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Indian Journal of Biochemistry & Biophysics Vol. 45, April 2008, pp. 75-90

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Cathepsins: Fundamental Effectors of Endolysosomal Proteolysis Sonia Guha and Harish Padh* Department of Cell and Molecular Biology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S.G. Highway, Thaltej, Ahmedabad 380054, India Received 21 August 2007; revised 7 March 2008 Intracellular protein degradation is a universal feature of eukaryotic cells and vital for nutrition, protein turnover, intracellular signaling, development and other major physiological processes like antigen presentation and immunity. One of the major compartments of intracellular proteolysis is the endosome-lysosome system. The latter offers a highly orchestrated, vesicular pathway for protein transport and ultimate degradation in lysosomes. Though lysosomes are the classical organelles of complex, multi-enzymatic degradation, it is increasingly evident that endosomes conduct much more than mere transport functions. Endosomes contain significant levels of proteases like cathepsins and are sites of potent intracellular proteolysis. Further, discrete classes of endosomes harbor specific cathepsins and perform selective and exclusive functions. Hence, extra-lysosomal proteolytic machinery within the endocytic pathway enjoys spatial and temporal control over proteolytic functions. The review outlines the structural association and function(s) of major endolysosomal cathepsins. Keywords: Endosome, Cathepsins, Intracellular proteolysis.

Introduction Endocytosis is the only cellular mechanism that allows direct uptake of small molecules and entire cells into the otherwise highly restricted intracellular milieu, all without compromising cellular integrity or energetics. Consequently, cells use a wide variety of endocytic mechanisms. Based on the size of target materials, the process is broadly classified under two categories-phagocytosis used by specialized mammalian cells like macrophages for uptake of large paticles like whole cells and pinocytosis used to target small molecules. Although pinocytic capacity is a more generic phenomenon present in most cells, at least four different pinocytic mechanisms are known to exist-macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, and __________ *Author for correspondence: Tel: 27439375; Fax: 079-27450449 E-mail: [email protected] Abbreviations: APC, antigen presenting cell; BSA, bovine serum albumin; CTLA-2 beta, cytotoxic T-lymphocyte antigen-2 beta; DC, dentritic cells; DPPI, dipeptidyl peptidase I; ECM, extracellular matrix; GTPase, guanosine-tri-phosphate; IFN, interferon; IGF-I, insulin-like growth factor-I; Ii, invarinant chain; LAMP, lysosome associated membrane protein; LPS, lipopolysaccharide; MHC, major histocompatibility complex; M6PR, mannose-6-phosphate receptors; PTH, pituitary hormone; TGN, trans-Golgi network.

clathrin and caveolae-independent endocytosis1. Despite their mechanistic differences, there are common features in the biogenesis and organization of endocytic pathway. Endosomes are formed by the inward vesicular excision of host plasma membrane around extracellular target(s), resulting in small intracellular membrane bound organelles containing the engulfed material. It is now well accepted that spatial and temporal axis of endosome biogenesis (which initiates with the nascent endosomes and terminates into the lysosomes) is spaced with several discrete stations of unique endosomal classes and function2. The two major classes of such vesicles are the early and late endosomes, however, at least two other types have also been reported — the intermediate class of recycling endosomes, which exist between early and late endosomes, and the hybrid endosomes which are result of endosome-lysosome fusions3. The mechanism of endosome biogenesis reconciles two competing models: one of gradual endosome maturation and the other based on endosomal carrier vesicles between the early and late endosomal organelles4. The transport across these vesicles is goverened by a myriad of Rab GTPases which have been reviewed elsewhere5.

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INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

Classical concept of endolysosomal proteolysis The concept of lysosome as sole designate of intracellular proteolysis was established by seminal descriptions of the organelle in early ’60s6. Traditionally, the definition of lysosome was based on two criteria: the existence of a limiting membrane and the predominant storage of mature acid hydrolases within the organelle. Subsequently, several independent lines of research indicated that other, essentially non-lysosomal pathways play important role in intracellular proteolysis, which ultimately lead to the discovery of ubiquitin-proteasome system7. For more than two decades following de Duve’s assessment6, lysosomes continued to be held as the singular organelle for intracellular proteolysis in the endocytic pathway. The endosomes were largely considered as ‘intracellular transport organelles’ that fed the lysosomes. This view started changing from ’80s with better tools for subcellular fractionation, which increasingly highlighted at least two kinds of vesicles in isolated lysosomal preparations, a lighter less dense vesicular fraction, termed as prelysosomes and a morphologically familiar dense, heavier, mature lysosomes. Despite evidence indicating the existence of acid protease-rich prelysosomal vesicles in many cell types, these vesicles were largely considered as immature or early lysosomes that matured into functioning lysosomes8. However, subsequently with better understanding of the endocytic pathway this view started changing. Pre-lysosomal proteolysis in endocytic pathway Ultimately, in late 80s several independent groups firmly established the concept of pre-lysosomal proteolysis. In their seminal work, Diment and Stahl9 showed complete degradation of the mannose-BSA ligand occurred as early as 6 min, following ligand uptake, indicating lysosomes did not participate in this proteolytic function. The enzyme contributing to this function was subsequently characterized as cathepsin D, a pepstatin sensitive, aspartic (acid) protease previously known to be associated with lysosomes. Purified endosome preparations, free from lysosomes successfully conducted proteolysis in vitro10. The ensuing concept of endocytic, pre-lysosomal proteolysis was substantiated by independent groups in the same period, who showed related enzymes like cathepsin B, carboxypeptidases and others were also present in endosomes11-14.

