Information

How are lysosome membranes protected from the attack of hydrolases?


Lysosomes are a bit like the suicidal bags of cells. They help to clean cells, have an acidic pH and contain a large number of hydrolyzing enzymes.

But why don't these hydrolyzing enzymes attack and destroy lysosomes' membranes?


Early histochemical work indicated that the internal surface of the lysosomal membrane has a glycocalyx - a layer of polysaccharide, presumed to have a protective role.

Neiss, W. F. (1984) A coat of glycoconjugates on the inner surface of the lysosomal membrane in the rat kidney. Histochemistry 80, 603-608

Subsequently it was found that major membrane proteins of the lysosomal membrane are protected from proteolysis by glycosylation:

Kundra, R. and Kornfeld, S. (1999) Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis. J. Biol. Chem. 274, 31039-31046

See this paper for a more recent study of another lysosomal membrane protein:

Schieweck O. et al. (2009) NCU-G1 is a highly glycosylated integral membrane protein of the lysosome. Biochem. J. 422: 83-90

NCU-G1 is the mouse orthologue of the human C1orf85 protein. The NCU-G1 gene encodes a 404 amino acid protein. This is a Type 1 transmembrane protein - i.e. the N terminal domain is directed to the extracytoplasmic surface of the membrane. In vivo the protein is detected in 70 kDa and 80 kDa forms. The discrepancy between the polypeptide size and the molecular mass is shown to be due to extensive glycosylation. The protein sequence includes 9 potential glycosylation sites.


BRAIN LYSOSOMES AND LYSOSOMAL ENZYMES

Biochemical, enzymatic and metabolic studies of purified lysosomal fractions from rat kidney and liver have shown that lysosomes contain two types of glycoproteins, enzyme glycoproteins, and soluble acidic lipoglycoproteins (SALGP) without catalytic properties. The SALGP comprise about 50% of the lysosol protein, are readily labelled by precursors of phospholipids, aminosugars, sialic acid and peptides, have a molecular weight of about 15,000 and a pI of about 4. Most or all of the acid hydrolases are glycoenzymes that are synthesized in a restricted portion of the rough endoplasmic reticulum in the form of basic glycoproteins containing N-acetylglucosamine and mannose. N-Acetylneuraminic acid (NANA) and additional N-acetylglucosamine are attached to the nascent glycoenzymes in the Golgi apparatus. The lysosomal enzymes are packaged in lysosomes exclusively as acidic sialoglycoproteins with pIs between 3.5 and 4.9. The basic forms of the lysosomal hydrolases apparently originate from the corresponding acidic forms during biodegradation through a partial autolytic cleavage of NANA, carbohydrate and (glyco)peptide. Similar changes occur during incubation of lysosomal extracts in vitro at an acid pH.

The molecular heterogeneity of lysosomal enzymes has been investigated in submitochondrial and submicrosomal fractions of rat brain. The solubility varies with the enzyme species and increases with increasing pH and Triton X-100 concentration of the medium. The hydrolases in the nerve ending (NE) fraction are generally less soluble than those in the mitochondrial-lysosomal (M-L) fraction, but are quantitatively solubilized in alkaline buffer containing 0.5% (v/v) Triton X-100. Polyacrylamide gel electrophoresis in an alkaline system (pH 8.8) resolves multiple components of acid phosphatase, organophosphorus-resistant acid esterase, β-glucuronidase, arylsulfatase and β-N-acetylhexosaminidase. The patterns vary with the subcellular fraction. The acidic forms of the enzymes are labile and are readily converted to more basic forms, probably owing to a partial autolytic cleavage of NANA, sugar and peptide residues. Gel electrophoretograms of a Triton X-100 extract of the lysosome-enriched, dense M-L and d fractions show distinctive patterns for the various enzymes. On the other hand, gel electropherograms of the lighter, membrane-rich fractions, including the myelin and NE fractions, reveal poor penetration of protein and enzymes, and the enzymes, and the enzyme patterns are single and stereotyped, usually consisting of a major cathodic and a minor anodic component. These enzyme components also stain for protein, carbohydrate, lipid and acidic groups, indicating that they are associated with acidic glycolipoglycoproteins of membranous origin as stable multienzyme-glycolipoglycoprotein complexes. Similar complexes have been identified by ultracentrifugal flotation of Triton X-100 extracts of membrane-rich fractions. Polyacrylamide gel electrophoresis in an acidic system (pH 4) permits adequate penetration of protein and enzyme into gels and yields uniform patterns of the hydrolases for the various subcellular fractions, while excluding the acidic glycolipoglycoproteins from the gels. However, the latter system suffers from the handicap that the acidic isoenzymes are labile at this low pH. Pretreatment of the NE fraction with cold acetone markedly increases the solubilization of protein and lysosomal hydrolases in detergent-free alkaline buffer, and sharply modifies the electrophoretic patterns of soluble acidic glycolipoglycoproteins and the hydrolases in Triton X-100 extracts. Thus, the partial binding of lysosmal hydrolases to membrane glycolipoglycoproteins may alter several properties of these enzymes including their solubility, buoyant density and electrophoretic mobilities in polyacrylamide gels. Solubilization studies involving purified liver and kidney lysosomal fractions have indicated that the “membrane-bound” acid hydrolases are, in fact, complexed with phospholipid-rich, SALGP aggregates present in the lysosomal matrix, and are not bound to the limiting membrane of lysosomes as commonly supposed. Further, lysosomal SALGP, prelabeled in vivo in the phospholipid moieties, form complexes with acid hydrolases that tend to persist during gel filtration in a Sephadex G-200 column but can be split by dissociating agents such as 6 N urea or 1 M KCl. Finally, an improved method is described for the partial purification of lysosomes from brain homogenates. This method involves sequential isopycnic centrifugations of a crude mitochondrial fraction on metrizamide and sucrose density gradients.


