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 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 like 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 clathrin and caveolae-independent endocytosis1. Endocytosis is the only cellular mechanism that Despite their mechanistic differences, there are allows direct uptake of small molecules and entire common features in the biogenesis and organization cells into the otherwise highly restricted intracellular of endocytic pathway. milieu, all without compromising cellular integrity or energetics. Consequently, cells use a wide variety of Endosomes are formed by the inward vesicular endocytic mechanisms. Based on the size of target excision of host plasma membrane around materials, the process is broadly classified under two extracellular target(s), resulting in small intracellular categories-phagocytosis used by specialized membrane bound organelles containing the engulfed mammalian cells like macrophages for uptake of large material. It is now well accepted that spatial and paticles like whole cells and pinocytosis used to target temporal axis of endosome biogenesis (which initiates small molecules. Although pinocytic capacity is a with the nascent endosomes and terminates into the more generic phenomenon present in most cells, at lysosomes) is spaced with several discrete stations of least four different pinocytic mechanisms are 2 unique endosomal classes and function . The two known to exist-macropinocytosis, clathrin-mediated major classes of such vesicles are the early and late endocytosis, caveolae-mediated endocytosis, and endosomes, however, at least two other types have ______also been reported — the intermediate class of *Author for correspondence: recycling endosomes, which exist between early and Tel: 27439375; Fax: 079-27450449 late endosomes, and the hybrid endosomes which are E-mail: [email protected] 3 result of endosome-lysosome fusions . The Abbreviations: APC, antigen presenting cell; BSA, bovine serum mechanism of endosome biogenesis reconciles two albumin; CTLA-2 beta, cytotoxic T-lymphocyte antigen-2 beta; DC, dentritic cells; DPPI, I; ECM, competing models: one of gradual endosome extracellular matrix; GTPase, guanosine-tri-phosphate; IFN, maturation and the other based on endosomal carrier interferon; IGF-I, insulin-like growth factor-I; Ii, invarinant chain; vesicles between the early and late endosomal LAMP, lysosome associated membrane protein; LPS, organelles4. The transport across these vesicles is lipopolysaccharide; MHC, major histocompatibility complex; goverened by a myriad of Rab GTPases which have M6PR, mannose-6-phosphate receptors; PTH, pituitary hormone; 5 TGN, trans-Golgi network. been reviewed elsewhere . 76 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

Classical concept of endolysosomal proteolysis Subsequent investigations implicated endosomal The concept of lysosome as sole designate of proteases in processing polypeptide hormones such as intracellular proteolysis was established by seminal glucagons15 and pituitary hormones (PTH)16, growth descriptions of the organelle in early ’60s6. factors13, plant and bacterial toxins such as ricin and Traditionally, the definition of lysosome was based on cholera toxin17,18 as well as antigens19. The two criteria: the existence of a limiting membrane and B was shown to cleave epidermal the predominant storage of mature acid growth factor (EGF) in early endosomes13. In a within the organelle. Subsequently, several related study, Blum et al14 radiolabelled lumenal independent lines of research indicated that other, contents of macrophage endosomes to confirm that essentially non-lysosomal pathways play important both cysteine B and aspartyl role in intracellular proteolysis, which ultimately lead protease cathepsin D were present in endosomal to the discovery of ubiquitin-proteasome system7. vesicles. Using the plant toxin ricin A chain, the group identified activity of endosomal proteases over For more than two decades following de Duve’s a broad pH range in macrophages. Cleavage of this assessment6, lysosomes continued to be held as the toxin was dependent upon its transit into early singular organelle for intracellular proteolysis in the endosomes and not observed on the plasma endocytic pathway. The endosomes were largely membrane. Once the toxin molecules were sorted into considered as ‘intracellular transport organelles’ that early endosomes, they encountered an increasing fed the lysosomes. This view started changing from gradient of proteolytic activity along the endocytic ’80s with better tools for subcellular fractionation, pathway. Both cysteine and aspartyl proteases were which increasingly highlighted at least two kinds of responsible for ricin A cleavage in early endosomes17. vesicles in isolated lysosomal preparations, a lighter Contemporary study20 in early and late endosomes less dense vesicular fraction, termed as prelysosomes of rat hepatocytes also found substantial enrichment and a morphologically familiar dense, heavier, mature of membrane bound cathepsin D and other associated lysosomes. Despite evidence indicating the existence acidic hydrolases. Significantly, Claus et al21 observed of acid protease-rich prelysosomal vesicles in many the distribution pattern and trafficking of the cell types, these vesicles were largely considered as lysosomal acid proteases in the isolated endocytic immature or early lysosomes that matured into compartments of J774 macrophages. Their work functioning lysosomes8. However, subsequently with provided the first biochemical and immunocyto- better understanding of the endocytic pathway this chemical evidence of enrichment of in view started changing. early endosomes. Later through successive experi- ments, Authier and colleagues investigated the Pre-lysosomal proteolysis in endocytic pathway endosomal proteolysis of EGFs in hepatocytes by Ultimately, in late 80s several independent groups cathepsin B22, insulin in hepatic parenchymal cells by firmly established the concept of pre-lysosomal cathepsin D23, IGF-I in human HepG2 hepatoma cells proteolysis. In their seminal work, Diment and Stahl9 by cathepsin B24 and internalized cholera toxin in the showed complete degradation of the mannose-BSA endosomal apparatus of rat liver by cathepsin D18. ligand occurred as early as 6 min, following ligand uptake, indicating lysosomes did not participate in Endolysosomal proteases this proteolytic function. The contributing to The vast arsenal of endolysosomal hydrolases this function was subsequently characterized as includes proteases, lipases, phosphatases, glyco- cathepsin D, a pepstatin sensitive, aspartic (acid) sidases and nucleases. Nearly all endolysosomal protease previously known to be associated with proteases are called cathepsins, which predominantly lysosomes. Purified endosome preparations, free includes a group of cysteine and aspartic proteases from lysosomes successfully conducted proteolysis sharing distribution specifically in the endocytic in vitro10. The ensuing concept of endocytic, pathway (thereby not all cysteine or aspartic pro- pre-lysosomal proteolysis was substantiated by teases are cathepsins). At least 13 different human independent groups in the same period, who showed cathepsins are known many with unique tissue related like , distributions, diverse substrate specificities and and others were also present in endosomes11-14. functions. They include cysteine proteases — GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 77

cathepsins B, H, L, S, C, K, O, F, V, X and W and cleft extending along the two-domain interface; the aspartic proteases — cathepsins D and E. left (L-) domain is dominated by three α-helices and Interestingly, though bulk of these cathepsins are the right (R-) domain is based on a β-barrel motif30. stored in lysosomes, the endosomes are the major These domains are highly conserved and all members functional centers of proteolysis2,25. The endocytic show common secondary structure elements in their pathway is not necessarily an intracellular system for respective domains. The essential catalytic residues incremental vesicular proteolysis, but more likely are Cys25 and His159 (Fig. 1), situated on the represents stations of distinct proteolytic environment opposite sides of cleft in L and R domains. and function. For example, proximal compartments Differences between different proteases are usually like early endosomes are functional centers of neutral due to deletions or insertions in the loop regions proteases like cathepsins H and D, while the later between the conserved structural elements comprising compartments are rich in acid active enzymes like the -fold31-33. cathepsins S, B, and D21. All members of mammalian aspartic cathepsin group share high degree of sequence similarity, which Cathepsins: Effectors of endolysosomal proteolysis includes well-described peptidases like cathepsin D Historical perspective and other related proteases like pepsinogen A and Cathepsins were discovered in the first half of the pepsin. Some of the conserved features include two 20th century. Gutman et al26. first reported the aspartic residues in the , which exist as a characterization of (then known as conserved Asp-Thr-Gly triad. Long stretches in the dipeptidyl peptidase I or DPPI). After a long gap, two sequence around these triads are also highly major cathepsins (B and H) were identified, and their conserved, particularly a Tyr and Cys in the so-called sequence was reported. Thereafter, ‘flap’ region around the active site and at least three cathepsins H, L, and S were discovered; however, not disulfide linkages. Significant similarities exist in the much was known about the physiological or primary, secondary and tertiary structures of aspartic pathological role of these proteases till the 1990s. In cathepsins as well. The latter exhibit a prominent 1990, the first crystal structure of a lysosomal bi-lobed structure consisting of a deep active site cleft human cathepsin B was determined, with the active site aspartic residues in the bottom of heralding rapid progress in cathepsin research27. the cleft34. Indeed, 1990s were the golden era of lysosomal cysteine protease research with six out of 11 known Targeting of cathepsins human enzymes (cathepsins B, H, L, S, C, K, O, F, V, Cathepsins are synthesized as inactive pre-pro- X and W) were identified. The 1990s also provided enzymes having a signal peptide and a multi- more clues on the functions of cysteine proteases: functional N-terminal proregion. The role of pre- several cathepsin knockouts highlighted the peptide is to drive cathepsin precursor in the lumen of physiological roles of cathepsins much more than rough endoplasmic reticulum, after which it is mere scavengers, which was long believed to be their subsequently hydrolyzed. The proregion on the other major function. With advent of novel concepts and hand folds the nascent cathepsin properly, inhibits its availability of sequences, this number proteolytic activity, preserves its three-dimensional might probably increase, especially since several new structure at neutral pH and maintains the precursor in mouse cathepsins without apparent human an inactive state until cleaved (Fig. 1). The counterparts have been discovered in 200028,29. procathepsin later undergoes carbohydrate processing and asparagine-linked glycosylation in the Golgi. The Structure enzymes N-acetylglucosaminylphosphotransferase The cysteine cathepsins comprise a group of and a-N-acetylglucosaminidase add a mannose-6- papain-like enzymes sharing similar amino acid phosphate label to the pro-cathepsin. The mannose-6- sequences and structural domains. The cathepsins are phosphate tagged pro-cathepsin subsequently binds to generally composed of disulfide-connected heavy and M6PR in the trans-Golgi network light chains. They are monomers with molecular mass There are two M6PRs; 275 kDa and 46 kDa which of ~30 kDa, except tetrameric cathepsin C. They have are cation-independent, with their bound ligands a two-domain structure with the V-shape active site subsequently translocated to late endosomes. 78 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