Subsequent investigations implicated endosomal proteases in processing polypeptide hormones such as glucagons15 and pituitary hormones (PTH)16, growth factors13, plant and bacterial toxins such as ricin and cholera toxin17,18 as well as antigens19. The carboxypeptidase B was shown to cleave epidermal growth factor (EGF) in early endosomes13. In a related study, Blum et al14 radiolabelled lumenal contents of macrophage endosomes to confirm that both cysteine protease cathepsin B and aspartyl protease cathepsin D were present in endosomal vesicles. Using the plant toxin ricin A chain, the group identified activity of endosomal proteases over a broad pH range in macrophages. Cleavage of this toxin was dependent upon its transit into early endosomes and not observed on the plasma membrane. Once the toxin molecules were sorted into early endosomes, they encountered an increasing gradient of proteolytic activity along the endocytic pathway. Both cysteine and aspartyl proteases were responsible for ricin A cleavage in early endosomes17. Contemporary study20 in early and late endosomes of rat hepatocytes also found substantial enrichment of membrane bound cathepsin D and other associated acidic hydrolases. Significantly, Claus et al21 observed the distribution pattern and trafficking of the lysosomal acid proteases in the isolated endocytic compartments of J774 macrophages. Their work provided the first biochemical and immunocytochemical evidence of enrichment of cathepsin H in early endosomes. Later through successive experiments, Authier and colleagues investigated the endosomal proteolysis of EGFs in hepatocytes by cathepsin B22, insulin in hepatic parenchymal cells by cathepsin D23, IGF-I in human HepG2 hepatoma cells by cathepsin B24 and internalized cholera toxin in the endosomal apparatus of rat liver by cathepsin D18. Endolysosomal proteases The vast arsenal of endolysosomal hydrolases includes proteases, lipases, phosphatases, glycosidases and nucleases. Nearly all endolysosomal proteases are called cathepsins, which predominantly includes a group of cysteine and aspartic proteases sharing distribution specifically in the endocytic pathway (thereby not all cysteine or aspartic proteases are cathepsins). At least 13 different human cathepsins are known many with unique tissue distributions, diverse substrate specificities and functions. They include cysteine proteases —

GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS

cathepsins B, H, L, S, C, K, O, F, V, X and W and aspartic proteases — cathepsins D and E. Interestingly, though bulk of these cathepsins are stored in lysosomes, the endosomes are the major functional centers of proteolysis2,25. The endocytic pathway is not necessarily an intracellular system for incremental vesicular proteolysis, but more likely represents stations of distinct proteolytic environment and function. For example, proximal compartments like early endosomes are functional centers of neutral proteases like cathepsins H and D, while the later compartments are rich in acid active enzymes like cathepsins S, B, and D21. Cathepsins: Effectors of endolysosomal proteolysis Historical perspective

Cathepsins were discovered in the first half of the 20th century. Gutman et al26. first reported the characterization of cathepsin C (then known as dipeptidyl peptidase I or DPPI). After a long gap, two major cathepsins (B and H) were identified, and their amino acid sequence was reported. Thereafter, cathepsins H, L, and S were discovered; however, not much was known about the physiological or pathological role of these proteases till the 1990s. In 1990, the first crystal structure of a lysosomal cysteine protease human cathepsin B was determined, heralding rapid progress in cathepsin research27. Indeed, 1990s were the golden era of lysosomal cysteine protease research with six out of 11 known human enzymes (cathepsins B, H, L, S, C, K, O, F, V, X and W) were identified. The 1990s also provided more clues on the functions of cysteine proteases: several cathepsin knockouts highlighted the physiological roles of cathepsins much more than mere scavengers, which was long believed to be their major function. With advent of novel concepts and availability of human genome sequences, this number might probably increase, especially since several new mouse cathepsins without apparent human counterparts have been discovered in 200028,29. Structure