Contents

Christian de Duve, the chairman of the Laboratory of Physiological Chemistry at the Catholic University of Louvain in Belgium, had been studying the mechanism of action of a pancreatic hormone insulin in liver cells. By 1949, he and his team had focused on the enzyme called glucose 6-phosphatase, which is the first crucial enzyme in sugar metabolism and the target of insulin. They already suspected that this enzyme played a key role in regulating blood sugar levels. However, even after a series of experiments, they failed to purify and isolate the enzyme from the cellular extracts. Therefore, they tried a more arduous procedure of cell fractionation, by which cellular components are separated based on their sizes using centrifugation.

They succeeded in detecting the enzyme activity from the microsomal fraction. This was the crucial step in the serendipitous discovery of lysosomes. To estimate this enzyme activity, they used that of the standardized enzyme acid phosphatase and found that the activity was only 10% of the expected value. One day, the enzyme activity of purified cell fractions which had been refrigerated for five days was measured. Surprisingly, the enzyme activity was increased to normal of that of the fresh sample. The result was the same no matter how many times they repeated the estimation, and led to the conclusion that a membrane-like barrier limited the accessibility of the enzyme to its substrate, and that the enzymes were able to diffuse after a few days (and react with their substrate). They described this membrane-like barrier as a "saclike structure surrounded by a membrane and containing acid phosphatase." [18]

It became clear that this enzyme from the cell fraction came from membranous fractions, which were definitely cell organelles, and in 1955 De Duve named them "lysosomes" to reflect their digestive properties. [19] The same year, Alex B. Novikoff from the University of Vermont visited de Duve's laboratory, and successfully obtained the first electron micrographs of the new organelle. Using a staining method for acid phosphatase, de Duve and Novikoff confirmed the location of the hydrolytic enzymes of lysosomes using light and electron microscopic studies. [20] [21] de Duve won the Nobel Prize in Physiology or Medicine in 1974 for this discovery.

Originally, De Duve had termed the organelles the "suicide bags" or "suicide sacs" of the cells, for their hypothesized role in apoptosis. [22] However, it has since been concluded that they only play a minor role in cell death. [23]

Lysosomes contain a variety of enzymes, enabling the cell to break down various biomolecules it engulfs, including peptides, nucleic acids, carbohydrates, and lipids (lysosomal lipase). The enzymes responsible for this hydrolysis require an acidic environment for optimal activity.

In addition to being able to break down polymers, lysosomes are capable of fusing with other organelles & digesting large structures or cellular debris through cooperation with phagosomes, they are able to conduct autophagy, clearing out damaged structures. Similarly, they are able to break down virus particles or bacteria in phagocytosis of macrophages.