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 two- fold 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 and the carbonyl carbon of scissile bond undergoes bind to M6PRs in the trans-Golgi network, they bind nucleophilic attack from the active-site thiol, resulting to M6PRs localized at the plasma membrane and in the release of amine product. The ensuing reach the late endosomes/lysosomes via early acylenzyme reacts with water to release the carboxyl endosomes and receptor-mediated endocytosis. In late product (deacylation), resulting in the regeneration of endosomes, mild acidic pH (6.0–6.5) dissociates free enzyme. In contrast to many other peptidases, pro-cathepsins from M6PRs and finally pro- cysteine cathepsins do not have a single specific cathepsins are processed into active proteases31. substrate, although they do differ considerably in their Interestingly, a distinct targeting pathway using preferred cleavage site. Binding sites for substrate aggregation has also been reported, where upregulated residues N-terminal to the cleaved peptide bond are procathepsin L is targeted to tetraspanin CD63 designated as S1, S2...etc.; those C-terminal are enriched multivesicular late endosomes35. 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 Mode of action residues31-33,37. The mode of action of cysteine cathepsins, especially the papain superfamily has been extensively characterized than their counterparts in Like cysteine cathepsins, aspartate cathepsins the aspargine protease family. Most of cysteine exhibit considerable sequence similarities and protease cathepsins require acidic pH for optimal common motifs. These include triades Asp-Thr-Gly activity. This is not surprising, since Cys25 at around two active sites aspartic residues (at number catalytic center exhibits a very low pKa value, which 32 and 215, numbering of human pepsin A) and helps formation of an thiolate ± imidazolium ion-pair relative position of Cys residues and of Tyr75 and its with hiatidine-159 required for catalysis 36. Substrates surroundings. Tyr75 is localized in a so-called flap bind into the active site in an extended conformation region, which is flexible and partially covers the GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 79

active site. The sequence similarities are also reflected from the protease, unfolds and is proteolytically in the significant resemblance in primary structures. degraded. The proteolytic removal is brought about Peptidases belonging to the A1 family are formed by by various proteases like pepsin, , bi-lobal structure and both lobes are structurally cathepsin D and different cysteine proteases. similar, which suggests a common ancestor. A Alternatively, the propeptide can be cleaved at several dimeric molecule consisting of two identical subunits, sites during activation, thus eliminating or by duplication and fusion evolves into diminishing its inhibitory function, as observed for monomeric bilobal structure38. cathepsins L and K31. Recently, cathepsin D has been shown to be processed in a manner independent of its Regulation catalytic function — by a combination of The cathepsins are regulated in many ways, i.e. at autoactivation and -assisted maturation47. the gene transcription, translation and expression level, by post-translational modifications, localization, Environmental pH environmental pH, zymogen activation, presence or An essential regulating factor of proteolytic activity absence of inhibitors, trafficking and by inactivation of endosomal cathepsins is the pH of environment. and degration31. Thus, decrease in pH occurring during maturation of endosomes presumably weakens the interaction Transcription and translation between the propeptide and proenzyme, favoring the Exposure to various inflammatory stimuli controls activation process. Furthermore, cathepsins B, S and the expression of cysteine proteases at the L are irreversibly denaturated in lysosomes towards transcriptional level. Interestingly, downregulation of the end of maturation process, when pH is decreased and B mRNA levels has been reported in to 3.8. Denatured cathepsin L is proteolytically primary alveolar macrophages, microglia and removed by acid-active cathepsin D. In addition, macrophage cell lines in response to pro- cathepsins L, B, H, K, V and F are unstable at neutral inflammatory cytokine IFN-α39. In contrast, pH and, therefore, are less active outside lysosomes, upregulation of cathepsin S has been observed in while others like cathepsins B and S are extremely human keratinocytes, bone marrow cells and lung stable, possibly accounting for their role outside 31,48 tissue40,41. In another study in peritoneal macrophages lysosomes . treated with IFN-γ, no change in the level of cathepsin S mRNA, but a marked decrease in the transcription Protein inhibitors of cathepsin L have been reported42. Similarly, LPS By far, the most important regulator for endosomal and transcription factor PU.1, in concert with IFN proteases is their endogenous protein inhibitors. The regulatory factors enhance cat S transcription in latter binds tightly to the enzyme, thereby preventing cervical smooth muscle cells and endosomes of substrate binding. Some of the common inhibitors are antigen-presenting cells (APC) respectively43,44. cystatins, thyropins, α2-macroglobulin, serpin, Additionally, alternative splicing of cathepsins B and squamous cell carcinoma antigen 1, CTLA-2 beta and L results in increased translation and protease chagasin. Cystatins are non-selective protein stability, along with enhanced secretion. inhibitors of the papain family of cysteine proteases. They inactivate target proteases reversibly and Zymogen activation competitively by indirect blockage of catalytic centers Most cathepsins are synthesized as inactive and form very stable bimolecular complexes with zymogens. The zymogen is activated by proteolytic proteases. Cystatin superfamily is traditionally removal of amino terminal pro-domain, which blocks sub-divided according to their sequence similarities access to the active catalytic site. The activation into three major families comprising mainly of process is triggered by a pH drop and/or by stefins, cystatins and kininogens. In contrast, glycosaminoglycans that substantially weaken the thyropins are more selective, probably reflecting their interactions between the propeptide and catalytic more specialized in vivo role, while the general part45,46. Consequently, the proenzyme adopts a looser inhibitors like α-macroglobulins unspecifically trap conformation, in which propeptide is less tightly of different types, blocking the access bound to the active site, while the secondary structure of protein substrates to the active site of trapped remains intact. Ultimately, the propeptide dissociates proteinases without inactivating them33,49,50. 80 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

Table 1—Cathepsins found within the endolysosomal system

Name Synonyms Catalytic Endo Operating Distribution Refs group peptidase Carboxy Amino pH peptidase peptidase

Cathepsin B Cathepsin B1 Cys + + _ 5.0-6.0 Ubiquitous 17, 54 Dipeptidyl Cathepsin J Cys + + dipeptidase 5.0-6.0 Ubiquitous 139 peptidase I Cathepsin C (DPPI) Cathepsin D _ Asp + _ _ 2.8-7.0 Ubiquitous 56 23 Cathepsin E _ Asp _ _ 3.0-3.5 Restricted 140, 141,142 _ Asp + _ _ 5.2- 6.8 Ubiquitous 143, 144 Cathepsin G _ Ser n.d n.d. n.d. 7.5 Neutrophils 105 Cathepsin H Cathepsin I Cys + _ + 6.8 Ubiquitous 21, 71 monopeptidase Cathepsin _ Cys n.d n.d. n.d. n.d. Placenta 145 J e(mouse) Cathepsin O2, Cys + _ _ 5.0-8.0 Osteoclasts 121, 123 OC2, O, X Chondroclast Cathepsin L _ Cys + _ _ 4.5-6.0 Ubiquitous 83 Cathepsin _ Cys n.d. n.d. n.d. n.d. Placenta 146 Me(mouse) _ Cys n.d. n.d. n.d. n.d. Ubiquitous 147