The cysteine cathepsins comprise a group of papain-like enzymes sharing similar amino acid sequences and structural domains. The cathepsins are generally composed of disulfide-connected heavy and light chains. They are monomers with molecular mass of ~30 kDa, except tetrameric cathepsin C. They have a two-domain structure with the V-shape active site

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cleft extending along the two-domain interface; the left (L-) domain is dominated by three α-helices and the right (R-) domain is based on a β-barrel motif30. These domains are highly conserved and all members show common secondary structure elements in their respective domains. The essential catalytic residues are Cys25 and His159 (Fig. 1), situated on the opposite sides of cleft in L and R domains. Differences between different proteases are usually due to deletions or insertions in the loop regions between the conserved structural elements comprising the papain-fold31-33. All members of mammalian aspartic cathepsin group share high degree of sequence similarity, which includes well-described peptidases like cathepsin D and other related proteases like pepsinogen A and pepsin. Some of the conserved features include two aspartic residues in the active site, which exist as a conserved Asp-Thr-Gly triad. Long stretches in the sequence around these triads are also highly conserved, particularly a Tyr and Cys in the so-called ‘flap’ region around the active site and at least three disulfide linkages. Significant similarities exist in the primary, secondary and tertiary structures of aspartic cathepsins as well. The latter exhibit a prominent bi-lobed structure consisting of a deep active site cleft with the active site aspartic residues in the bottom of the cleft34. Targeting of cathepsins

Cathepsins are synthesized as inactive pre-proenzymes having a signal peptide and a multifunctional N-terminal proregion. The role of prepeptide is to drive cathepsin precursor in the lumen of rough endoplasmic reticulum, after which it is subsequently hydrolyzed. The proregion on the other hand folds the nascent cathepsin properly, inhibits its proteolytic activity, preserves its three-dimensional structure at neutral pH and maintains the precursor in an inactive state until cleaved (Fig. 1). The procathepsin later undergoes carbohydrate processing and asparagine-linked glycosylation in the Golgi. The enzymes N-acetylglucosaminylphosphotransferase and a-N-acetylglucosaminidase add a mannose-6phosphate label to the pro-cathepsin. The mannose-6phosphate tagged pro-cathepsin subsequently binds to M6PR in the trans-Golgi network There are two M6PRs; 275 kDa and 46 kDa which are cation-independent, with their bound ligands subsequently translocated to late endosomes.

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Fig. 1—Structure of cathepsins [(A): Human cathepsin B, a typical member of the cysteine cathepsin family (i) Procathepsin B: The single chain of procathepsin B entails the propeptide sequence from ArgP1 to LysP62 and mature peptide from Leu1 to Asp254. The propeptide folds on the cathepsin B surface, shielding the enzyme active site from exposure to solvent. (ii) Mature cathepsin B: The maturation process includes removal of the N-terminal propeptide, the C-terminal extension and a dipeptide between residues 45 and 50 (mature enzyme numbering). The product is an enzymatically active molecule with two chains co-valently crosslinked by a disulfide bridge; and (iii) Interaction between cathepsin B and its inhibitor, dipeptidyl nitrile: The reversible nitrile inhibitor binds through the formation of a thioimidate ester with the Cys29 of cathepsin B active site155,156. (B): Human cathepsin D, a typical member of the aspartic cathepsin family complexed with inhibitor pepstatin. The native enzyme reveals two identical molecules that are related by a pseudo twofold rotation. There are three topologically distinct regions: N-terminal domain (residues 1-188), C-terminal domain (residues 189-346), and an interdomain, anti-parallel/3sheet composed of the N terminus (residues 1-7), the C terminus (residues 330-346), and the interdomain-linking residues (160-200). The latter region links the pseudo-twofold-related N and C domains, each of which contributes an aspartic acid, Asp33 and Asp231 to the active site. Binding of pepstatin to cathepsin D induces small structural changes in the "flap" region (the hairpin structure composed of residues 72-87). Residues 79 and 80 at the tip of the flap moves in toward the inhibitor by about 1.7 angstorm157. The white and red arrows denote active site and inhibitor binding respectively.

However, if newly synthesized pro-cathepsins fail to bind to M6PRs in the trans-Golgi network, they bind to M6PRs localized at the plasma membrane and reach the late endosomes/lysosomes via early endosomes and receptor-mediated endocytosis. In late endosomes, mild acidic pH (6.0–6.5) dissociates pro-cathepsins from M6PRs and finally procathepsins are processed into active proteases31. Interestingly, a distinct targeting pathway using aggregation has also been reported, where upregulated procathepsin L is targeted to tetraspanin CD63 enriched multivesicular late endosomes35. Mode of action