The size of lysosomes varies from 0.1 μm to 1.2 μm. [24] With a pH ranging from

4.5–5.0, the interior of the lysosomes is acidic compared to the slightly basic cytosol (pH 7.2). The lysosomal membrane protects the cytosol, and therefore the rest of the cell, from the degradative enzymes within the lysosome. The cell is additionally protected from any lysosomal acid hydrolases that drain into the cytosol, as these enzymes are pH-sensitive and do not function well or at all in the alkaline environment of the cytosol. This ensures that cytosolic molecules and organelles are not destroyed in case there is leakage of the hydrolytic enzymes from the lysosome.

The lysosome maintains its pH differential by pumping in protons (H + ions) from the cytosol across the membrane via proton pumps and chloride ion channels. Vacuolar-ATPases are responsible for transport of protons, while the counter transport of chloride ions is performed by ClC-7 Cl − /H + antiporter. In this way a steady acidic environment is maintained. [25] [26]

It sources its versatile capacity for degradation by import of enzymes with specificity for different substrates cathepsins are the major class of hydrolytic enzymes, while lysosomal alpha-glucosidase is responsible for carbohydrates, and lysosomal acid phosphatase is necessary to release phosphate groups of phospholipids.

Many components of animal cells are recycled by transferring them inside or embedded in sections of membrane. For instance, in endocytosis (more specifically, macropinocytosis), a portion of the cell's plasma membrane pinches off to form vesicles that will eventually fuse with an organelle within the cell. Without active replenishment, the plasma membrane would continuously decrease in size. It is thought that lysosomes participate in this dynamic membrane exchange system and are formed by a gradual maturation process from endosomes. [27] [28]

The production of lysosomal proteins suggests one method of lysosome sustainment. Lysosomal protein genes are transcribed in the nucleus in a process that is controlled by transcription factor EB (TFEB). [14] mRNA transcripts exit the nucleus into the cytosol, where they are translated by ribosomes. The nascent peptide chains are translocated into the rough endoplasmic reticulum, where they are modified. Lysosomal soluble proteins exit the endoplasmic reticulum via COPII-coated vesicles after recruitment by the EGRESS complex (ER-to-Golgi relaying of enzymes of the lysosomal system), which is composed of CLN6 and CLN8 proteins. [9] [10] COPII vesicles then deliver lysosomal enzymes to the Golgi apparatus, where a specific lysosomal tag, mannose 6-phosphate, is added to the peptides. The presence of these tags allow for binding to mannose 6-phosphate receptors in the Golgi apparatus, a phenomenon that is crucial for proper packaging into vesicles destined for the lysosomal system. [29]

Upon leaving the Golgi apparatus, the lysosomal enzyme-filled vesicle fuses with a late endosome, a relatively acidic organelle with an approximate pH of 5.5. This acidic environment causes dissociation of the lysosomal enzymes from the mannose 6-phosphate receptors. The enzymes are packed into vesicles for further transport to established lysosomes. [29] The late endosome itself can eventually grow into a mature lysosome, as evidenced by the transport of endosomal membrane components from the lysosomes back to the endosomes. [27]

As the endpoint of endocytosis, the lysosome also acts as a safeguard in preventing pathogens from being able to reach the cytoplasm before being degraded. Pathogens often hijack endocytotic pathways such as pinocytosis in order to gain entry into the cell. The lysosome prevents easy entry into the cell by hydrolyzing the biomolecules of pathogens necessary for their replication strategies reduced Lysosomal activity results in an increase in viral infectivity, including HIV. [30] In addition, AB5 toxins such as cholera hijack the endosomal pathway while evading lysosomal degradation. [30]

Lysosomes are involved in a group of genetically inherited deficiencies, or mutations called lysosomal storage diseases (LSD), inborn errors of metabolism caused by a dysfunction of one of the enzymes. The rate of incidence is estimated to be 1 in 5,000 births, and the true figure expected to be higher as many cases are likely to be undiagnosed or misdiagnosed. The primary cause is deficiency of an acid hydrolase. Other conditions are due to defects in lysosomal membrane proteins that fail to transport the enzyme, non-enzymatic soluble lysosomal proteins. The initial effect of such disorders is accumulation of specific macromolecules or monomeric compounds inside the endosomal–autophagic–lysosomal system. [15] This results in abnormal signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking, ultimately leading to pathogenetic disorders. The organs most affected are brain, viscera, bone and cartilage. [31] [32]

There is no direct medical treatment to cure LSDs. [33] The most common LSD is Gaucher's disease, which is due to deficiency of the enzyme glucocerebrosidase. Consequently, the enzyme substrate, the fatty acid glucosylceramide accumulates, particularly in white blood cells, which in turn affects spleen, liver, kidneys, lungs, brain and bone marrow. The disease is characterized by bruises, fatigue, anaemia, low blood platelets, osteoporosis, and enlargement of the liver and spleen. [34] [35] As of 2017, enzyme replacement therapy is available for treating 8 of the 50-60 known LDs. [36]

The most severe and rarely found, lysosomal storage disease is inclusion cell disease. [37]

Metachromatic leukodystrophy is another lysosomal storage disease that also affects sphingolipid metabolism.