Cathepsin _ Cys n.d. n.d. n.d. n.d. Placenta 148 Qe(rat), R(mouse) Cathepsin S _ Cys + _ _ 5.0-8.0 Macrophages/ 51, 106 Monocytes, dendritic cells, microglial cells Cathepsin Vc Cys + _ _ 5.7 Thymus, 149, 150 Cathepsin U testis, corneal epithelium Lymphopain Cys n.d. n.d. n.d. _ CD8 + T cells 151, 152 and NK cells Cathepsin X , Pd, Cys _ + _ 5-5.5 Ubiquitous 153, 154 Y monopeptidase dipeptidase cKnown only in primates; dRat ortholog; eKnown only in rodents; n.d., not detected

Oxidation states H, S, D and C are distributed between late endosomes The cysteine protease cathepsins are readily and lysosomes51. The cathepsin activity is increased in inactivated by oxidation of the active site cysteine and late endosomes after LPS-induced stimulation52. require a reducing environment for full activity. Major cathepsins Trafficking The enormous diversity of cathepsin types, their Cathepsin activity is also modulated by the cellular cellular distribution and functions (see Table 1 and environment. In human B lymphoblastoid cells, Fig. 2) make it difficult to prioretise them on the basis distinct patterns of cathepsin activity are concentrated of physiological importance. However, a few of the in specific endocytic compartments. Cathepsins B and most well characterized representative members of Z are most prominent in early and late endosomes, cysteine and aspartate families are briefly described suggesting initial proteolytic attack, while cathepsins below. GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 81

as the occluding loop which is connected via His110 and Arg116 residues to the Asp22 and Asp224 of the enzyme, respectively. When an 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 carboxy- termini. This leads to an increase in the number of new termini, allowing the 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 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 Fig. 2—Mutiple sequence alignment of (human) endosomal cultured periosteal explants. Intracellular cathepsin B cysteine cathepsins using ClustalW [Though the sequences also processes and activates prorenin58, brings about indicate considerable divergence, the active sites Cys25 and His159 are conserved (highlighted in bold). The sequences were histolysis of silk gland during metamorphosis in 59 taken from the SWISS- PROT or GenBank databases] Bombyx mori along with cathepsin L , and processes yolk during maturation of killifish oocytes60. Significant cathepsin B activity in the intestine of Widely expressed cathepsins parasitic nematode Haemonchus contortus supports its Cathepsin B protease role in nutrient digestion61. Cathepsin B is by far the most abundantly expressed mammalian cathepsin and exhibits multiple Recent studies have identified cathepsin B as the tissue/cell distribution including skeletal tissues, main enzyme in degradation of insulin-like growth plasma membrane caveolae of differentiating factor-1 (IGF-I), operative within endosomes at a pH myoblasts53, epithelial cells and APC’s like dendritic range of 5.5-7.024. The proteolytic activity of cells (DCs) and macrophages. It is synthesized as a cathepsin B is also crucial for the activation of 45-39 kDa precursor and processed to a 32 kDa and trypsinogen to trypsin in the development of acute 28 kDa active forms, prior to sorting into the pancreatitis62,63. Increased expression of cathepsin B endocytic pathway. It can catalyze cleavage of at protein and mRNA levels in patients with heart peptide bonds by two mechanisms: endoproteolytic failure suggests its role in the genesis and attack with a pH optimum around 7.4 and an development of cardiac disease64. Furthermore, exopeptidase attack from the C-terminus with a pH cathepsin B is negatively correlated with other optimum at 4.5-5.554. pathological conditions like osteoarthritis65, gastric Unlike other members of cysteine protease family, cancer66, oral cancer metastasis67, colorectal cancer68 cathepsin B has a large flexible insertion loop termed and ovarian cancer69,70. 82 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

Cathepsin H factor, platelet-derived growth factor and hormones Although ubiquitous, cathepsin H is present at a like follicle stimulating and leuteinizing hormones84. relatively high concentration in non-skeletal tissues In Pichia pastoris, intermolecular processing event such as kidney, spleen and macrophages. Two mature within a conserved GXNXFXD motif in the 37 kDa forms (22 and 28 kDa) and an immature form 71 cathepsin L proform generates a 30 kDa intermediate (37 kDa) of cathepsin H have been reported . It acts that is finally processed to a 24.5 kDa mature form at as an endopeptidase as well as an pH 5.085. Active site-directed labeling in resting with a pH optimum of about 6.5-6.8. Its human DC also confirms two enzymatically active aminopeptidase activity is essentially due to the isoforms 25 kDa and 30 kDa of cathepsin L. The two presence of a mini chain (residual octapeptide chain forms of cathepsin L in the late endocytic EPQNCSAT of the propeptide) present at 72,73 compartments of antigen-presenting cells are C-terminal . Recently, the mini-chain of human maintained by the chaperonic activity of p41. In the cathepsin H has been identified as the major structural absence of p41, the mature chain forms are degraded element determining its substrate specificity. The by other endocytic proteases86. deletion of mini-chain switches the substrate specificity from aminopaptidase to endopeptidase74. Functionally, cathepsin L is an endopeptidase and Unlike other cysteine proteinases, cathepsin H is not active at pH 3.0-6.5 with optimum activity at pH 5.5. inhibited by its own free propeptides. During Cathepsins L and B are suggested to play a role in maturation and mini-chain formation the propeptide lysosomal protein turnover at acidic pH in vitro. Since binding region of the parent peptide is rearranged, both cathepsins L and B have overlapping substrate which disables the propeptides to recognize the specificities, validation of their in vivo role has proven mature cathepsin75. difficult. Although, both cathepsins L and B could Cathepsin H has been detected as an early cleave the Nipah virus fusion protein, only cathepsin endosomal acid protease in J774 macrophages21. In L mediated cleavge results in the physiologically human dendritic cells (DCs), the enzyme is found to relevant size of the products87. be concentrated in late endosomes in response to Functionally, cathepsin L is one of the widely maturation stimulus like LPS, indicating more expressed peptidase with potent elastase and efficient local proteolysis52. Cathepsin H acts as a 76 collagenase activity. Both cathepsins L and B are neuropeptidase in human brain and degrades a essential for maturation and integrity of the post-natal variety of components of the extracellular matrix central nervous system and both compensate for each (ECM), such as proteoglycans, laminin, and collagens other in vivo. In brief, cathepsin L appears to be II, IX, and XI. It is involved in the first N-terminal critically involved in epidermal homeostasis, normal processing step of a 21 kDa pro-surfactant protein to a tissue remodeling, phagocytosis, regulation of hair 4.2 kDa dipalmitoylated in electron-dense 77 cycle, follicular wall degradation during oogenesis multivesicular bodies of type II pneumocytes . It is and MHC class II-mediated antigen presentation in also associated with various pathological conditions 78 79 cortical epithelial cells of thymus. like human fibrous meningioma , colorectal cancer , juvenile idiopathic arthritis80, human prostrate tumor81 Cathepsin L has been implicated in the proteolytic and lung cancer82. processing of Hendra virus precursor F protein F(0) to the active F(1) + F(2) disulfide-linked heterodimer. Cathepsin L The latter is essential for the generation of a mature Cathepsin L is constitutively expressed in many and fusogenic form of the F protein88. In a number of tissues, and has highly expressed levels in tissues that pathological conditions, cathepsins L and B are turnover more rapidly, such as the liver, kidney and secreted out of the vesicular lumen and are involved ovary as well as in the lysosomes of stimulated in degradation of ECM components. More specific macrophages, fibroblasts, DCs, kupffer cells, and functions have also been postulated in a wide range of endotheliocytes83. Upon stimulation with LPS, the physiological and pathological processes. These lysosomal enzymes show a shift to late endocytic include antigen presentation89, prohormone vesicles in mature DC’s. Interestingly, expression processing90, turnover of amyloid in Alzheimer’s level of cathepsin L is enhanced by several growth disease91, tumor invasion92-94, inflammatory skin factors like epidermal growth factor, fibroblast growth disease95, apoptosis96, and auto-immune diabetes97. GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 83