The mode of action of cysteine cathepsins, especially the papain superfamily has been extensively characterized than their counterparts in the aspargine protease family. Most of cysteine protease cathepsins require acidic pH for optimal activity. This is not surprising, since Cys25 at catalytic center exhibits a very low pKa value, which helps formation of an thiolate ± imidazolium ion-pair with hiatidine-159 required for catalysis 36. Substrates bind into the active site in an extended conformation

and the carbonyl carbon of scissile bond undergoes nucleophilic attack from the active-site thiol, resulting in the release of amine product. The ensuing acylenzyme reacts with water to release the carboxyl product (deacylation), resulting in the regeneration of free enzyme. In contrast to many other peptidases, cysteine cathepsins do not have a single specific substrate, although they do differ considerably in their preferred cleavage site. Binding sites for substrate residues N-terminal to the cleaved peptide bond are designated as S1, S2...etc.; those C-terminal are designated as S1_, S2_ (where S1 and S1_ are proximal to the cleaved bond). Similarly, P1, P2......, etc. are used to designate the corresponding substrate residues31-33,37. Like cysteine cathepsins, aspartate cathepsins exhibit considerable sequence similarities and common motifs. These include triades Asp-Thr-Gly around two active sites aspartic residues (at number 32 and 215, numbering of human pepsin A) and relative position of Cys residues and of Tyr75 and its surroundings. Tyr75 is localized in a so-called flap region, which is flexible and partially covers the


active site. The sequence similarities are also reflected in the significant resemblance in primary structures. Peptidases belonging to the A1 family are formed by bi-lobal structure and both lobes are structurally similar, which suggests a common ancestor. A dimeric molecule consisting of two identical subunits, by gene duplication and fusion evolves into monomeric bilobal structure38. Regulation The cathepsins are regulated in many ways, i.e. at the gene transcription, translation and expression level, by post-translational modifications, localization, environmental pH, zymogen activation, presence or absence of inhibitors, trafficking and by inactivation and degration31. Transcription and translation

Exposure to various inflammatory stimuli controls the expression of cysteine proteases at the transcriptional level. Interestingly, downregulation of cathepsin S and B mRNA levels has been reported in primary alveolar macrophages, microglia and macrophage cell lines in response to proinflammatory cytokine IFN-α39. In contrast, upregulation of cathepsin S has been observed in human keratinocytes, bone marrow cells and lung tissue40,41. In another study in peritoneal macrophages treated with IFN-γ, no change in the level of cathepsin S mRNA, but a marked decrease in the transcription of cathepsin L have been reported42. Similarly, LPS and transcription factor PU.1, in concert with IFN regulatory factors enhance cat S transcription in cervical smooth muscle cells and endosomes of antigen-presenting cells (APC) respectively43,44. Additionally, alternative splicing of cathepsins B and L genes results in increased translation and protease stability, along with enhanced secretion. Zymogen activation

Most cathepsins are synthesized as inactive zymogens. The zymogen is activated by proteolytic removal of amino terminal pro-domain, which blocks access to the active catalytic site. The activation process is triggered by a pH drop and/or by glycosaminoglycans that substantially weaken the interactions between the propeptide and catalytic part45,46. Consequently, the proenzyme adopts a looser conformation, in which propeptide is less tightly bound to the active site, while the secondary structure remains intact. Ultimately, the propeptide dissociates

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from the protease, unfolds and is proteolytically degraded. The proteolytic removal is brought about by various proteases like pepsin, neutrophil elastase, cathepsin D and different cysteine proteases. Alternatively, the propeptide can be cleaved at several sites during activation, thus eliminating or diminishing its inhibitory function, as observed for cathepsins L and K31. Recently, cathepsin D has been shown to be processed in a manner independent of its catalytic function — by a combination of autoactivation and cathepsin L-assisted maturation47. Environmental pH

An essential regulating factor of proteolytic activity of endosomal cathepsins is the pH of environment. Thus, decrease in pH occurring during maturation of endosomes presumably weakens the interaction between the propeptide and proenzyme, favoring the activation process. Furthermore, cathepsins B, S and L are irreversibly denaturated in lysosomes towards the end of maturation process, when pH is decreased to 3.8. Denatured cathepsin L is proteolytically removed by acid-active cathepsin D. In addition, cathepsins L, B, H, K, V and F are unstable at neutral pH and, therefore, are less active outside lysosomes, while others like cathepsins B and S are extremely stable, possibly accounting for their role outside lysosomes31,48. Protein inhibitors

By far, the most important regulator for endosomal proteases is their endogenous protein inhibitors. The latter binds tightly to the enzyme, thereby preventing substrate binding. Some of the common inhibitors are cystatins, thyropins, α2-macroglobulin, serpin, squamous cell carcinoma antigen 1, CTLA-2 beta and chagasin. Cystatins are non-selective protein inhibitors of the papain family of cysteine proteases. They inactivate target proteases reversibly and competitively by indirect blockage of catalytic centers and form very stable bimolecular complexes with proteases. Cystatin superfamily is traditionally sub-divided according to their sequence similarities into three major families comprising mainly of stefins, cystatins and kininogens. In contrast, thyropins are more selective, probably reflecting their more specialized in vivo role, while the general inhibitors like α-macroglobulins unspecifically trap endopeptidases of different types, blocking the access of protein substrates to the active site of trapped proteinases without inactivating them33,49,50.


