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Proteolytic activity is of import for normal operation of an being and must be strictly controlled to avoid potentially unsafe extra protein debasement. Failure in biological control mechanisms of proteolytic activities causes a broad scope of diseases, among them malignant neoplastic disease, rheumatoid arthritis and degenerative arthritis, Alzheimer ‘s disease, multiple induration and muscular dystrophy ( reviewed in Turk et al. , 2000 ) . For many diseases ensuing from extra proteolysis, no inhibitors have yet been identified with the necessary profile for curative usage. Therefore, research into the physiological functions of peptidases and into the find of substances to modulate them will stay a precedence of both scientific discipline and the pharmaceutical industry for the foreseeable hereafter. For now, drug design returns manus in manus with the find of the biological functions of enzymes ; when a specific function has been identified by an inhibitor, this compound is already a drug campaigner.

About 500-600 peptidases have been identified in the human genome ( Lopez-Otin & A ; Overall, 2002 ) . Of these, approximately 60 are lysosomal peptidases ( Mason, 1995 ) , which include a group of about a twelve papain-like lysosomal cysteine peptidases. For historical grounds, intracellular peptidases were named cathepsins ( the root of the word originates from the Grecian linguistic communication, where it means to digest ) ; nevertheless, there is no rigorous regulation that links the reactive mechanism and localisation of cathepsins with their name. All known lysosomal cysteine peptidases are cathepsins, but non all cathepsins are lysosomal or cysteine peptidases. Cathepsins D and E are aspartic peptidases, whereas cathepsins A and G are serine peptidases ; cathepsins E and G are non lysosomal peptidases. Discovery of legumain ( Chen et al. , 1997, 1998 ) , besides a lysosomal cysteine peptidase belonging to clan CD, added to the confusion in terminology. This reappraisal focuses on the group of papain-like cysteine peptidases, which are omnipresent among populating beings ( including bacteriums, viruses and workss, and lower and higher animate beings, including parasites ) . Particular attending is paid to human lysosomal enzymes ( cathepsins ) and their mammalian homologues. The comparatively little size of the group, the unambiguously reactive cysteine sulfohydryl group ( pKa in the scope 2.5-3.5 ; Pinitglang et al. , 1997 ) and their alone reactive mechanism make these enzymes attractive marks for drug design. There are 11 human enzymes presently known ( cathepsins B, C, F, H, L, K, O, S, V, X and W ; Turk et al. , 2000 ; Turk, Turk et al. , 2001 ) and it is rather likely that the list has already been completed. Human cistron informations bank hunts have non indicated any new members of the household ( Sali, personal communicating ) .

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The classical cathepsins ( B, C, H, L and S ) were discovered by biochemical techniques, get downing with cathepsin C ( Gutman & A ; Fruton, 1948 ) . Other cathepsins ( F, K, O, V, X and W ) were found in the 1990s by agencies of DNA-manipulation techniques. The scientific community still awaits studies on the biochemical word picture of cathepsins O and W. The papain-like crease was revealed in the early yearss of crystallography ( Drenth et al. , 1968 ) ; nevertheless, structural word picture of cathepsins began in earnest in the early 1990s with the cathepsin B construction ( Musil et al. , 1991 ) . Crystal constructions of all human representatives or their mammalian parallels except cathepsins O and W have now been determined and are available from the PDB ( Table 1 ) . The relevancy of cathepsins as possible drug marks is best indicated by the fact that four ( K, S, V and late F ) of the nine constructions of cathepsins were published by industrial research groups. Structures of cathepsins L ( Fujishima et al. , 1997 ) and S ( McGrath et al. , 1998 ) have been besides reported by industrial groups but are non yet publicly available. An extra publication depicting the composites of cathepsin S with man-made inhibitors is in readying ( Cygler & A ; Rath, personal communicating ) . So far, constructions of four proenzymes are known ( Table 2 ) . Their construction finding by and large followed the constructions of the active enzymes. An exclusion was the procathepsin L construction ( Coulombe et al. , 1996 ) , which preceded the mature enzyme construction by 3 old ages.