Cathepsin D of cathepsin D-deficient mice, it is shown that It is most extensively characterized enzyme among cathepsin D is involved in the limited and specific cathepsins and is demonstrated to be exclusively rather than bulk proteolysis. Cathepsin D deficient 98 associated with endosome functions . It causes mice are born normal, but die at post-natal day 26 due complete degradation of the mannose-BSA ligand in to massive necrosis, thromboembolia, lymphopenia. 9 early endosome preparations free from lyososomes . The enzyme also takes part in antigen processing, in Cathepsin D is a pepstatin sensitive aspartate protease generation of peptide hormones, intracellular protein belonging to the pepsin superfamily. It is present in degradation, tissue remodeling and apoptosis. During endosomes of smooth muscle cells, epithelial cells, apoptosis, the endolysosomal enzyme translocates 99 macrophages, and fibroblasts and in multivesi- into the cytosol and the cytosolic enzyme triggers cular endosomes in macrophages, hepatocytes and apoptotic pathways by interacting with members of B-lymphoblastoid cells. the apoptotic machinery rather than cleaving 103,104 Cathepsin D is synthesized as an 52 kDa inactive proteolytic substrates . precursor and transported from the golgi complex to the acidic endosomes100. In acidic endosomal lumen, Tissue-specific cathepsins the proenzyme is processed and activated to its Cathepsin S corresponding 48 kDa intermediate and finally to Cathepsin S was first purified from bovine lymph node and spleen and cDNA was cloned from bovine 34 kDa and 14 kDa mature forms. Earlier hypothesis 105,106 emphasized on partial auto-activation generating a spleen by RT-PCR . Subsequently, human cathepsin S was independently cloned by two 51 kDa pseudo-cathepsin D, followed by enzyme- 107 assisted maturation involving cysteine and/or aspartic groups . Expression of cathepsin S is tissue-specific proteases. However, recent studies with cathepsin D and is preferentially expressed in phagocytic and deficient fibroblast cell lines have shown that APCs. High levels of cathepsin S are found in maturation of endocytosed pro-cath-D is independent alveolar macrophages of human and rat lung, DCs, of its catalytic function and requires cysteine spleen, lymph nodes, vascular smooth muscle cells, 47 while moderate to low levels have been detected in proteases like cathepsin L and B . 108,109 appendix, bone marrow and thymus . The mature In vitro cathepsin D acts at different pH ranging protein is found to be a single-chain polypeptide of from 2.8-6.0, depending on the substrate and In vivo, around 28-30 kDa52. It is a potent endopeptidase it requires an acidic pH (3.0–5.0), known to be cleaving a range of proteins and synthetic substrates present in the different endosomal compartments. It over a broad pH range of 5-8106. acts as a ligand in its zymogen state and as an The enzyme shows elastinolytic activity at pH effective endosomal protease in its mature form101. It 4.5-5.5 (comparable with pancreatic elastase), but is implicated in processing of the 23 kDa prolactin unlike other cathepsins retains about 20-25% of pituitary hormone to 16 kDa fragments in rat maximal activity at neutral pH (where it is mammary epithelial cells102. In related studies, Merlen comparable with leukocyte elastase in activity)107. At and colleagues documented co-localization of neutral pH, it degrades fibronectin, laminin and internalized cholera toxin and cathepsin D in early myelin basic protein and proteoglycans110. Further, endosomes in rat liver endosomes, HepG2 and cathepsin S, unlike cathepsins B, H, and L is stable at intestinal Caco-2 cell lines, where cholera toxin is pH 7.0. Consistent with its broad pH optimum, subsequently degraded in a pH-dependent manner. cathepsins S and B are found to be the most potent These results strongly confirm the role of endocytic proteolytic enzymes in endosomes of human DCs. cathepsin D in proteolytic activation of cholera Consequently, it has been detected all along the toxin18. Similarly, cathepsin D is involved in endocytic pathway with a high concentration in proteolytic degradation of ricin A chain in early lysosomes111. However, within hours of addition of a endosomes of macrophage98. maturation stimulus in DCs, its local activity in the Membrane association may influence the pH late endosomes is increased, implying its important dependence of cathepsin D activity, accounting role in stable antigenic processing. In contrast, in J774 for the observation of some aspartyl protease activity macrophages, bulk of the enzyme is localized to late in endosomes at neutral pH. Through the generation endosomes. 84 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

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 significant amount of cathepsin K123. Like most other presentation112. Professional as well as non- enzymes in its class, cathepsin K is synthesized as professional MHC class II-expressing APCs use a ~42 kDa proform, which is activated to a ~27 kDa cathepsin S for MHC class II-mediated antigen mature form123,124. Enzyme activities of cathepsin K presentation113. Absence of lysosomal cathepsin S are similar to those of cathepsin S105,106, which leads to an impaired degradation of invariant chain. includes a broad pH optimum between 5-8. At neutral Subsequently, invariant chain remnant accumulates in pH, it is more stable than cathepsin L, but less class II MHC-positive endosomal compartments, so than cathepsin S. Being expressed significantly in which are enlarged in size and lack multivesicular bone cells, it is actively engaged in cartilage morphology114. Furthermore, microvascular resorption124-126. It is a potent collagenase and endothelial cells from cathepsin S-deficient mice gelatinase; besides also being active against non- shows reduced collagenolytic activity and defective collagenous matrix proteins, such as aggrecan, microvessel development during wound repair, osteopontin, and osteonectin127-129. implying an essential role of cathepsin S in ECM degradation and angiogenesis115. Cathepsin K is essential for normal bone Alternatively, its role in angiogenic islet formation resorption; humans lacking it exhibit pycnodysostosis, and growth of solid tumors in mouse model of which is characterized by short stature and 130-132 multi-stage murine pancreatic islet cell carcinogenesis osteosclerosis . In a pioneering work, the direct is also reported116. The enzyme functions both evidence of physiological importance of this enzyme 129 intracellularly in degradation of endosomal protein has been demonstrated in lung matrix homeostasis . cargo117, and extracellularly in tissue, matrix, elastin Using a chemically induced lung fibrosis in cathepsin -/- and bone remodelling. Activated macrophages release K-deficient mice (CTSK ), the study showed reduced substantial amount of cathepsin S and hence the latter collagenolytic activity and consequent higher fibrosis. +/+ is responsible for various pathological conditions like Both in control (CTSK ) fibroblasts and lung atherosclerosis118, astrocytoma progression119, and specimens obtained from patients with lung fibrosis autoimmune diseases120. fibroblasts, capthesin K levels are significantly upregulated, indicating its protective role in fighting 129 Cathepsin K excessive collagen deposition in diseased lungs . It It was first cloned from osteoclasts and described is also involved in kinin degradation133. A direct role as OC-2121. Subsequently, its cDNA was of cathepsin K is implicated in atherosclerosis134, characterized and designated as cathepsin K and osteoarthritis135, osteoporosis136, and rheumatoid highest expression was found in bone tissues122. arthritis137. The macrophages in human atheromas and Besides osteoclasts and chondroclasts and their giant tumor cells of bones express high levels of precursors, epithelial cells in various organs express cathepsin K138. GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 85