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Table 1—Cathepsins found within the endolysosomal system Name

Synonyms

Catalytic Endo group peptidase

Exopeptidase Carboxy Amino peptidase peptidase

Operating pH

Distribution

Refs

_

5.0-6.0

Ubiquitous

17, 54

+ dipeptidase

5.0-6.0

Ubiquitous

139

Cathepsin B

Cathepsin B1

Cys

+

Dipeptidyl peptidase I (DPPI) Cathepsin D Cathepsin E

Cathepsin J Cathepsin C

Cys

+

_ _

Asp Asp

+

_ _

_ _

2.8-7.0 3.0-3.5

Ubiquitous Restricted

Cathepsin F Cathepsin G Cathepsin H

_ _ Cathepsin I

Asp Ser Cys

+ n.d +

_ n.d. _

5.2- 6.8 7.5 6.8

Ubiquitous Neutrophils Ubiquitous

Cathepsin J e(mouse) Cathepsin K

_

Cys

n.d

n.d.

_ n.d. + monopeptidase n.d.

56 23 140, 141,142 143, 144 105 21, 71

n.d.

Placenta

145

Cathepsin O2, OC2, O, X _ _

Cys

+

_

_

5.0-8.0

121, 123

Cys Cys

+ n.d.

_ n.d.

_ n.d.

4.5-6.0 n.d.

Osteoclasts Chondroclast Ubiquitous Placenta

_

Cys

n.d.

n.d.

n.d.

n.d.

Ubiquitous

147

Cathepsin Qe(rat), R(mouse) Cathepsin S

_

Cys

n.d.

n.d.

n.d.

n.d.

Placenta

148

_

Cys

+

_

_

5.0-8.0

Cathepsin Vc

Cathepsin L2 Cathepsin U

Cys

+

_

_

5.7

Cathepsin W

Lymphopain

Cys

n.d.

n.d.

n.d.

_

Cathepsin X

Cathepsin Z, Pd, Y

Cys

_

+ monopeptidase dipeptidase

_

5-5.5

Cathepsin L Cathepsin Me(mouse) Cathepsin O

+ dipeptidase

83 146

Macrophages/ 51, 106 Monocytes, dendritic cells, microglial cells Thymus, 149, 150 testis, corneal epithelium CD8 + T cells 151, 152 and NK cells Ubiquitous 153, 154

c

Known only in primates; dRat ortholog; eKnown only in rodents; n.d., not detected

Oxidation states

The cysteine protease cathepsins are readily inactivated by oxidation of the active site cysteine and require a reducing environment for full activity.

H, S, D and C are distributed between late endosomes and lysosomes51. The cathepsin activity is increased in late endosomes after LPS-induced stimulation52. Major cathepsins

Trafficking

Cathepsin activity is also modulated by the cellular environment. In human B lymphoblastoid cells, distinct patterns of cathepsin activity are concentrated in specific endocytic compartments. Cathepsins B and Z are most prominent in early and late endosomes, suggesting initial proteolytic attack, while cathepsins

The enormous diversity of cathepsin types, their cellular distribution and functions (see Table 1 and Fig. 2) make it difficult to prioretise them on the basis of physiological importance. However, a few of the most well characterized representative members of cysteine and aspartate families are briefly described below.


Fig. 2—Mutiple sequence alignment of (human) endosomal cysteine cathepsins using ClustalW [Though the sequences indicate considerable divergence, the active sites Cys25 and His159 are conserved (highlighted in bold). The sequences were taken from the SWISS- PROT or GenBank databases]

Widely expressed cathepsins Cathepsin B

Cathepsin B is by far the most abundantly expressed mammalian cathepsin and exhibits multiple tissue/cell distribution including skeletal tissues, plasma membrane caveolae of differentiating myoblasts53, epithelial cells and APC’s like dendritic cells (DCs) and macrophages. It is synthesized as a 45-39 kDa precursor and processed to a 32 kDa and 28 kDa active forms, prior to sorting into the endocytic pathway. It can catalyze cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4 and an exopeptidase attack from the C-terminus with a pH optimum at 4.5-5.554. Unlike other members of cysteine protease family, cathepsin B has a large flexible insertion loop termed