Table 1

Primary commendations and the PDB codifications of cathepsin constructions

The parallel entries indicate constructions that were determined at the same time by several groups.

Cathepsin PDB codification Citation Species

B 1huc Musil et Al. ( 1991 ) Human

C 1jqp Olsen et Al. ( 2001 ) Rat

1k3b Turk, Janjic et Al. ( 2001 ) Human

F 1m6d Somoza et Al. ( 2002 ) Human

H 8pch Guncar et Al. ( 1998 ) Porcine

L 1icf Guncar et Al. ( 1999 ) Human

K 1mem McGrath et Al. ( 1997 ) Human

1atk Zhao et Al. ( 1997 ) Human

S 1glo Turkenburg et Al. ( 2002 ) Human

V 1fh0 Somoza et Al. ( 2000 ) Human

X 1ef7 Guncar et Al. ( 2000 ) Human

Table 2

Primary commendations and the PDB codifications of zymogen cathepsin constructions

The parallel entries indicate constructions that were determined at the same time by several groups.

Zymogen PDB codification Citation Species

B 1mir Cygler et Al. ( 1996 ) Rat

1pbh Turk et Al. ( 1996 ) Human

2pbh

3pbh Podobnik et Al. ( 1997 ) Human

L 1cjl Coulombe et Al. ( 1996 ) Human

K 7pck Sivaraman et Al. ( 1999 ) Human

1by8 LaLonde et Al. ( 1999 ) Porcine

X 1deu Sivaraman et Al. ( 2000 ) Human

2. Physiological functions and localisation

The papain-like lysosomal cysteine peptidases have long been believed to be responsible for protein debasement in lysosomes ( Kirschke et al. , 1995 ) . Analysiss of cistron smashers suggested that this map is non entirely dependent on any individual cathepsin ( Saftig et al. , 1998 ; Shi et al. , 1999 ; Pham & A ; Ley, 1999 ; Deussing et al. , 1998 ; Nakagawa et al. , 1998, 1999 ; Roth et al. , 2000 ) . However, analyses of cistron smashers and the locations of mutants on cistrons of lysosomal cysteine proteases responsible for some familial diseases revealed several specific biological maps. These maps are a effect of limited proteolysis of their mark substrates and to boot trust on co-localization and timing.

i?? ( I ) Cathepsin K was found to be important in bone remodelling ( Chapman et al. , 1997 ; Saftig et al. , 1998 ) .

i?? ( two ) Cathepsin S is the major processing enzyme of the MHC category II associated invariant concatenation and is therefore indispensable for the normal operation of the MHC category II associated antigen processing and presentation ( Nakagawa et al. , 1998, 1999 ; Shi et al. , 1999 ) . Cathepsins L and F were shown to take part in the same procedure, chiefly in tissues or cells non showing cathepsin S ( Nakagawa et al. , 1998 ; Shi et al. , 2000 ) , although the function of the former has likely been taken by cathepsin V in worlds ( Bri??mme et al. , 1999 ) .

i?? ( three ) Cathepsin L-deficient mice developed periodic hair loss and cuticular hyperplasia, bespeaking that cathepsin L is involved in cuticular homeostasis and regular hair-follicle morphogenesis and cycling ( Roth et al. , 2000 ) . One-year-old cathepsin L-deficient mice ( Stypmann et al. , 2002 ) exhibited histomorphological and functional changes of the bosom, ensuing in dilated myocardiopathy, which is a frequent cause of bosom failure.

i?? ( four ) Cells derived from cathepsin C-deficient mice fail to trip groups of serine peptidases from granules of immune ( cytotoxic T lymph cells, natural killer cells ) and inflammatory ( neutrophils, mast cells ) cells chiefly involved in the defense mechanism of the being, showing that cathepsin C is involved in their activation ( Pham & A ; Ley, 1999 ; Wolters et al. , 2001 ) . The current list of unrefined proenzymes of peptidases in cathepsin C smasher mice contains granzymes A, B and C, cathepsin G, neutrophil elastase and a chymase.