Dysfunctional lysosome activity is also heavily implicated in the biology of aging, and age-related diseases such as Alzheimer's, Parkinson's, and cardiovascular disease. [38] [39]

Sr. No Enzymes Substrate
1 Phosphates
A- Acid phosphatase Most phosphomonoesters
B- Acid phosphodiesterase Oligonucleotides and phosphodiesterase
2 Nucleases
A- Acid ribonuclease RNA
B- Acid deoxyribonuclease DNA
3 Polysaccharides/ mucopolysaccharides hydrolyzing enzymes
A- beta Galactosidase Galactosides
B- alfa Glucosidase Glycogen
C- alfa Mannosidase Mannosides, glycoproteins
D- beta Glucoronidase Polysaccharides and mucopolyssacharides
E- Lysozymes Bacterial cell walls and mucopolyssacharides
F- Hyaluronidase Hyaluronic acids, chondroitin sulphates
H- Arylsulphatase Organic sulfates
4 Proteases
A- Cathepsin(s) Proteins
B- Collagenase Collagen
C- Peptidase Peptides
5 Lipid degrading enzymes
A- Esterase Fatty acyl esters
B- Phospolipase Phospholipids

Lysosomotropism Edit

Weak bases with lipophilic properties accumulate in acidic intracellular compartments like lysosomes. While the plasma and lysosomal membranes are permeable for neutral and uncharged species of weak bases, the charged protonated species of weak bases do not permeate biomembranes and accumulate within lysosomes. The concentration within lysosomes may reach levels 100 to 1000 fold higher than extracellular concentrations. This phenomenon is called lysosomotropism, [41] "acid trapping" or "proton pump" effect. [42] The amount of accumulation of lysosomotropic compounds may be estimated using a cell-based mathematical model. [43]

A significant part of the clinically approved drugs are lipophilic weak bases with lysosomotropic properties. This explains a number of pharmacological properties of these drugs, such as high tissue-to-blood concentration gradients or long tissue elimination half-lifes these properties have been found for drugs such as haloperidol, [44] levomepromazine, [45] and amantadine. [46] However, high tissue concentrations and long elimination half-lives are explained also by lipophilicity and absorption of drugs to fatty tissue structures. Important lysosomal enzymes, such as acid sphingomyelinase, may be inhibited by lysosomally accumulated drugs. [47] [48] Such compounds are termed FIASMAs (functional inhibitor of acid sphingomyelinase) [49] and include for example fluoxetine, sertraline, or amitriptyline.

Ambroxol is a lysosomotropic drug of clinical use to treat conditions of productive cough for its mucolytic action. Ambroxol triggers the exocytosis of lysosomes via neutralization of lysosomal pH and calcium release from acidic calcium stores. [50] Presumably for this reason, Ambroxol was also found to improve cellular function in some disease of lysosomal origin such as Parkinson's or lysosomal storage disease. [51] [52]

Systemic lupus erythematosus Edit

Impaired lysosome function is prominent in systemic lupus erythematosus preventing macrophages and monocytes from degrading neutrophil extracellular traps [53] and immune complexes. [54] [55] [56] The failure to degrade internalized immune complexes stems from chronic mTORC2 activity, which impairs lysosome acidification. [57] As a result, immune complexes in the lysosome recycle to the surface of macrophages causing an accumulation of nuclear antigens upstream of multiple lupus-associated pathologies. [54] [58] [59]