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as the occluding loop which is connected via His110 and Arg116 residues to the Asp22 and Asp224 of the enzyme, respectively. When an endopeptidase substrate binds to the enzyme, this loop moves, resulting in an increased endopeptidase activity55. The endopeptidase attack initiates protein breakdown by cleaving peptide bonds within the sequence of substrate protein away from the amino and carboxytermini. This leads to an increase in the number of new termini, allowing the exopeptidases to work with greater efficiency. With this dual action as an endopeptidase and a peptidyldipeptidase (an exopeptidase that removes dipeptides from the C-terminus of proteins and peptides), cathepsin B is equipped to participate in both the early and late stages of the endosomal protein breakdown54. The highest concentration of cathepsin B is found in the early endosomes and less in the lysosomes. Cathepsin B is usually involved in the turnover of proteins and plays various roles in maintaining the normal metabolism of cells. In macrophage endosomes, it causes proteolytic cleavage of ricin A, mannose-bovine serum albumin (BSA), pituitary hormone, glucagon, thyroglobulin and vitellogenin, both at neutral and acidic pH14,17,56. Creemers et al57 used specific inhibitor of cathepsin B and implicated cathepsin B involvement in the intracellular degradation of soft connective tissue collagen in cultured periosteal explants. Intracellular cathepsin B also processes and activates prorenin58, brings about histolysis of silk gland during metamorphosis in Bombyx mori along with cathepsin L59, and processes yolk during maturation of killifish oocytes60. Significant cathepsin B activity in the intestine of parasitic nematode Haemonchus contortus supports its protease role in nutrient digestion61. Recent studies have identified cathepsin B as the main enzyme in degradation of insulin-like growth factor-1 (IGF-I), operative within endosomes at a pH range of 5.5-7.024. The proteolytic activity of cathepsin B is also crucial for the activation of trypsinogen to trypsin in the development of acute pancreatitis62,63. Increased expression of cathepsin B at protein and mRNA levels in patients with heart failure suggests its role in the genesis and development of cardiac disease64. Furthermore, cathepsin B is negatively correlated with other pathological conditions like osteoarthritis65, gastric cancer66, oral cancer metastasis67, colorectal cancer68 and ovarian cancer69,70.

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Cathepsin H

Although ubiquitous, cathepsin H is present at a relatively high concentration in non-skeletal tissues such as kidney, spleen and macrophages. Two mature forms (22 and 28 kDa) and an immature form (37 kDa) of cathepsin H have been reported71. It acts as an endopeptidase as well as an aminopeptidase with a pH optimum of about 6.5-6.8. Its aminopeptidase activity is essentially due to the presence of a mini chain (residual octapeptide EPQNCSAT of the propeptide) present at C-terminal72,73. Recently, the mini-chain of human cathepsin H has been identified as the major structural element determining its substrate specificity. The deletion of mini-chain switches the substrate specificity from aminopaptidase to endopeptidase74. Unlike other cysteine proteinases, cathepsin H is not inhibited by its own free propeptides. During maturation and mini-chain formation the propeptide binding region of the parent peptide is rearranged, which disables the propeptides to recognize the mature cathepsin75. Cathepsin H has been detected as an early endosomal acid protease in J774 macrophages21. In human dendritic cells (DCs), the enzyme is found to be concentrated in late endosomes in response to maturation stimulus like LPS, indicating more efficient local proteolysis52. Cathepsin H acts as a neuropeptidase in human brain76 and degrades a variety of components of the extracellular matrix (ECM), such as proteoglycans, laminin, and collagens II, IX, and XI. It is involved in the first N-terminal processing step of a 21 kDa pro-surfactant protein to a 4.2 kDa dipalmitoylated in electron-dense multivesicular bodies of type II pneumocytes77. It is also associated with various pathological conditions like human fibrous meningioma78, colorectal cancer79, juvenile idiopathic arthritis80, human prostrate tumor81 and lung cancer82. Cathepsin L

Cathepsin L is constitutively expressed in many tissues, and has highly expressed levels in tissues that turnover more rapidly, such as the liver, kidney and ovary as well as in the lysosomes of stimulated macrophages, fibroblasts, DCs, kupffer cells, and endotheliocytes83. Upon stimulation with LPS, the lysosomal enzymes show a shift to late endocytic vesicles in mature DC’s. Interestingly, expression level of cathepsin L is enhanced by several growth factors like epidermal growth factor, fibroblast growth