More informations about the procedures in which the lysosomal papain-like cysteine peptidases take part can be found elsewhere ( Kirschke et al. , 1995 ; Chapman et al. , 1997 ; Barrett et al. , 1998 ; McGrath, 1999 ; Turk et al. , 2000 ; Turk, Turk et al. , 2001 ; Bri??mme & A ; Kaleta, 2002 ) .

3. Pathology

Lysosomal cysteine peptidases have been found to be associated with a figure of pathologies, including malignant neoplastic disease, redness, arthritic arthritis and degenerative arthritis, Alzheimer ‘s disease, multiple induration, muscular dystrophy, pancreatitis, liver upsets, lung upsets, lysosomal upsets, Batten ‘s disease, diabetes and myocardial upsets. In many of these diseases, the lysosomal enzymes have been found in the extracellular and extralysosomal environment in their ( zymogenic ) `pro ‘ signifiers, which are well more stable than the mature enzymes ( reviewed in Kirschke et al. , 1995 ; Chapman et al. , 1997 ; Barrett et al. , 1998 ; Kos & A ; Lah, 1998 ; Turk, Turk et al. , 2001 ) . Cathepsins besides participate in programmed cell death, although the exact mechanism is non yet clear ( Stoka et al. , 2001 ; Salvesen, 2001 ; Leist & A ; Ji??i??tteli?? , 2001 ; Turk et al. , 2002 ) .

Several familial upsets have been traced to cistrons of lysosomal cysteine peptidases. Pycnodysostosis, an autosomal recessionary osteochondrodysplasia characterized in worlds by terrible bone abnormalcies, was found to be associated with the loss-of-function mutant of cathepsin K ( Gelb et al. , 1996 ) , while loss-of-function mutant in the cathepsin C cistron leads to Papillon-Lefevre syndrome, an autosomal recessionary upset characterized in patients by palmoplantar keratosis and terrible early-onset periodontal disease ( Toomes et al. , 1999 ; Hart et al. , 1999 ; Hart, Hart, Michalec, Zhang, Firatli et al. , 2000 ; Hart, Hart, Michalec, Zhang, Marazita et al. , 2000 ; Allende et al. , 2001 ) . These effects are rather likely to be a consequence of uncomplete processing of some as yet unidentified peptidases presumptively involved in set uping or keeping the structural organisation of the cuticle of the appendages and the unity of the tissues environing the dentition and in the processing of proteins such as ceratins ( Nuckolls & A ; Slavkin, 1999 ) . In add-on, cathepsin C may be involved in chronic air passage diseases such as asthma ( Wolters et al. , 2000 ) .

Similarly, down-regulation of natural inhibitors, as demonstrated by a mutant in the cistron for stefin B, predisposes affected persons to a familial signifier of myoclonal epilepsy ( Pennacchio et al. , 1996 ; Lalioti et al. , 1997 ) .

4. Fold and specificity

The papain-like lysosomal cysteine peptidases are monomeric proteins with MW between 22 and 28 kDa. The lone exclusion is cathepsin C, which is a tetrameric molecule with an MW of 200 kDa ( Dolenc et al. , 1995 ) . They all portion the common crease of a papain-like construction. Cathepsin L, as a typical endopeptidase, has been chosen as a representative of the household ( Fig. 1 ) . A papain-like crease consists of two spheres, reminiscent of a closed book with the spinal column at the forepart. The spheres separate at the top in a V-shaped active-site cleft, in the center of which the residues Cys25 and His159, one from each sphere, organize the catalytic site of the enzyme. The most outstanding characteristic of the left ( L ) sphere is cardinal -helix of about 30 residues in length ; the right ( R ) sphere forms a sort of -barrel, which includes a shorter -helical motive. ( The footings left and right sphere refer to the standard position shown in Fig. 1. )

Figure 1

Fold of cathepsin L. Cathepsin L ( 1icf ) is shown as a thread in its standard orientation, viewed along the two-domain interface with the cardinal -helix in a perpendicular orientation and the active site at the top. The side ironss of the catalytic residues Cys25 and His159 are shown as yellow and green atom balls, severally. This figure and Fig. 2 were prepared utilizing the plan RIBBONS ( Carson, 1997 ) .