By scientific convention, the term lysosome is applied to these vesicular organelles only in animals, and the term vacuole is applied to those in plants, fungi and algae (some animal cells also have vacuoles). Discoveries in plant cells since the 1970s started to challenge this definition. Plant vacuoles are found to be much more diverse in structure and function than previously thought. [60] [61] Some vacuoles contain their own hydrolytic enzymes and perform the classic lysosomal activity, which is autophagy. [62] [63] [64] These vacuoles are therefore seen as fulfilling the role of the animal lysosome. Based on de Duve's description that "only when considered as part of a system involved directly or indirectly in intracellular digestion does the term lysosome describe a physiological unit", some botanists strongly argued that these vacuoles are lysosomes. [65] However, this is not universally accepted as the vacuoles are strictly not similar to lysosomes, such as in their specific enzymes and lack of phagocytic functions. [66] Vacuoles do not have catabolic activity and do not undergo exocytosis as lysosomes do. [67]

The word lysosome ( / ˈ l aɪ s oʊ s oʊ m / , / ˈ l aɪ z ə z oʊ m / ) is New Latin that uses the combining forms lyso- (referring to lysis and derived from the Latin lysis, meaning "to loosen", via Ancient Greek λύσις [lúsis]), and -some, from soma, "body", yielding "body that lyses" or "lytic body". The adjectival form is lysosomal. The forms *lyosome and *lyosomal are much rarer they use the lyo- form of the prefix but are often treated by readers and editors as mere unthinking replications of typos, which has no doubt been true as often as not.


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Opposite roles of cathepsins in tumor progression localization matters

Cathepsins are powerful hydrolases that can be divided into three subgroups depending on the amino acid located in their catalytic dyad: aspartate cathepsins (cathepsins D and E), cysteine cathepsins (cathepsins B, C, F, H, K, L, O, S, V/U/L2, W, X/Z/Y) and serine cathepsins (cathepsins A and G). 34 Akin to most other lysosomal hydrolases, they function optimally at pH around 4.5 or lower. With respect to their secretion to the extracellular space and leakage to the cytosol, it is important to note that many cysteine cathepsins can also be active at higher pH, albeit often with changed functional parameters such as enzyme kinetics and substrate specificity. 35

Increased expression, activity and secretion of cathepsins lead to enhanced tumor growth, invasion and angiogenesis. 4, 36, 37 Accordingly, several cathepsins are overexpressed in variety of cancers, often both in the tumor cells and in tumor-associated leukocytes, fibroblasts, osteoclasts, myoepithelial cells and endothelial cells. 4 The tumor promoting effects of cathepsins have been mainly assigned to their proteolytic activities outside the cell, whereas increased cysteine cathepsin activity in the lumen of lysosomes enhances the proteolysis of LAMP1 and LAMP2 and leads to the destabilization of lysosomal membranes thereby sensitizing cells to various stresses and limiting cell survival. 26 Upon destabilization of lysosomal membranes, cathepsins leak to the cytosol where they can activate either the mitochondrial apoptosis pathway or trigger non-apoptotic cell death, a mode of cell demise that can effectively kill even highly apoptosis resistant cancer cells. 7, 38 Below we highlight the cancer-associated roles of individual cathepsins.

Cathepsin D (encoded by CTSD)

Cathepsin D is an aspartate cathepsin and belongs to the pepsin superfamily of proteases. 39 First reports of increased levels of cathepsin D in human cancer date back to the 1980s. 40, 41 Since then plentiful studies have confirmed these findings in the majority of solid cancers. 37, 42 Furthermore, positive correlations between cathepsin D expression and tumor size, tumor grade, metastasis, poor prognosis, chemoresistance and risk of recurrence have been reported. 42, 43 Accordingly, cathepsin D plays an essential role in the multiple steps of tumor progression by stimulating cancer cell proliferation and survival, fibroblast outgrowth and angiogenesis. 37, 43 Interestingly, these cancer-promoting functions of cathepsin D appear to be largely independent of its proteolytic activity suggesting that the protein or its unprocessed precursors have cytokine or growth factor-like activities. 37, 43 Fittingly, pro-cathepsin D and its propeptide can induce the activation of the mitogen-activated protein kinase (MAPK-ERK) signaling pathway, cytokine secretion and expression of genes encoding for proteins supporting proliferation, metastasis and angiogenesis. 42, 43, 44 The importance of the pro-form rather than the active form of cathepsin D in cancer progression challenges the development of cathepsin D targeting anti-cancer drugs since small molecule enzyme inhibitors are not likely to be effective.