Conclusion 13 Schaudies R P, Gorman R M, Savage C R, Jr. & Poretz R D It is increasingly evident that cathepsins are not (1987) Proteolytic processing of epidermal growth factor within endosomes. Biochem Biophys Res Commun 143, exclusively lysosomal but enjoy specific distribution 710-715 within a diverse of endosomal classes that differ in 14 Blum J S, Diaz R, Diment S, Fiani M, Mayorga L, Rodman J morphology, biochemistry and function (Fig. 3). S & Stahl P D (1989) Proteolytic processing in endosomal Togther, this generates the enormous catabolic vesicles. Cold Spring Harb Symp Quant Biol 54, 287-292 repertoire required to process multifarious endocytic 15 Authier F, Mort J S, Bell A W, Posner B I & Bergeron J J (1995) Proteolysis of glucagon within hepatic endosomes by cargo. Accurate knowledge of endosome-cathepsin membrane-associated cathepsins B and D. J Biol Chem 270, relationships is essential to expand our current 15798-15807 understanding of intracellular proteolysis that plays 16 Doherty J J, 2nd, Kay D G, Lai W H, Posner B I & Bergeron important roles in health and disease. J J (1990) Selective degradation of insulin within rat liver endosomes. J Cell Biol 110, 35-42 17 Blum J S, Fiani M L & Stahl P D (1991) Proteolytic cleavage Acknowledgement of ricin A chain in endosomal vesicles. Evidence for the We are thankful to Commissionerate of Industry of action of endosomal proteases at both neutral and acidic pH. the Government of Gujarat, Department of Science J Biol Chem 266, 22091-22095 and Technology (DST) and Lady Tata Memorial 18 Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin Trust for funding the research project and awarding A & Authier F (2005) Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D. Febs fellowship (to SG). J 272, 4385-4397 19 Blum J S & Cresswell P (1988) Role for intracellular References proteases in the processing and transport of class II HLA 1 Conner S D & Schmid S L (2003) Regulated portals of entry antigens. Proc Natl Acad Sci (USA) 85, 3975-3979 into the cell. Nature 422, 37-44 20 Runquist E A & Havel R J (1991) Acid hydrolases in early 2 Tjelle T E, Brech A, Juvet L K, Griffiths G & Berg T (1996) and late endosome fractions from rat liver. J Biol Chem 266, Isolation and characterization of early endosomes, late 22557-22563 endosomes and terminal lysosomes: their role in protein 21 Claus V, Jahraus A, Tjelle T, Berg T, Kirschke H, Faulstich degradation. J Cell Sci 109, 2905-2914 H & Griffiths G (1998) Lysosomal enzyme trafficking 3 Luzio J P, Rous B A, Bright N A, Pryor P R, Mullock B M & between phagosomes, endosomes, and lysosomes in J774 Piper R C (2000) Lysosome-endosome fusion and lysosome macrophages. Enrichment of cathepsin H in early biogenesis. J Cell Sci 113, 1515-1524 endosomes. J Biol Chem 273, 9842-9851 4 Rink J, Ghigo E, Kalaidzidis Y & Zerial M (2005) Rab 22 Authier F, Metioui M, Bell A W & Mort J S (1999) Negative conversion as a mechanism of progression from early to late regulation of epidermal growth factor signaling by selective endosomes. Cell 122, 735-749 proteolytic mechanisms in the endosome mediated by 5 Zerial M & McBride H (2001) Rab proteins as membrane cathepsin B. J Biol Chem 274, 33723-33731 organizers. Nat Rev Mol Cell Biol 2, 107-117 23 Authier F, Metioui M, Fabrega S, Kouach M & Briand G 6 De Duve C (1963) The lysosome. Sci Am 208, 64-72 (2002) Endosomal proteolysis of internalized insulin at the 7 Ciechanover A (2005) Proteolysis: from the lysosome to C-terminal region of the B chain by cathepsin D. J Biol ubiquitin and the proteasome. Nat Rev Mol Cell Biol 6, 79-87 Chem 277, 9437-9446 8 Mayorek N, Pinson A & Mayer M (1979) Intracellular 24 Authier F, Kouach M & Briand G (2005) Endosomal proteolysis in rat cardiac and skeletal muscle cells in culture. proteolysis of insulin-like growth factor-I at its C-terminal D- J Cell Physiol 98, 587-595 domain by cathepsin B. FEBS Lett 579, 4309-4316 9 Diment S & Stahl P (1985) Macrophage endosomes contain 25 Pillay C S, Elliott E & Dennison C (2002) Endolysosomal proteases which degrade endocytosed protein ligands. J Biol proteolysis and its regulation. Biochem J 363, 417-419 Chem 260, 15311-15317 26 Takio K, Towatari T, Katunuma N, Teller D C & Titani K 10 Mayorga L S, Diaz R & Stahl P D (1989) Reconstitution of (1983) Homology of amino acid sequences of rat liver endosomal proteolysis in a cell-free system. Transfer of cathepsins B and H with that of papain. Proc Natl Acad Sci immune complexes internalized via Fc receptors to an (USA) 80, 3666-3670 endosomal proteolytic compartment. J Biol Chem 264, 27 Lee X, Ahmed F R, Hirama T, Huber C P, Rose D R, To R, 5392-5399 Hasnain S, Tam A & Mort J S (1990) Crystallization of 11 Wiley H S, VanNostrand W, McKinley D N & Cunningham recombinant rat cathepsin B. J Biol Chem 265, 5950-5951 D D (1985) Intracellular processing of epidermal growth 28 Sol-Church K, Frenck J, Bertenshaw G & Mason R W factor and its effect on ligand-receptor interactions. J Biol (2000) Characterization of mouse cathepsin R, a new Chem 260, 5290-5295 member of a family of placentally expressed cysteine 12 Roederer M, Bowser R & Murphy R F (1987) Kinetics and proteases. Biochim Biophys Acta 1492, 488-492 temperature dependence of exposure of endocytosed material 29 Sol-Church K, Frenck J & Mason R W (2000) Mouse to proteolytic enzymes and low pH: evidence for a cathepsin M, a placenta-specific lysosomal cysteine protease maturation model for the formation of lysosomes. J Cell related to cathepsins L and P. Biochim Biophys Acta 1491, Physiol 131, 200-209 289-294 86 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

30 McGrath M E (1999) The lysosomal cysteine proteases. is independent of its catalytic function and auto-activation: Annu Rev Biophys Biomol Struct 28, 181-204 involvement of cathepsins L and B. J Biochem (Tokyo) 139, 31 Turk V, Turk B & Turk D (2001) Lysosomal cysteine 363-371 proteases: facts and opportunities. Embo J 20, 4629-4633 48 Turk B, Dolenc I, Lenarcic B, Krizaj I, Turk V, Bieth J G & 32 Turk V, Turk B, Guncar G, Turk D & Kos J (2002) Bjork I (1999) Acidic pH as a physiological regulator of Lysosomal cathepsins: structure, role in antigen processing human cathepsin L activity. Eur J Biochem 259, 926-932 and presentation, and cancer. Adv Enzyme Regul 42, 285-303 49 Turk B, Turk D & Salvesen G S (2002) Regulating cysteine 33 Turk D, Turk B & Turk V (2003) Papain-like lysosomal protease activity: essential role of protease inhibitors as cysteine proteases and their inhibitors: drug discovery guardians and regulators. Curr Pharm Des 8, 1623-1637 targets? Biochem Soc Symp 70, 15-30 50 Turk B & Fritz H (2003) Vito Turk--30 years of research on 34 van der Drift A C, van Noort J M & Kruse J (1990) Catheptic cysteine proteases and their inhibitors. Biol Chem 384, 833- processing of protein antigens: enzymic and molecular 836 aspects. Semin Immunol 2, 255-271 51 Lautwein A, Kraus M, Reich M, Burster T, Brandenburg J, 35 Collette J, Bocock J P, Ahn K, Chapman R L, Godbold G, Overkleeft H S, Schwarz G, Kammer W, Weber E, Yeyeodu S & Erickson A H (2004) Biosynthesis and Kalbacher H, Nordheim A & Driessen C (2004) Human B alternate targeting of the lysosomal cysteine protease lymphoblastoid cells contain distinct patterns of cathepsin cathepsin L. Int Rev Cytol 241, 1-51 activity in endocytic compartments and regulate MHC class 36 Brocklehurst K (1994) A sound basis for pH-dependent II transport in a cathepsin S-independent manner. J Leukoc kinetic studies on enzymes. Protein Eng 7, 291-299 Biol 75, 844-855 37 Turk B, Turk V & Turk D (1997) Structural and functional 52 Lautwein A, Burster T, Lennon-Dumenil A M, Overkleeft H aspects of papain-like cysteine proteinases and their protein S, Weber E, Kalbacher H & Driessen C (2002) Inflammatory inhibitors. Biol Chem 378, 141-150 stimuli recruit cathepsin activity to late endosomal 38 Ostermann N, Gerhartz B, Worpenberg S, Trappe J & Eder J compartments in human dendritic cells. Eur J Immunol 32, (2004) Crystal structure of an activation intermediate of 3348-3357 cathepsin E. J Mol Biol 342, 889-899 53 Jane D T, Morvay L, Dasilva L, Cavallo-Medved D, Sloane 39 Liuzzo J P, Petanceska S S, Moscatelli D & Devi L A (1999) B F & Dufresne M J (2006) Cathepsin B localizes to plasma Inflammatory mediators regulate cathepsin S in macrophages membrane caveolae of differentiating myoblasts and is and microglia: A role in attenuating heparan sulfate secreted in an active form at physiological pH. Biol cell 387, interactions. Mol Med 5, 320-333 223-234 40 Schwarz G, Boehncke W H, Braun M, Schroter C J, Burster 54 Stachowiak K, Tokmina M, Karpinska A, Sosnowska R & T, Flad T, Dressel D, Weber E, Schmid H & Kalbacher H Wiczk W (2004) Fluorogenic peptide substrates for (2002) Cathepsin S activity is detectable in human carboxydipeptidase activity of cathepsin B. Acta Biochim Pol keratinocytes and is selectively upregulated upon stimulation 51, 81-92 with interferon-gamma. J Invest Dermatol 119, 44-49 55 Nagler D K, Storer A C, Portaro F C, Carmona E, Juliano L 41 Storm van's Gravesande K, Layne M D, Ye Q, Le L, Baron & Menard R (1997) Major increase in endopeptidase activity R M, Perrella M A, Santambrogio L, Silverman E S & Riese of human cathepsin B upon removal of occluding loop R J (2002) IFN regulatory factor-1 regulates IFN-gamma- contacts. Biochemistry 36, 12608-12615 dependent cathepsin S expression. J Immunol 168, 4488- 56 Blum J S, Fiani M L & Stahl P D (1991) Localization of 4494 cathepsin D in endosomes: characterization and biological 42 Beers C, Honey K, Fink S, Forbush K & Rudensky A (2003) importance. Adv Exp Med Biol 306, 281-287 Differential regulation of cathepsin S and cathepsin L in 57 Creemers L B, Hoeben K A, Jansen D C, Buttle D J, interferon gamma-treated macrophages. J Exp Med 197, Beertsen W & Everts V (1998) Participation of intracellular 169-179 cysteine proteinases, in particular cathepsin B, in degradation 43 Wang Y, Baron R M, Zhu G, Joo M, Christman J W, of collagen in periosteal tissue explants. Matrix Biol 16, 575- Silverman E S, Perrella M A, Riese R J & Cernadas M 584 (2006) PU.1 regulates cathepsin S expression in professional 58 Almeida P C, Oliveira V, Chagas J R, Meldal M, Juliano M APCs. J Immunol 176, 275-283 A & Juliano L (2000) Hydrolysis by cathepsin B of 44 Watari M, Watari H, Nachamkin I & Strauss J F (2000) fluorescent peptides derived from human prorenin. Lipopolysaccharide induces expression of genes encoding Hypertension 35, 1278-1283 pro-inflammatory cytokines and the elastin-degrading 59 Shiba H, Uchida D, Kobayashi H & Natori M (2001) enzyme, cathepsin S, in human cervical smooth-muscle cells. Involvement of cathepsin B- and L-like proteinases in silk J Soc Gynecol Investig 7, 190-198 gland histolysis during metamorphosis of Bombyx mori. Arch 45 Rozman J, Stojan J, Kuhelj R, Turk V & Turk B (1999) Biochem Biophys 390, 28-34 Autocatalytic processing of recombinant human procathepsin 60 Raldua D, Fabra M, Bozzo M G, Weber E & Cerda J (2006) B is a bimolecular process. FEBS Lett 459, 358-362 Cathepsin B-mediated yolk protein degradation during 46 Jerala R, Zerovnik E, Kidric J & Turk V (1998) pH-induced killifish oocyte maturation is blocked by an H+-ATPase conformational transitions of the propeptide of human inhibitor: effects on the hydration mechanism. Am J Physiol cathepsin L. A role for a molten globule state in zymogen Regul Integr Comp Physiol 290, R 456-466 activation. J Biol Chem 273, 11498-11504 61 Shompole S & Jasmer D P (2001) Cathepsin B-like cysteine 47 Laurent-Matha V, Derocq D, Prebois C, Katunuma N & proteases confer intestinal cysteine protease activity in Liaudet-Coopman E (2006) Processing of human cathepsin d Haemonchus contortus. J Biol Chem 276, 2928-2934 GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 87