Lysosomal cathepsins are encoded as `pre-proenzymes ‘ . Following cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic Reticulum membrane ( Erickson, 1989 ) , procathepsins undergo proteolytic processing to the active mature enzyme signifier in the acidic environment of late endosomes or lysosomes ( Nishimura et al. , 1988 ; Kominami et al. , 1988 ) . The crystal constructions of zymogens ( Table 2 ) showed that the construction of the mature enzyme is already formed in the proenzyme signifier. Propeptide concatenation builds a -helical sphere, which continues along the active-site cleft towards the N-terminus of the mature enzyme in a preponderantly extended conformation in the way antonym to substrate binding, barricading entree to the active site. The procathepsin L construction ( Coulombe et al. , 1996 ) has been chosen as a representative of the household ( Fig. 2 ) . Propeptides are in fact inhibitors of their blood relation enzymes, as demonstrated by kinetic informations ( Guay et al. , 2000 ) . Among them, cathepsin K ( Sivaraman et al. , 1999 ; LaLonde et al. , 1999 ) has the longest N-terminal peptide and cathepsin X ( Sivaraman et al. , 2000 ) the shortest. The propeptide of cathepsin X is besides the lone one covalently attached to the reactive-site cysteine via a disulfide bond.

Figure 2

Fold of procathepsin L ( 1cjl ) . The mature enzyme portion of cathepsin L is shown in bluish and and the propeptide is shown in ruddy.

4.1. Substrate-binding sites

When in 1967 Schechter and Berger reported their cardinal work on the substrate-binding sites of papain they had to trust entirely on kinetic informations ( Schechter & A ; Berger, 1967 ) . They studied the dependance of substrate dynamicss on the length of a polyalanine concatenation and discovered the dynamicss to be influenced by the polypeptide-chain length up to a length of seven aminic acids and concluded that there are seven substrate-binding sites on the papain molecule. Their definition of substrate-enzyme interactions and their terminology became the criterions ( Fig. 3 ) for the assignment of interaction sites of a polypeptide substrate and a proteolytic enzyme.

Figure 3

Schechter and Berger ‘s definition of substrate-binding sites ( Schechter & A ; Berger, 1967 ) .

Three decennaries subsequently, when a sufficient figure of protease-inhibitor constructions became available, the definition of Schechter and Berger substrate-binding sites on the papain-like enzymes was revisited and redefined ( Turk et al. , 1998 ) . The base and walls of the substrate-binding sites are formed by four concatenation sections consisting two shorter cringles in the L-domain ( 19-25, 61-69 ) and two longer cringles in the R-domain ( 136-162, 182-213 ; Fig. 4 ) . A 3rd cringle from the L-domain might besides be named if the disulfide ( Cys22-Cys65 ) which connects the two L-domain cringles at the top is considered to be an extra cringle closing.