Cathepsin B (encoded by CTSB)

Cathepsin B is a cysteine cathepsin of the papain superfamily, whose increased expression has been widely reported in most cancer types, often both in cancer cells themselves as well as in tumor-associated macrophages and fibroblasts. 4, 45, 46 In line with the idea that cathepsin B enhances invasion and metastasis, it is often localized at the surface of tumor cells, and its expression is predominantly increased in the cells at the invasive edge of the tumors. In general, increased cathepsin B levels detected by immunohistochemistry correlate well with higher mRNA levels pointing to a transcriptional upregulation of cathepsin B encoding CTSB gene. 47 Accordingly, transformation of fibroblast and/or epithelial cells by activated Ras, Src or ERBB2 oncogenes increases the levels of CTSB/CtsB mRNAs, protein and proteolytic activity and promotes pericellular distribution of lysosomes. 26, 48, 49, 50 Moreover, ErbB2-induced upregulation of cathepsins B (and L) renders non-invasive breast cancer cells highly invasive in a three-dimensional Matrigel invasion model. 50

The ErbB2-induced expression of cathepsin B-encoding CTSB gene appears to be independent of TFEB, 50 the major regulator of the transcription of lysosomal genes. 31 Instead, ErbB2 activates a signaling network consisting of PAK4, cdc42bpβ, PKCα and MAPK-ERK2 serine–threonine kinases that activates a myeloid zinc-finger transcription factor 1 (MZF1), which in turn binds to an ErbB2-induced enhancer element in the first intron of CTSB gene in vivo. 50 Interestingly, MAPK-ERK2 activity, which is essential for the ErbB2-induced activation of myeloid zinc-finger transcription factor 1 and expression of CTSB, inhibits the nuclear import of TFEB. 51 It remains to be studied whether similar transcriptional switch occurs in response to other CTSB-activating oncogenes and whether the transcription of other lysosomal genes also switches from TFEB-dependency to dependency on myeloid zinc-finger transcription factor 1 or other oncogene-activated transcription factors.

The active role of cathepsin B in tumorigenesis is strongly supported by murine cancer models. In the RIP1-Tag2/RT2 (RT2) pancreatic islet cell carcinogenesis model, the multiple pancreatic islet tumors driven by the expression of oncogenic SV40 T-antigen in insulin producing β-cells express high levels of cathepsin B. 52, 53 Crossing of these mice to a Ctsb-deficient background results in strongly impaired tumor formation, tumor cell proliferation, angiogenesis and invasiveness. The suggested molecular basis in this model includes secretion of cathepsin B from tumor cells and tumor-associated macrophages and direct cathepsin B-mediated cleavage of extracellular matrix components, E-cadherin and urokinase plasminogen activator propeptide, the latter leading to the activation of a potent proteolytic cascade involving sequential activation of urokinase plasminogen activator, plasmin and various matrix metalloproteases. 52, 54, 55, 56, 57 Similarly, Ctsb deficiency significantly delays the onset and growth of primary mammary tumors and their lung metastases in murine mammary tumor virus-polyoma middle T antigen (PyMT) transgenic mice. 58 Vice versa, cathepsin B overexpression in PyMT mice promotes mammary tumor growth and metastasis. 59 These data reveal extracellular cathepsin B as an attractive target for cancer therapy. Supporting this notion, a non-cell permeable cathepsin B specific inhibitor CA-074 has given promising results in murine models of metastatic melanoma and breast cancer. 60, 61

Contrary to the strong tumor-promoting properties of cathepsin B, increased cathepsin B (and L) activity in the lumen of the lysosomes may form an Achilles heal for the cancer cells via the cleavage of LAMP1 and LAMP2, and subsequent destabilization of lysosomal membranes, decreased stress tolerance and sensitization to drugs that activate the lysosomal cell death pathway. 26, 62 Thus, high cathepsin B expression in tumor cells may prove to be a useful biomarker for the responsiveness to lysosome-targeting therapies.

Cathepsin L (encoded by CTSL1)

Cathepsin L is another ubiquitously expressed cysteine cathepsin that is frequently overexpressed in various human tumors. 46 It enhances migration and invasion by decreasing cell–cell adhesion and increasing extracellular matrix degradation. 4, 36 Its identified substrates that include extracellular matrix proteins, such as laminin, fibronectin and collagens as well as E-cadherin and proheparanase. 52, 63, 64, 65, 66 Studies on the role of cathepsin L in angiogenesis are somewhat contradicting. 65 Supporting its role in angiogenesis, cathepsin L expression is significantly associated with the neovasculature of astrocytic tumors, 67 and cathepsin L specific inhibitor NSITC displays potent antiangiogenic activity in chick chorioallantoic membrane and mouse matrigel models of angiogenesis in vitro. 68 Nevertheless, in the RT2 mouse model of pancreatic islet cell carcinogenesis Ctsl deletion has no effect in the angiogenic switch even though it effectively impairs tumor growth and invasiveness. 52