62 Lindkvist B, Fajardo I, Pejler G & Borgstrom A (2006) C in type II pneumocytes. Am J Respir Cell Mol Biol 26, Cathepsin B Activates Human Trypsinogen 1 but Not 659-670 Proelastase 2 or Procarboxypeptidase B. Pancreatology 6, 78 Chornaya V & Lyannaya O (2004) Some physicochemical 224-231 properties of cathepsin H from human meningioma. Exp 63 Van Acker G J, Saluja A K, Bhagat L, Singh V P, Song A M Oncol 26, 278-281 & Steer M L (2002) Cathepsin B inhibition prevents 79 Schweiger A, Christensen I J, Nielsen H J, Sorensen S, trypsinogen activation and reduces pancreatitis severity. Am Brunner N & Kos J (2004) Serum cathepsin H as a potential J Physiol Gastrointest Liver Physiol 283, G794-800 prognostic marker in patients with colorectal cancer. Int J 64 Ge J, Zhao G, Chen R, Li S, Wang S, Zhang X, Zhuang Y, Biol Markers 19, 289-294 Du J, Yu X, Li G & Yang Y (2006) Enhanced myocardial 80 Taubert H, Riemann D, Kehlen A, Meye A, Bartel F, John V, cathepsin B expression in patients with dilated Brandt J, Bache M, Wurl P, Schmidt H & Weber E (2002) cardiomyopathy. Eur J Heart Fail 8, 284-289 Expression of cathepsin B, D and L protein in juvenile 65 Baici A, Lang A, Zwicky R & Muntener K (2005) Cathepsin idiopathic arthritis. Autoimmunity 35, 221-224 B in osteoarthritis: uncontrolled proteolysis in the wrong 81 Waghray A, Keppler D, Sloane B F, Schuger L & Chen Y Q place. Semin Arthritis Rheum 34, 24-28 (2002) Analysis of a truncated form of cathepsin H in human 66 Ebert M P, Kruger S, Fogeron M L, Lamer S, Chen J, Pross prostate tumor cells. J Biol Chem 277, 11533-11538 M, Schulz H U, Lage H, Heim S, Roessner A, Malfertheiner 82 Schweiger A, Staib A, Werle B, Krasovec M, Lah T T, Ebert P & Rocken C (2005) Overexpression of cathepsin B in W, Turk V & Kos J (2000) Cysteine proteinase cathepsin H gastric cancer identified by proteome analysis. Proteomics 5, in tumours and sera of lung cancer patients: relation to 1693-1704 prognosis and cigarette smoking. Br J Cancer 82, 782-788 67 Wickramasinghe N S, Nagaraj N S, Vigneswaran N & 83 Ryvnyak V V, Ryvnyak E I & Tudos R V (2004) Electron Zacharias W (2005) Cathepsin B promotes both motility and histochemical localization of cathepsin L in the liver. Bull invasiveness of oral carcinoma cells. Arch Biochem Biophys Exp Biol Med 137, 90-91 436, 187-195 84 Robker R L, Russell D L, Espey L L, Lydon J P, O'Malley B 68 Guzinska-Ustymowicz K, Zalewski B, Kasacka I, Piotrowski W & Richards J S (2000) Progesterone-regulated genes in Z & Skrzydlewska E (2004) Activity of cathepsin B and D in the ovulation process: ADAMTS-1 and cathepsin L colorectal cancer: relationships with tumour budding. proteases. Proc Natl Acad Sci (USA) 97, 4689-4694 Anticancer Res 24, 2847-2851 85 Collins P R, Stack C M, O'Neill S M, Doyle S, Ryan T, 69 Nishikawa H, Ozaki Y, Nakanishi T, Blomgren K, Tada T, Brennan G P, Mousley A, Stewart M, Maule A G, Dalton J P Arakawa A & Suzumori K (2004) The role of cathepsin B & Donnelly S (2004) , the major protease and cystatin C in the mechanisms of invasion by ovarian involved in liver fluke (Fasciola hepatica) virulence: cancer. Gynecol Oncol 92, 881-886 propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells. J Biol Chem 279, 17038- 70 Podgorski I & Sloane B F (2003) Cathepsin B and its role(s) 17046 in cancer progression. Biochem Soc Symp 70, 263-276 86 Lennon-Dumenil A M, Roberts R A, Valentijn K, Driessen 71 Haraguchi C M, Ishido K, Kominami E & Yokota S (2003) C, Overkleeft H S, Erickson A, Peters P J, Bikoff E, Ploegh Expression of cathepsin H in differentiating rat spermatids: H L & Wolf Bryant P (2001) The p41 isoform of invariant immunoelectron microscopic study. Histochem Cell Biol chain is a chaperone for cathepsin L. Embo J 20, 4055-4064 120, 63-71 87 Craft W W, Jr., Patch J & Dutch R E (2006) A mature and 72 Guncar G, Podobnik M, Pungercar J, Strukelj B, Turk V & fusogenic form of the Nipah virus fusion protein requires Turk D (1998) Crystal structure of porcine cathepsin H proteolytic processing by cathepsin L. Virology 346, 251-257 determined at 2.1 A resolution: location of the mini-chain C- 88 Pager C T & Dutch R E (2005) Cathepsin L is involved in terminal carboxyl group defines cathepsin H aminopeptidase proteolytic processing of the Hendra virus fusion protein. J function. Structure 6, 51-61 Virol 79, 12714-12720 73 Vasiljeva O, Dolinar M, Turk V & Turk B (2003) 89 Hsieh C S, deRoos P, Honey K, Beers C & Rudensky A Y Recombinant human cathepsin H lacking the mini chain is an (2002) A role for cathepsin L and cathepsin S in peptide endopeptidase. Biochemistry 42, 13522-13528 generation for MHC class II presentation. J Immunol 168, 74 Dodt J & Reichwein J (2003) Human cathepsin H: deletion 2618-2625 of the mini-chain switches substrate specificity from 90 Yasothornsrikul S, Greenbaum D, Medzihradszky K F, aminopeptidase to endopeptidase. Biol Chem 384, 1327-1332 Toneff T, Bundey R, Miller R, Schilling B, Petermann I, 75 Horn M, Doleckova-Maresova L, Rulisek L, Masa M, Dehnert J, Logvinova A, Goldsmith P, Neveu J M, Lane W Vasiljeva O, Turk B, Gan-Erdene T, Baudys M & Mares M S, Gibson B, Reinheckel T, Peters C, Bogyo M & Hook V (2005) Activation processing of cathepsin H impairs (2003) Cathepsin L in secretory vesicles functions as a recognition by its propeptide. Biol Chem 386, 941-947 prohormone-processing enzyme for production of the 76 Brguljan P M, Turk V, Nina C, Brzin J, Krizaj I & Popovic T enkephalin peptide neurotransmitter. Proc Natl Acad Sci (2003) Human brain cathepsin H as a neuropeptide and (USA) 100, 9590-9595 bradykinin metabolizing enzyme. Peptides 24, 1977-1984 91 Yoshiyama Y, Arai K, Oki T & Hattori T (2000) Expression 77 Brasch F, Ten Brinke A, Johnen G, Ochs M, Kapp N, Muller of invariant chain and pro-cathepsin L in Alzheimer's brain. K M, Beers M F, Fehrenbach H, Richter J, Batenburg J J & Neurosci Lett 290, 125-128 Buhling F (2002) Involvement of cathepsin H in the 92 Kirschke H, Eerola R, Hopsu-Havu V K, Bromme D & processing of the hydrophobic surfactant-associated protein Vuorio E (2000) Antisense RNA inhibition of cathepsin L 88 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