factor, platelet-derived growth factor and hormones like follicle stimulating and leuteinizing hormones84. In Pichia pastoris, intermolecular processing event within a conserved GXNXFXD motif in the 37 kDa cathepsin L proform generates a 30 kDa intermediate that is finally processed to a 24.5 kDa mature form at pH 5.085. Active site-directed labeling in resting human DC also confirms two enzymatically active isoforms 25 kDa and 30 kDa of cathepsin L. The two chain forms of cathepsin L in the late endocytic compartments of antigen-presenting cells are maintained by the chaperonic activity of p41. In the absence of p41, the mature chain forms are degraded by other endocytic proteases86. Functionally, cathepsin L is an endopeptidase and active at pH 3.0-6.5 with optimum activity at pH 5.5. Cathepsins L and B are suggested to play a role in lysosomal protein turnover at acidic pH in vitro. Since both cathepsins L and B have overlapping substrate specificities, validation of their in vivo role has proven difficult. Although, both cathepsins L and B could cleave the Nipah virus fusion protein, only cathepsin L mediated cleavge results in the physiologically relevant size of the products87. Functionally, cathepsin L is one of the widely expressed peptidase with potent elastase and collagenase activity. Both cathepsins L and B are essential for maturation and integrity of the post-natal central nervous system and both compensate for each other in vivo. In brief, cathepsin L appears to be critically involved in epidermal homeostasis, normal tissue remodeling, phagocytosis, regulation of hair cycle, follicular wall degradation during oogenesis and MHC class II-mediated antigen presentation in cortical epithelial cells of thymus. Cathepsin L has been implicated in the proteolytic processing of Hendra virus precursor F protein F(0) to the active F(1) + F(2) disulfide-linked heterodimer. The latter is essential for the generation of a mature and fusogenic form of the F protein88. In a number of pathological conditions, cathepsins L and B are secreted out of the vesicular lumen and are involved in degradation of ECM components. More specific functions have also been postulated in a wide range of physiological and pathological processes. These include antigen presentation89, prohormone processing90, turnover of amyloid in Alzheimer’s disease91, tumor invasion92-94, inflammatory skin disease95, apoptosis96, and auto-immune diabetes97.


Cathepsin D

It is most extensively characterized enzyme among cathepsins and is demonstrated to be exclusively associated with endosome functions98. It causes complete degradation of the mannose-BSA ligand in early endosome preparations free from lyososomes9. Cathepsin D is a pepstatin sensitive aspartate protease belonging to the pepsin superfamily. It is present in endosomes of smooth muscle cells, epithelial cells, macrophages, and fibroblasts99 and in multivesicular endosomes in macrophages, hepatocytes and B-lymphoblastoid cells. Cathepsin D is synthesized as an 52 kDa inactive precursor and transported from the golgi complex to the acidic endosomes100. In acidic endosomal lumen, the proenzyme is processed and activated to its corresponding 48 kDa intermediate and finally to 34 kDa and 14 kDa mature forms. Earlier hypothesis emphasized on partial auto-activation generating a 51 kDa pseudo-cathepsin D, followed by enzymeassisted maturation involving cysteine and/or aspartic proteases. However, recent studies with cathepsin D deficient fibroblast cell lines have shown that maturation of endocytosed pro-cath-D is independent of its catalytic function and requires cysteine proteases like cathepsin L and B47. In vitro cathepsin D acts at different pH ranging from 2.8-6.0, depending on the substrate and In vivo, it requires an acidic pH (3.0–5.0), known to be present in the different endosomal compartments. It acts as a ligand in its zymogen state and as an effective endosomal protease in its mature form101. It is implicated in processing of the 23 kDa prolactin pituitary hormone to 16 kDa fragments in rat mammary epithelial cells102. In related studies, Merlen and colleagues documented co-localization of internalized cholera toxin and cathepsin D in early endosomes in rat liver endosomes, HepG2 and intestinal Caco-2 cell lines, where cholera toxin is subsequently degraded in a pH-dependent manner. These results strongly confirm the role of endocytic cathepsin D in proteolytic activation of cholera toxin18. Similarly, cathepsin D is involved in proteolytic degradation of ricin A chain in early endosomes of macrophage98. Membrane association may influence the pH dependence of cathepsin D activity, accounting for the observation of some aspartyl protease activity in endosomes at neutral pH. Through the generation

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of cathepsin D-deficient mice, it is shown that cathepsin D is involved in the limited and specific rather than bulk proteolysis. Cathepsin D deficient mice are born normal, but die at post-natal day 26 due to massive necrosis, thromboembolia, lymphopenia. The enzyme also takes part in antigen processing, in generation of peptide hormones, intracellular protein degradation, tissue remodeling and apoptosis. During apoptosis, the endolysosomal enzyme translocates into the cytosol and the cytosolic enzyme triggers apoptotic pathways by interacting with members of the apoptotic machinery rather than cleaving proteolytic substrates103,104. Tissue-specific cathepsins Cathepsin S