Cathepsin L has an additional tumorigenic activity in the nucleus. An isoform of cathepsin L that lacks the signal peptide necessary for the lysosomal targeting migrates to the nucleus where it cleaves histone H3 and transcription factors such as the CCAAT-displacement protein/cut homeobox (CDP/Cux), thereby altering cell cycle progression and gene expression patterns and contributing to oncogenic transformation. 69, 70, 71, 72 In colorectal cancer increased nuclear versus lysosomal localization of cathepsin L correlates with advanced tumor stage and poor prognosis. 73 As for cathepsin B, specific inhibitors have also been developed for cathepsin L. 74, 75 Of these, the most promising metastasis and/or invasion blocking compounds include CLIK-148 and FF-FMK. 65 FF-FMK, which can also inhibit cathepsin B at higher concentrations, decreases the invasion of human oral squamous cell carcinoma cell lines in vitro, 76 and oral administration of CLIK-148 inhibits human melanoma A375 cell metastasis to bone but not to liver or muscle in a xenograft mouse model. 77 Several additional studies support the bone degradative function of cathepsin L, making it a possible target molecule in the treatment of metastatic bone disease. 78

The usefulness of cathepsin L as a therapeutic target for cancer treatment has, however, been questioned by studies showing that its deficiency or inactivation promotes tumor progression in mouse skin cancer models 79, 80, 81 as well as in intestinal epithelia. 82 The molecular explanation for this tumor suppressive role of cathepsin L may be its essential role in the negative regulation of the recycling of growth factors and their receptors from the endocytic compartment back to the plasma membrane. 83

Cathepsin K (encoded by CTSK)

Cysteine cathepsin K has high collagenolytic and osteolytic activity. It is predominantly expressed in osteoclasts, where it is the key protease responsible for bone resorption and bone metabolism. 84 Correspondingly, inactivating mutations in human cathepsin K gene (CTSK) result in pycnodysostosis characterized by short stature, skull deformities and skeletal abnormalities. 85 Cathepsin K expression is activated in breast and prostate carcinomas with higher expression levels in bone metastases as compared with primary tumors. 86, 87, 88 Cathepsin K inhibitors (CKI, AFG495 and Odanacatib), which have been successfully used to treat osteoporosis-associated bone loss, 84, 89 reduce breast cancer-induced osteolysis and skeletal tumor burden in a mouse model of skeletal metastasis. 87 Notably Odanacatib is currently on phase III trial as a treatment to reduce the risk of breast cancer-induced bone metastasis (http://www.cancer.gov/drugdictionary). In melanoma, cathepsin K expression correlates with advanced metastatic disease, 90 and pharmacological inhibition of cathepsin K with Boc-I reduces melanoma cell invasion through the Matrigel basement membrane matrix. 90 Inhibition of cathepsin K in this model results in the accumulation of internalized collagen in the lysosomal compartment, suggesting that cathepsin K is essential for the effective intracellular degradation of phagocytosed matrix proteins also outside the bone.

Other cysteine cathepsins

The expression of cysteine cathepsins S, F, H, O, V (also known as L2 or U) and Z (also known as Y or X) is also increased in human cancers. However, relatively little is known about their regulation or function. Cathepsin S may function in angiogenesis by degrading anti-angiogenic, type IV collagen derived peptides and generating pro-angiogenic peptides by cleavage of laminin-5. 91, 92 Accordingly, genetic inactivation of cathepsin S encoding CTSS gene in the RT2 mouse model of pancreatic cancer leads to impaired tumor formation and impaired angiogenesis. 52 In the same model system, genetic inactivation of CTSH (cathepsin H) also significantly impaired angiogenic switching of the pre-malignant hyperplastic islets and resulted in a reduction in the subsequent number of tumors. 93 Moreover, the tumor burden in CTSH null RT2 mice is significantly reduced, in association with defects in the blood vasculature and increased apoptosis. Finally, simultaneous depletion of cathepsins B and Z results in synergistic antitumor effects in the murine PyMT breast cancer model, suggesting that most, if not all, cysteine cathepsins may posess tumor-promoting features. 94