expression reduces tumorigenicity of malignant cells. Eur J macrophage cathepsin S, an elastinolytic cysteine protease. Cancer 36, 787-795 J Biol Chem 267, 7258-7262 93 Nasu K, Kai K, Fujisawa K, Takai N, Nishida Y & 108 Cheng X W, Kuzuya M, Nakamura K, Di Q, Liu Z, Sasaki T, Miyakawa I (2001) Expression of cathepsin L in normal Kanda S, Jin H, Shi G P, Murohara T, Yokota M & Iguchi A endometrium and endometrial cancer. Eur J Obstet Gynecol (2006) Localization of cysteine protease, cathepsin S, to the Reprod Biol 99, 102-105 surface of vascular smooth muscle cells by association with 94 Niedergethmann M, Wostbrock B, Sturm J W, Willeke F, integrin alphanubeta3. Am J Pathol 168, 685-694 Post S & Hildenbrand R (2004) Prognostic impact of 109 Shi G P, Webb A C, Foster K E, Knoll J H, Lemere C A, cysteine proteases cathepsin B and cathepsin L in pancreatic Munger J S & Chapman H A (1994) Human cathepsin S: adenocarcinoma. Pancreas 29, 204-211 chromosomal localization, gene structure, and tissue 95 Bylaite M, Moussali H, Marciukaitiene I, Ruzicka T & Walz distribution. J Biol Chem 269, 11530-11536 M (2006) Expression of cathepsin L and its inhibitor hurpin 110 Liuzzo J P, Petanceska S S & Devi L A (1999) Neurotrophic in inflammatory and neoplastic skin diseases. Exp Dermatol factors regulate cathepsin S in macrophages and microglia: A 15, 110-118 role in the degradation of myelin basic protein and amyloid 96 Wille A, Gerber A, Heimburg A, Reisenauer A, Peters C, beta peptide. Mol Med 5, 334-343 Saftig P, Reinheckel T, Welte T & Buhling F (2004) 111 Driessen C, Bryant R A, Lennon-Dumenil A M, Villadangos Cathepsin L is involved in cathepsin D processing and J A, Bryant P W, Shi G P, Chapman H A & Ploegh H L regulation of apoptosis in A549 human lung epithelial cells. (1999) Cathepsin S controls the trafficking and maturation of Biol Chem 385, 665-670 MHC class II molecules in dendritic cells. J Cell Biol 147, 97 Maehr R, Mintern J D, Herman A E, Lennon-Dumenil A M, 775-790 Mathis D, Benoist C & Ploegh H L (2005) Cathepsin L is 112 Liu W & Spero D M (2004) Cysteine protease cathepsin S as essential for onset of autoimmune diabetes in NOD mice. a key step in antigen presentation. Drug News Perspect 17, J Clin Invest 115, 2934-2943 357-363 98 Diment S, Leech M S & Stahl P D (1988) Cathepsin D is 113 Beers C, Burich A, Kleijmeer M J, Griffith J M, Wong P & membrane-associated in macrophage endosomes. J Biol Rudensky A Y (2005) Cathepsin S controls MHC class II- Chem 263, 6901-6907 mediated antigen presentation by epithelial cells in vivo. J 99 Ryvnyak V V, Gudumak V S & Dulgieru O F (2003) Immunol 174, 1205-1212 Electron-histochemical location of cathepsin D and elastase 114 Boes M, van der Wel N, Peperzak V, Kim Y M, Peters P J & in the uterus. Bull Exp Biol Med 136, 199-201 Ploegh H (2005) In vivo control of endosomal architecture 100 Hasilik A, von Figura K, Conzelmann E, Nehrkorn H & by class II-associated invariant chain and cathepsin S. Eur Sandhoff K (1982) Lysosomal enzyme precursors in human J Immunol 35, 2552-2562 fibroblasts. Activation of cathepsin D precursor in vitro and 115 Shi G P, Sukhova G K, Kuzuya M, Ye Q, Du J, Zhang Y, activity of beta-hexosaminidase A precursor towards Pan J H, Lu M L, Cheng X W, Iguchi A, Perrey S, Lee A M, ganglioside GM2. Eur J Biochem 125, 317-321 Chapman H A & Libby P (2003) Deficiency of the cysteine 101 Fusek M & Vetvicka V (2005) Dual role of cathepsin D: protease cathepsin S impairs microvessel growth. Circ Res ligand and protease. Biomed Pap Med Fac Univ Palacky 92, 493-500 Olomouc Czech Repub 149, 43-50 116 Wang B, Sun J, Kitamoto S, Yang M, Grubb A, Chapman H 102 Lkhider M, Castino R, Bouguyon E, Isidoro C & Ollivier- A, Kalluri R & Shi G P (2006) Cathepsin S controls Bousquet M (2004) Cathepsin D released by lactating rat angiogenesis and tumor growth via matrix-derived mammary epithelial cells is involved in prolactin cleavage angiogenic factors. J Biol Chem 281, 6020-6029 under physiological conditions. J Cell Sci 117, 5155-5164 117 Beck H, Schwarz G, Schroter C J, Deeg M, Baier D, 103 Beaujouin M, Baghdiguian S, Glondu-Lassis M, Berchem G Stevanovic S, Weber E, Driessen C & Kalbacher H (2001) & Liaudet-Coopman E (2006) Overexpression of both Cathepsin S and an asparagine-specific endoprotease catalytically active and -inactive cathepsin D by cancer cells dominate the proteolytic processing of human myelin basic enhances apoptosis-dependent chemo-sensitivity. Oncogene protein in vitro. Eur J Immunol 31, 3726-3736 25, 1967-1973 118 Liu J, Ma L, Yang J, Ren A, Sun Z, Yan G, Sun J, Fu H, Xu 104 Bidere N, Lorenzo H K, Carmona S, Laforge M, Harper F, W, Hu C & Shi G P (2005) Increased serum cathepsin S in Dumont C & Senik A (2003) Cathepsin D triggers Bax patients with atherosclerosis and diabetes. Atherosclerosis activation, resulting in selective apoptosis-inducing factor 186, 411-419 (AIF) relocation in T lymphocytes entering the early 119 Flannery T, McQuaid S, McGoohan C, McConnell R S, commitment phase to apoptosis. J Biol Chem 278, McGregor G, Mirakhur M, Hamilton P, Diamond J, Cran G, 31401-31411 Walker B, Scott C, Martin L, Ellison D, Patel C, Nicholson 105 Kirschke H, Barrett A J & Rawlings N D (1995) Proteinases C, Mendelow D, McCormick D & Johnston P G (2006) 1: lysosomal cysteine proteinases. Protein Profile 2, Cathepsin S expression: An independent prognostic factor in 1581-1643 glioblastoma tumours-a pilot study. Int J Cancer 119, 106 Kirschke H & Wiederanders B (1994) Cathepsin S and 854-860 related lysosomal endopeptidases. Methods Enzymol 244, 120 Saegusa K, Ishimaru N, Yanagi K, Arakaki R, Ogawa K, 500-511 Saito I, Katunuma N & Hayashi Y (2002) Cathepsin S 107 Shi G P, Munger J S, Meara J P, Rich D H & Chapman H A inhibitor prevents autoantigen presentation and (1992) Molecular cloning and expression of human alveolar autoimmunity. J Clin Invest 110, 361-369 GUHA & PADH: CATHEPSINS: EFFECTORS OF ENDOLYSOSOMAL PROTEOLYSIS 89

