-lysosome budding during ALR. Our data not only uncover a molecular pathway by which lysosome homeostasis is maintained through the ALR process, but also reveal unexpected functions of clathrin and PtdIns(4,5)P(2) in lysosome homeostasis.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.     

Lysosomal metabolism of glycoproteins

The lysosomal catabolism of glycoproteins is part of the normal turnover of cellular constituents and the cellular homeostasis of glycosylation. Glycoproteins are delivered to lysosomes for catabolism either by endocytosis from outside the cell or by autophagy within the cell. Once inside the lysosome, glycoproteins are broken down by a combination of proteases and glycosidases, with the characteristic properties of soluble lysosomal hydrolases. The proteases consist of a mixture of endopeptidases and exopeptidases, which act in concert to produce a mixture of amino acids and dipeptides, which are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Although the glycans of all mature glycoproteins are probably degraded in lysosomes, the breakdown of N-linked glycans has been studied most intensively. The catabolic pathways for high-mannose, hybrid, and complex glycans have been established. They are bidirectional with concurrent sequential removal of monosaccharides from the nonreducing end by exoglycosidases and proteolysis and digestion of the carbohydrate-polypeptide linkage at the reducing end. The process is initiated by the removal of any core and peripheral fucose, which is a prerequisite for the action of the peptide N-glycanase aspartylglucosaminidase, which hydrolyzes the glycan-peptide bond. This enzyme also requires free alpha carboxyl and amino groups on the asparagine residue, implying extensive prior proteolysis. The catabolism of O-linked glycans has not been studied so intensively, but many lysosomal glycosidases appear to act on the same linkages whether they are in N- or O-linked glycans, glycosaminoglycans, or glycolipids. The monosaccharides liberated during the breakdown of N- and O-linked glycans are transported across the lysosomal membrane into the cytosol by a combination of diffusion and carrier-mediated transport. Defects in these pathways lead to lysosomal storage diseases. The structures of some of the oligosaccharides that accumulate in these diseases are not digestion intermediates in the lysosomal catabolic pathways but correspond to intermediates in the biosynthetic pathway for N-linked glycans, suggesting another route of delivery of glycans to the lysosome. Incorrectly folded or glycosylated proteins that are rejected by the quality control mechanism are broken down in the ER and cytoplasm and the end product of the cytosolic degradation of N-glycans is delivered to the lysosomes. This route is enhanced in cells actively secreting glycoproteins or producing increased amounts of aberrant glycoproteins. Thus interaction between the lysosome and proteasome is important for the regulation of the biosynthesis and distribution of N-linked glycoproteins. Another example of the extralysosomal function of lysosomal enzymes is the release of lysosomal proteases into the cytosol to initiate the lysosomal pathway of apoptosis.     

Loss of functional MYO1C/myosin 1c,a motor protein involved in lipid raft trafficking,disrupts autophagosome-lysosome fusion

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.     

Loss of functional MYO1C/myosin 1c,a motor protein involved in lipid raft trafficking,disrupts autophagosome-lysosome fusion

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.     

Distinct mechanisms of ferritin delivery to lysosomes in iron-depleted and iron-replete cells

Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by cells. Ferritin was targeted for degradation in the lysosome even under iron-replete conditions in primary cells; however, the mechanisms underlying lysosomal targeting of ferritin were distinct under depleted and replete conditions. In iron-depleted cells, ferritin was targeted to the lysosome via a mechanism that involved autophagy. In contrast, lysosomal targeting of ferritin in iron-replete cells did not involve autophagy. The autophagy-independent pathway of ferritin delivery to lysosomes was deficient in several cancer-derived cells, and cancer-derived cell lines are more resistant to iron toxicity than primary cells. Collectively, these results suggest that ferritin trafficking may be differentially regulated by cell type and that loss of ferritin delivery to the lysosome under iron-replete conditions may be related to oncogenic cellular transformation.     

Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

Destination 'lysosome': a target organelle for tumour cell killing?

Lysosomes and lysosome-related organelles constitute a system of acid compartments that interconnect the inside of the cell with the extracellular environment via endocytosis, phagocytosis and exocytosis. In recent decades it has been recognized that lysosomes are not just wastebaskets for disposal of unused cellular constituents, but that they are involved in several cellular processes such as post-translational maturation of proteins, degradation of receptors and extracellular release of active enzymes. By complementing the autophagic process, lysosomes actively contribute to the maintenance of cellular homeostasis. Proteolysis by lysosomal cathepsins has been shown to mediate the death signal of cytotoxic drugs and cytokines, as well as the activation of pro-survival factors. Secreted lysosomal cathepsins have been shown to degrade protein components of the extracellular matrix, thus contributing actively to its re-modelling in physiological and pathological processes. The malfunction of lysosomes can, therefore, impact on cell behaviour and fate. Here we review the role of lysosomal hydrolases in several aspects of the malignant phenotype including loss of cell growth control, altered regulation of cell death, acquisition of chemoresistance and of metastatic potential. Based on these observations, the lysosome is proposed as a potential target organelle for the chemotherapy of tumours. We will also present some recent data concerning the technologies for delivering chemotherapeutic drugs to the endosomal-lysosomal compartment and the strategies to improve their efficacy.     

AMDE-1 Is a Dual Function Chemical for Autophagy Activation and Inhibition

Autophagy is the process by which cytosolic components and organelles are delivered to the lysosome for degradation. Autophagy plays important roles in cellular homeostasis and disease pathogenesis. Small chemical molecules that can modulate autophagy activity may have pharmacological value for treating diseases. Using a GFP-LC3-based high content screening assay we identified a novel chemical that is able to modulate autophagy at both initiation and degradation levels. This molecule, termed as Autophagy Modulator with Dual Effect-1 (AMDE-1), triggered autophagy in an Atg5-dependent manner, recruiting Atg16 to the pre-autophagosomal site and causing LC3 lipidation. AMDE-1 induced autophagy through the activation of AMPK, which inactivated mTORC1 and activated ULK1. AMDE-1did not affect MAP kinase, JNK or oxidative stress signaling for autophagy induction. Surprisingly, treatment with AMDE-1 resulted in impairment in autophagic flux and inhibition of long-lived protein degradation. This inhibition was correlated with a reduction in lysosomal degradation capacity but not with autophagosome-lysosome fusion. Further analysis indicated that AMDE-1 caused a reduction in lysosome acidity and lysosomal proteolytic activity, suggesting that it suppressed general lysosome function. AMDE-1 thus also impaired endocytosis-mediated EGF receptor degradation. The dual effects of AMDE-1 on autophagy induction and lysosomal degradation suggested that its net effect would likely lead to autophagic stress and lysosome dysfunction, and therefore cell death. Indeed, AMDE-1 triggered necroptosis and was preferentially cytotoxic to cancer cells. In conclusion, this study identified a new class of autophagy modulators with dual effects, which can be explored for potential uses in cancer therapy.     

LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.     

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.