Figure 4

Substrate-binding sites. ( a ) A position from the top: a polyalanine substrate theoretical account edge in the active-site cleft of cathepsin L. ( Modelling of the adhering geometry of a substrate is based on information gained from the crystal constructions of substrate-analogue inhibitors and their interactions with a papain-like peptidase active site ; see Figs. 5a, 5b and 5c ) . Substrate residues are shown as green sticks and are denoted utilizing the Schechter and Berger terminology. Cathepsin L is shown with a gray surface representation. The surface of the catalytic cysteine side concatenation is xanthous. ( B ) The same as Fig. 4 ( a ) , merely that in this instance cathepsin L is shown as a concatenation hint. Most of the concatenation is gray, whereas the cringles constructing the substrate-binding sites are colour-coded: the L-domain cringles ( 19-25 and 61-69 ) are violet and xanthous and the R-domain cringles ( 136-162 and 182-213 ) are bluish and ruddy. The loops constructing the substrate-binding sites do non merely incorporate the residues straight lending to the surface, but besides include those that provide the foundation for it. ( hundred ) Structure-based amino-acid alliance of sequences of papain-like spheres of all known human cathepsins. Structural alliance was made utilizing the plan Modeller ( Sali and Blundell, 1993 ) , so the sequences of cathepsins F, O and W were aligned to the templet with the ClustalW plan ( Higgins et al. , 1996 ) . The sequences were taken from SWISS-PROT or GENBANK databases and the constructions from the PDB. The loops constructing the substrate-binding sites are marked at the top marked with chevrons and utilizing the same coloring material codification as in Fig. 4 ( B ) . Figs. 4 ( a ) , 4 ( B ) , 5, 8 and 9 were prepared with MAIN ( Turk, 1992 ) and rendered with Raster3D ( Merritt & A ; Bacon, 1997 ) . The cathepsin L surface was generated with GRASP ( Nicholls et al. , 1991 ) .

The overlying constructions of composites of substrate-analogue inhibitors and cathepsins ( Figs. 5a and 5c ) have revealed that substrate residues bind along the active-site cleft in an drawn-out conformation, with the side chains alternately oriented toward the L- and R-domains. Residues P2, P1 and P1 ‘ bind into good defined binding sites. Positioning of these residues is governed by interactions which involve both main-chain and side-chain atoms. The S2 binding site is a deep pocket, whereas the S1 and S1 ‘ sites provide a binding surface. The placement of the P3 residue is mediated merely by side-chain interactions. For this ground, the adhering geometries of the latter are scattered over a wide country and are alone for each substrate. On the premier side of the binding cleft, the P2 ‘ residue-binding site appears to be rather good defined. However, current cognition is based on specific interactions between CA030 and the parts of cathepsin B construction responsible for its carboxydipeptidase activity ( Fig. 5c ) ( Turk et al. , 1995 ) . It therefore remains possible that interactions within the S2 ‘ site of an endopeptidase would be different.

Figure 5

Low-molecular-weight inhibitor-binding geometry. The inhibitors ( shown as sticks ) from constructions of composites with papain-like cysteine peptidases are superimposed on top of the cathepsin L surface. The catalytic site Cys25 surface is coloured xanthous. PDB codifications are given in parentheses. Complexs with works enzymes are besides included. ( a ) Substrate-analogue inhibitors: fluoro- and chloromethylketone-based inhibitors and leupeptin are shown in light blue. Inhibitors are taken from constructions of composites with the undermentioned enzymes: cruzipain ( 1aim, 2aim ) , papain ( 1pad, 1pop, 5pad, 6pad ) , glycyl endopeptidase ( 1gec ) and cathepsin B ( 1the, 1cte ) . ( B ) E-64 and derivative are shown in magenta. Inhibitors are taken from constructions of composites with the undermentioned enzymes: actinidin ( 1aec ) , caricain ( 1meg ) , cathepsin K ( 1atk ) and papain ( 1pe6, 1ppp ) . ( degree Celsius ) CA030 inhibitor from the complex with cathepsin B ( 1csb ) is shown in blue. ( vitamin D ) Vinylsulfone-based inhibitors taken from construction of composites with cathepsin K ( 1mem ) and cathepsin V ( 1fh0 ) are shown in green. ( vitamin E ) A group of non-covalent cathepsin K inhibitors are shown in ruddy ( 1ayu, 1ayv, 1ayw, 1au0, 1bgo, 1au2, 1au3, 1au4 ) .