Pharmacological targeting of cysteine cathepsins

More than one cathepsin is usually upregulated upon oncogene-driven transformation in vitro in pre-clinical murine cancer models as well as in human cancer. Their highly overlapping substrate specificities and functions in tumorigenesis suggest thus that a combined inhibition of their activities might be required for an efficient cancer therapy. Accordingly, a single genetic inactivation of Ctsb or Ctsz in the PyMT oncogene-induced mammary carcinomas delays only slightly the appearance of tumors and lung metastasis, whereas their combined inactivation results in a significant delay in tumor growth and reduction in the number and size of metastases. 94, 95 In line with this, a cell-permeable broad-spectrum cysteine cathepsin inhibitor JPM-OEt is much more effective than single depletions of Ctsb, Ctsl or Ctss in reducing angiogenic switching in the murine RT2 pancreatic carcinoma model. 52, 92 By contrast, JPM-OEt shows poor efficacy in the PyMT murine breast cancer model, 96 and fails to reduce metastasis in the breast cancer bone metastasis model in which the non cell-permeable CA-074 is effective. 61 These results may be simply due to differential bioavailability of the drugs in different tissues. 96 Alternatively, different cysteine cathepsins may have opposing roles in some cancers, or the ability of JPM-OEt to enter the cell and inhibit intracellular cathepsins may stabilize lysosomal membranes and provide cancer cells with a survival advantage that counteracts the effects of extracellular inhibition of cathepsins. However, the available preclinical data strongly encourage the continuing development of broad-spectrum cysteine cathepsin inhibitors as cancer therapeutics, but clearly more basic research on the anticancer effects of individual cathepsins and their localization is needed to find the optimal treatment modalities.


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Lysosomal Physiology

Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases.


How are lysosome membranes protected from the attack of hydrolases? - Biology

Cholesterol is delivered to lysosomes by receptor-mediated delivery of low density lipoproteins (LDLs) from the extracellular space.

In lysosomes, cholesterol esters are hydrolyzed and subject to binding to specialized proteins such as NPC2.

The transport of cholesterol to the limiting membrane of the lysosome requires that the dense layer of carbohydrates has to be overcome by hydrophobic tunnel systems found in NPC1 as well as in LIMP-2/SCARB2.

At the cytosolic side of the limiting membrane of the lysosome, protein interactions between lysosomal membrane proteins and organelle membrane proteins mediate membrane contact sites and transfer of cholesterol.

The lysosomal cholesterol efflux is sensed and translated to the regulation of cell proliferation and autophagy.

Lysosomes are of major importance for the regulation of cellular cholesterol homeostasis. Food-derived cholesterol and cholesterol esters contained within lipoproteins are delivered to lysosomes by endocytosis. From the lysosomal lumen, cholesterol is transported to the inner surface of the lysosomal membrane through the glycocalyx this shuttling requires Niemann–Pick C (NPC) 1 and NPC2 proteins. The lysosomal membrane proteins lysosomal-associated membrane protein (LAMP)-2 and lysosomal integral membrane protein (LIMP)-2/SCARB2 also bind cholesterol. LAMP-2 may serve as a cholesterol reservoir, whereas LIMP-2, like NPC1, is able to transport cholesterol through a transglycocalyx tunnel. Contact sites and fusion events between lysosomes and other organelles mediate the distribution of cholesterol. Lysosomal cholesterol content is sensed thereby regulating mammalian target of rapamycin complex (mTORC)-dependent signaling. This review summarizes our understanding of the major steps in cholesterol handling from the moment it enters the lysosome until it leaves this compartment.


Conflict of interest

A. Ballabio is co-founder of CASMA Therapeutics and of Next Generation Diagnostics (NGD).

For more information

Pending Issues

The current understanding of lysosome biology and function is still evolving. There is a need for further characterization of these aspects that may provide critical information on the pathophysiology of lysosomal storage diseases.

New methodologies should be exploited to improve our knowledge on lysosomal biology and on lysosomal disease pathophysiology. These methodologies may also have a major impact of patient care, with more efficient diagnostic pathways and availability of biomarkers to follow disease progression and effects of therapies.

Current therapies for the treatment of lysosomal storage disease have significant limitations. Particularly, biodistribution in target organs, such as brain, is a critical issue as many of these disorders are associated with central nervous system involvement.

The understanding of disease pathophysiology is critical as it has the potential to identify novel therapeutic targets and to indicate new strategies for the treatment of these disorders.


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