Cathepsin S was first purified from bovine lymph node and spleen and cDNA was cloned from bovine spleen by RT-PCR105,106. Subsequently, human cathepsin S was independently cloned by two groups107. Expression of cathepsin S is tissue-specific and is preferentially expressed in phagocytic and APCs. High levels of cathepsin S are found in alveolar macrophages of human and rat lung, DCs, spleen, lymph nodes, vascular smooth muscle cells, while moderate to low levels have been detected in appendix, bone marrow and thymus108,109. The mature protein is found to be a single-chain polypeptide of around 28-30 kDa52. It is a potent endopeptidase cleaving a range of proteins and synthetic substrates over a broad pH range of 5-8106. The enzyme shows elastinolytic activity at pH 4.5-5.5 (comparable with pancreatic elastase), but unlike other cathepsins retains about 20-25% of maximal activity at neutral pH (where it is comparable with leukocyte elastase in activity)107. At neutral pH, it degrades fibronectin, laminin and myelin basic protein and proteoglycans110. Further, cathepsin S, unlike cathepsins B, H, and L is stable at pH 7.0. Consistent with its broad pH optimum, cathepsins S and B are found to be the most potent proteolytic enzymes in endosomes of human DCs. Consequently, it has been detected all along the endocytic pathway with a high concentration in lysosomes111. However, within hours of addition of a maturation stimulus in DCs, its local activity in the late endosomes is increased, implying its important role in stable antigenic processing. In contrast, in J774 macrophages, bulk of the enzyme is localized to late endosomes.

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Fig. 3—Distribution and functions of cathepsins along the endocytic pathway [The schematic summarizes our current understanding about the roles of various cathepsins encountered in the endocytic pathway]

Cathepsin S plays a key role in antigen presentation112. Professional as well as nonprofessional MHC class II-expressing APCs use cathepsin S for MHC class II-mediated antigen presentation113. Absence of lysosomal cathepsin S leads to an impaired degradation of invariant chain. Subsequently, invariant chain remnant accumulates in class II MHC-positive endosomal compartments, which are enlarged in size and lack multivesicular morphology114. Furthermore, microvascular endothelial cells from cathepsin S-deficient mice shows reduced collagenolytic activity and defective microvessel development during wound repair, implying an essential role of cathepsin S in ECM degradation and angiogenesis115. Alternatively, its role in angiogenic islet formation and growth of solid tumors in mouse model of multi-stage murine pancreatic islet cell carcinogenesis is also reported116. The enzyme functions both intracellularly in degradation of endosomal protein cargo117, and extracellularly in tissue, matrix, elastin and bone remodelling. Activated macrophages release substantial amount of cathepsin S and hence the latter is responsible for various pathological conditions like atherosclerosis118, astrocytoma progression119, and autoimmune diseases120. Cathepsin K

It was first cloned from osteoclasts and described as OC-2121. Subsequently, its cDNA was characterized and designated as cathepsin K and highest expression was found in bone tissues122. Besides osteoclasts and chondroclasts and their precursors, epithelial cells in various organs express

significant amount of cathepsin K123. Like most other enzymes in its class, cathepsin K is synthesized as a ~42 kDa proform, which is activated to a ~27 kDa mature form123,124. Enzyme activities of cathepsin K are similar to those of cathepsin S105,106, which includes a broad pH optimum between 5-8. At neutral pH, it is more stable than cathepsin L, but less so than cathepsin S. Being expressed significantly in bone cells, it is actively engaged in cartilage resorption124-126. It is a potent collagenase and gelatinase; besides also being active against noncollagenous matrix proteins, such as aggrecan, osteopontin, and osteonectin127-129. Cathepsin K is essential for normal bone resorption; humans lacking it exhibit pycnodysostosis, which is characterized by short stature and osteosclerosis130-132. In a pioneering work, the direct evidence of physiological importance of this enzyme has been demonstrated in lung matrix homeostasis129. Using a chemically induced lung fibrosis in cathepsin K-deficient mice (CTSK-/-), the study showed reduced collagenolytic activity and consequent higher fibrosis. Both in control (CTSK+/+) fibroblasts and lung specimens obtained from patients with lung fibrosis fibroblasts, capthesin K levels are significantly upregulated, indicating its protective role in fighting excessive collagen deposition in diseased lungs129. It is also involved in kinin degradation133. A direct role of cathepsin K is implicated in atherosclerosis134, osteoarthritis135, osteoporosis136, and rheumatoid arthritis137. The macrophages in human atheromas and giant tumor cells of bones express high levels of cathepsin K138.


Conclusion It is increasingly evident that cathepsins are not exclusively lysosomal but enjoy specific distribution within a diverse of endosomal classes that differ in morphology, biochemistry and function (Fig. 3). Togther, this generates the enormous catabolic repertoire required to process multifarious endocytic cargo. Accurate knowledge of endosome-cathepsin relationships is essential to expand our current understanding of intracellular proteolysis that plays important roles in health and disease. Acknowledgement We are thankful to Commissionerate of Industry of the Government of Gujarat, Department of Science and Technology (DST) and Lady Tata Memorial Trust for funding the research project and awarding fellowship (to SG).

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