The location of the substrate-binding sites beyond S3 and S2 ‘ is non constrained by main-chain interactions. Each substrate residue docks on the surface of an enzyme in its ain manner ( Fig. 5a ) . In peculiar, for the non-primed binding sites, there is grounds that a common S4 binding site and besides an S3 ‘ site do non be. Therefore, it was suggested that the substrate residue-binding parts beyond S2 and S2 ‘ should non be called sites but countries ( Turk et al. , 1998 ) . The papain-like peptidases therefore represent a particular category of proteolytic enzymes with the smallest figure of substrate-binding sites, as opposed to chymotrypsin-like serine peptidases which have six ( Bode & A ; Huber, 1992 ) and aspartic peptidases which have eight adhering sites ( Wlodawer & A ; Gustchina, 2000 ) .

4.2. Binding of low-molecular-weight inhibitors

The instead short adhering country seems to ease covalent interactions with low-molecular-weight inhibitors. Covalent interactions, nevertheless, enforce difficult restraints on the binding geometry. It therefore took some clip to plan inhibitors that bind into the primed every bit good as non-primed side of the active-site cleft.

The constructions of the first composites of substrate-analogue inhibitors, based on the chloromethyl reactive group, with papain clarified the substrate binding in the non-primed binding sites in the 1970s ( Drenth et al. , 1976 ) . Other constructions followed subsequently ( Fig. 5a ) . At about the same clip, a natural cysteine peptidase inhibitor named E-64 was discovered ( Aoyagi & A ; Umezawa, 1975 ; Hanada et al. , 1978 ) . E-64 utilizes an epoxysuccinyl group to covalently interact with the reactive-site cysteine ( Fig. 6 ) . Structures of E-64 ( Varughese et al. , 1992 ) and its parallels ( Yamamoto et al. , 1991 ) revealed that they bind into the non-primed part of the active site, but in the way of propeptide binding and opposite to substrate binding ( Fig. 5b ) .

Figure 6

Schemes of three most frequent reactive groups before and after adhering to the reactive-site cysteine. ( a ) Chloromethylketone, ( B ) epoxysuccinyl, ( degree Celsius ) vinylsulfone.

The crystal construction of CA030 in complex with human cathepsin B ( Turk et al. , 1995 ) showed that E-64 derived functions can besides adhere into the fit binding side in the way of a substrate binding. Switch of the binding side was made possible by the specific interactions. The carboxylic group of the C-terminal residue of CA030 mimics the C-terminus of a substrate and docks against the obstructing cringle residues His110 and His111 ( Fig. 5c ) . Alliance of the E-64 and CA030 binding geometries showed ( Fig. 7 ; Turk et al. , 1995 ) that the epoxysuccinyl group possesses internal symmetricalness with two carboxylic caputs, miming a polypeptide C-terminus to which amino-acid residues can be attached. The synthesis of double-head inhibitors followed ( Schaschke et al. , 1997, 2000 ; Katunuma et al. , 1999 ) . The adhering geometry of the double-head inhibitor design has late been confirmed by the crystal constructions of cathepsin L and cathepsin B-inhibitor composites ( Tsuge et al. , 1999 ; Stern et al. , unpublished consequences ) .

Figure 7

Alignement of epoxysuccinyl derived functions.

The S1 ‘ adhering site can besides be reached with inhibitor concepts utilizing the vinylsulfone reactive group ( Fig. 6c ; McGrath et al. , 1997 ; Somoza et al. , 2000, 2002 ) and exceptionally even by a long side concatenation of a P1-mimicking residue of a chloromethyl inhibitor ( Figs. 5a and 5d ; Jia et al. , 1995 ) .

The covalent interaction with the reactive-site cysteine is non compulsory as shown by a series of `Smith-Kline ‘ compounds ( Fig. 5e ) , which utilize assorted concepts to tightly barricade the reactive site, but are non engaged in covalent interactions ( Thompson et al. , 1997 ) .

Extra information sing inhibitors and their chemical science can be found elsewhere ( Shaw, 1990 ; Otto & A ; Schirmeister, 1997 ; Bri??mme & A ; Kaleta, 2002 ) .