121 Tezuka K, Tezuka Y, Maejima A, Sato T, Nemoto K, 136 Deaton D N & Kumar S (2004) 6. Cathepsin K inhibitors: Kamioka H, Hakeda Y & Kumegawa M (1994) Molecular their potential as anti-osteoporosis agents. Prog Med Chem cloning of a possible cysteine proteinase predominantly 42, 245-375 expressed in osteoclasts. J Biol Chem 269, 1106-1109 137 Wang D, Li W, Pechar M, Kopeckova P, Bromme D & 122 Inaoka T, Bilbe G, Ishibashi O, Tezuka K, Kumegawa M & Kopecek J (2004) Cathepsin K inhibitor-polymer conjugates: Kokubo T (1995) Molecular cloning of human cDNA for potential drugs for the treatment of osteoporosis and cathepsin K: novel cysteine proteinase predominantly rheumatoid arthritis. Int J Pharm 277, 73-79 expressed in bone. Biochem Biophys Res Commun 206, 138 Lindeman J H, Hanemaaijer R, Mulder A, Dijkstra P D, 89-96 Szuhai K, Bromme D, Verheijen J H & Hogendoorn P C 123 Buhling F, Gerber A, Hackel C, Kruger S, Kohnlein T, (2004) Cathepsin K is the principal protease in giant cell Bromme D, Reinhold D, Ansorge S & Welte T (1999) tumor of bone. Am J Pathol 165, 593-600 Expression of cathepsin K in lung epithelial cells. Am 139 McGuire M J, Lipsky P E & Thiele D L (1997) Cloning and J Respir Cell Mol Biol 20, 612-619 characterization of the cDNA encoding mouse dipeptidyl 124 Mandelin J, Hukkanen M, Li T F, Korhonen M, Liljestrom peptidase I (cathepsin C). Biochim Biophys Acta 1351, 267- M, Sillat T, Hanemaaijer R, Salo J, Santavirta S & Konttinen 273 Y T (2005) Human osteoblasts produce cathepsin K. Bone 140 Sastradipura D F, Nakanishi H, Tsukuba T, Nishishita K, 38, 769-777 Sakai H, Kato Y, Gotow T, Uchiyama Y & Yamamoto K (1998) Identification of cellular compartments involved in 125 Dodds R A (2003) A cytochemical assay for osteoclast processing of cathepsin E in primary cultures of rat cathepsin K activity. Cell Biochem Funct 21, 231-234 microglia. J Neurochem 70, 2045-2056 126 Kafienah W, Bromme D, Buttle D J, Croucher L J & 141 Chain B M, Free P, Medd P, Swetman C, Tabor A B & Hollander A P (1998) Human cathepsin K cleaves native Terrazzini N (2005) The expression and function of type I and II collagens at the N-terminal end of the triple cathepsin E in dendritic cells. J Immunol 174, 1791-1800 helix. Biochem J 331, 727-732 142 Athauda S B & Takahashi K (2002) Distinct cleavage 127 Buhling F, Waldburg N, Gerber A, Hackel C, Kruger S, specificity of human cathepsin E at neutral pH with special Reinhold D, Bromme D, Weber E, Ansorge S & Welte T preference for Arg-Arg bonds. Protein Pept Lett 9, 15-22 (2000) Cathepsin K expression in human lung. Adv Exp Med 143 Santamaria I, Velasco G, Pendas A M, Paz A & Lopez-Otin Biol 477, 281-286 C (1999) Molecular cloning and structural and functional 128 Atley L M, Mort J S, Lalumiere M & Eyre D R (2000) characterization of human cathepsin F, a new cysteine Proteolysis of human bone collagen by cathepsin K: proteinase of the papain family with a long propeptide characterization of the cleavage sites generating by domain. J Biol Chem 274, 13800-13809 cross-linked N-telopeptide neoepitope. Bone 26, 241-247 144 Wang B, Shi G P, Yao P M, Li Z, Chapman H A & Bromme 129 Buhling F, Rocken C, Brasch F, Hartig R, Yasuda Y, Saftig D (1998) Human cathepsin F. Molecular cloning, functional P, Bromme D & Welte T (2004) Pivotal role of cathepsin K expression, tissue localization, and enzymatic in lung fibrosis. Am J Pathol 164, 2203-2216 characterization. J Biol Chem 273, 32000-32008 130 Troen B R (2004) The role of cathepsin K in normal bone 145 Tisljar K, Deussing J & Peters C (1999) Cathepsin J, a novel resorption. Drug News Perspect 17, 19-28 murine cysteine protease of the papain family with a placenta-restricted expression. FEBS Lett 459, 299-304 131 Motyckova G & Fisher D E (2002) Pycnodysostosis: role 146 Bode S, Peters C & Deussing J M (2005) Placental cathepsin and regulation of cathepsin K in osteoclast function and M is alternatively spliced and exclusively expressed in human disease. Curr Mol Med 2, 407-421 the spongiotrophoblast layer. Biochim Biophys Acta 1731, 132 Everts V, Hou W S, Rialland X, Tigchelaar W, Saftig P, 160-167 Bromme D, Gelb B D & Beertsen W (2003) Cathepsin K 147 Santamaria I, Pendas A M, Velasco G & Lopez-Otin C deficiency in pycnodysostosis results in accumulation of (1998) Genomic structure and chromosomal localization of non-digested phagocytosed collagen in fibroblasts. Calcif the human cathepsin O gene (CTSO). Genomics 53, 231-234 Tissue Int 73, 380-386 148 Sol-Church K, Frenck J & Mason R W (2000) Cathepsin Q, 133 Godat E, Lecaille F, Desmazes C, Duchene S, Weidauer E, a novel lysosomal cysteine protease highly expressed in Saftig P, Bromme D, Vandier C & Lalmanach G (2004) placenta. Biochem Biophys Res Commun 267, 791-795 Cathepsin K: a cysteine protease with unique kinin- 149 Bromme D, Li Z, Barnes M & Mehler E (1999) Human degrading properties. Biochem J 383, 501-506 functional expression, tissue distribution, 134 Lutgens E, Lutgens S P, Faber B C, Heeneman S, Gijbels M electrostatic surface potential, enzymatic characterization, M, de Winther M P, Frederik P, van der Made I, Daugherty and chromosomal localization. Biochemistry 38, 2377-2385 A, Sijbers A M, Fisher A, Long C J, Saftig P, Black D, 150 Tolosa E, Li W, Yasuda Y, Wienhold W, Denzin L K, Daemen M J & Cleutjens K B (2006) Disruption of the Lautwein A, Driessen C, Schnorrer P, Weber E, Stevanovic cathepsin K gene reduces atherosclerosis progression and S, Kurek R, Melms A & Bromme D (2003) Cathepsin V is induces plaque fibrosis but accelerates macrophage foam cell involved in the degradation of invariant chain in human formation. Circulation 113, 98-107 thymus and is overexpressed in myasthenia gravis. J Clin 135 Morko J P, Soderstrom M, Saamanen A M, Salminen H J & Invest 112, 517-526 Vuorio E I (2004) Up regulation of cathepsin K expression in 151 Ondr J K & Pham C T (2004) Characterization of murine articular chondrocytes in a transgenic mouse model for cathepsin W and its role in cell-mediated cytotoxicity. J Biol osteoarthritis. Ann Rheum Dis 63, 649-655 Chem 279, 27525-27533 90 INDIAN J. BIOCHEM. BIOPHYS., VOL. 45, APRIL 2008

152 Wex T, Levy B, Smeekens S P, Ansorge S, Desnick R J & Angstroms resolution reveal an interaction motif between a Bromme D (1998) Genomic structure, chromosomal papain-like cysteine protease and its propeptide. FEBS Lett localization, and expression of human cathepsin W. Biochem 384, 211-214 Biophys Res Commun 248, 255-261 156 Greenspan P D, Clark K L, Tommasi R A, Cowen S D, 153 Klemencic I, Carmona A K, Cezari M H, Juliano M A, McQuire L W, Farley D L, van Duzer J H, Goldberg R L, Juliano L, Guncar G, Turk D, Krizaj I, Turk V & Turk B Zhou H, Du Z, Fitt J J, Coppa D E, Fang Z, Macchia W, Zhu (2000) Biochemical characterization of human cathepsin X L, Capparelli M P, Goldstein R, Wigg A M, Doughty J R, revealed that the enzyme is an exopeptidase, acting as Bohacek R S & Knap A K (2001) Identification of dipeptidyl carboxymonopeptidase or carboxydipeptidase. Eur nitriles as potent and selective inhibitors of cathepsin B J Biochem 267, 5404-5412 through structure-based drug design. J Med Chem 44, 154 Guncar G, Klemencic I, Turk B, Turk V, Karaoglanovic- 4524-4534 Carmona A, Juliano L & Turk D (2000) Crystal structure of cathepsin X: a flip-flop of the ring of His23 allows carboxy- 157 Baldwin E T, Bhat T N, Gulnik S, Hosur M V, Sowder R C, monopeptidase and carboxy-dipeptidase activity of the 2nd, Cachau R E, Collins J, Silva A M & Erickson J W protease. Structure 8, 305-313 (1993) Crystal structures of native and inhibited forms of 155 Turk D, Podobnik M, Kuhelj R, Dolinar M & Turk V (1996) human cathepsin D: implications for lysosomal targeting and Crystal structures of human procathepsin B at 3.2 and 3.3 drug design. Proc Natl Acad Sci (USA) 90, 6796-6800