4.3. Exopeptidases

Whereas in endopeptidases ( cathepsins F, L, K, O, S and V ) the active-site cleft extends along the whole length of the two-domain interface, the exopeptidases ( cathepsin B, C, H and X ) possess extra characteristics that cut down the figure of substrate-binding sites ( Fig. 8 ) . The function of these characteristics is double: they prevent the binding of longer peptidyl substrates and they dock with charged N or C concatenation end point of substrates by using selective electrostatic interactions.

Figure 8

Features of exopeptidases. Chain hints of cathepsins H ( 8pch ) , C ( 1k3b ) , B ( 1huc ) and X ( 1ef7 ) , coloured orange, ruddy, dark blue and light blue, severally, are shown superimposed on the cathepsin L construction viewed from the top. The surface of cathepsin L is shown in Grey ; a xanthous coloring material denotes the surface of the catalytic residue Cys25. Structural elements easing the exopeptidase activity are labelled. Residues that play a important function in exopeptidase specificity are shown in stick representation.

Carboxydipeptidase cathepsin B ( Musil et al. , 1991 ) has an interpolation of about 20 residues, termed the obstructing cringle, which blocks the active-site cleft on the fit binding side beyond S2 ‘ and provides two histidine residues, His110 and His111, that bind to the charged main-chain carboxylic group of the C-terminal residue of a substrate. Cathepsin B besides exhibits an endopeptidase activity that is made possible by the flexible occluding cringle, which can be displaced from the active-site cleft ( Illy et al. , 1997 ; Podobnik et al. , 1997 ; Nagler et al. , 1997 ) .

Cathepsin X is chiefly a carboxymonopeptidase ( Nagler et al. , 1999 ) , which can besides move as a carboxydipeptidase ( Klemencic et al. , 2000 ) . The crystal construction showed that a histidine residue, His23, positioned within a short cringle termed a mini-loop ( Nagler et al. , 1999 ) , is the ground tackle for the carboxylic group of the C-terminal substrate residue ( Guncar et al. , 2000 ) . In the free-enzyme construction, the histidine ring occupies the place which is the S2 ‘ substrate-binding site in related cathepsins. This construction therefore corresponds to the carboxymonopeptidase manner of cathepsin X. A simple modeling survey ( manual rotary motion about the side-chain bonds ) suggested that the histidine ring can accommodate a place equivalent to His110 of cathepsin B. This cathepsin B-like place would therefore match to the carboxydipeptidase manner.

Cathepsin H is an aminomonopeptidase. The crystal construction of the porcine enzyme ( Guncar et al. , 1998 ) revealed that an eight-residue section of the propeptide, called the mini-chain, binds in the active-site cleft of the enzyme in the way of a edge substrate. The negatively charged carboxylic group of its C-terminal residue, Thr83P, attracts the positively charged N-terminus of a substrate and thereby facilitates the aminopeptidase activity of cathepsin H. Thr83P mimics a substrate P2 residue by busying the place that is the S2 binding site in related enzymes. The mini-chain is to boot fastened to the enzyme surface by a four-residue interpolation ( Lys155A-Asp155D ) and a saccharide concatenation attached to Asn112. The placement of the cathepsin H mini-chain closely resembles the placement of the C-terminus of a distant homologue, bleomycin hydrolase ( Joshua-Tor et al. , 1995 ) .

Cathepsin C ( besides termed dipeptidyl protease I or DPPI ) is an aminodipeptidase. The four independent active sites of cathepsin C are located on the external surface of the tetrahedral molecule. In contrast, oligomeric proteolytic machineries such as 20S proteasome ( Lowe et al. , 1995 ; Groll et al. , 1997 ) , bleomycin hydrolase ( Joshua-Tor et al. , 1995 ) , tryptase ( Pereira et al. , 1998 ) and tricorn peptidase ( Brandstetter et al. , 2001 ) have their active sites on the inside surface. Proteasomes are barrel-like constructions composed of four rings of – and -subunits, which cleave unfolded proteins captured in the cardinal pit into short peptides. Tryptases are level tetramers with a cardinal pore in which the active sites reside. The pore restricts the size of accessible substrates and inhibitors. Similarly, the active sites of bleomycin hydrolase and tricorn peptidase are besides located within the hexameric construction. The open active sites make cathepsin C a alone oligomeric peptidase capable of the hydrolysis of protein substrates in their native province regardless of their size. Its design, supported by the oligomeric construction, confines the activity of the enzyme to an aminodipeptidase and thereby makes it suited for usage in many different environments, where cathepsin C can selectively trip a group of chymotrypsin-like peptidases and presumptively besides other proteins.

The active site of cathepsin C is blocked beyond the S2 binding site by the monolithic organic structure of the exclusion sphere ( Turk, Janjic et al. , 2001 ; Olsen et al. , 2001 ) . An open -hairpin, the first N-terminal residues of the exclusion sphere and the saccharide ring attached to Asn5 block undesired entree, while Asp1 with its carboxylic group side concatenation controls entry into the S2 adhering pocket by repairing the N-terminal amino group of the substrate. Asp1 at the same time prevents the positively charged side ironss of arginine and lysine residues from adhering in the S2 binding pocket. An extra particular characteristic of cathepsin C is the dependance of its activity on chloride ions. One was located at the underside of the really long S2 binding pocket.

Interestingly, structural comparing and similar interactions within the active-site cleft ( Turk, Janjic et al. , 2001 ) suggested that the exclusion sphere of cathepsin C was adapted from a metalloprotease inhibitor ( Baumann et al. , 1995 ) . The N-terminus of the exclusion sphere merely blocks entree to a part of the active-site cleft, whereas the N-terminus of the metalloprotease inhibitor binds along the fit binding sites and interacts with the reactive-site Zn ion.

4.4. Specificity

Papain-like cathepsins are instead non-specific enzymes with no clear substrate-recognition site. This does non connote that specific inhibitors can non be designed. It merely suggests that specificity is non an issue affecting a individual binding site, but is instead a cumulative part of all interactions. This suggests that inhibitor concepts interacting with parts on both sides of reactive site can be advantageous compared with those which bind to merely one side. The specificity of exopeptidases is, nevertheless, more a affair of sole interactions of free concatenation end point than side-chain acknowledgment. The design of exopeptidase inhibitors hence seems easier, as such inhibitors can trust on covalent interactions with the reactive site and electrostatic interactions with negatively charged carboxylic groups or positively charged histidines in the instances of aminopeptidases and carboxypeptidases, severally.

5. Hints from interactions with protein inhibitors

Stefins and cystatins are instead non-specific endogenous inhibitors of cysteine peptidases. They are merely able to know apart between endo- and exopeptidases. Whereas the suppression of endopeptidases is rapid and tight, about being pseudo-irreversible, with Ki values in the picomolar scope, the suppression of exopeptidases is much weaker with Ki values in the millimolar to nanomolar scope ( reviewed in Turk & A ; Bode, 1991 ; Turk et al. , 2000 ) . Similar to stefins, an repressive fragment of the p41 signifier of MHC category II associated invariant concatenation ( termed the p41 fragment ) inhibits endopeptidase cathepsin L ( Ki = 1.7 autopsy ) and exopeptidase cathepsin H ( Ki = 5.3 nanometer ) ; nevertheless, it does non suppress endopeptidase cathepsin S and exopeptidase cathepsin B ( Bevec et al. , 1996 ) . How can this be explained on a structural footing?

Two crystal constructions provided insight into the interactions between a papain-like cysteine peptidase and its protein inhibitor: those of the composites of papain-stefin B ( Stubbs et al. , 1990 ) and cathepsin L-p41 fragment ( Fig. 9 ; Guncar et al. , 1999 ) .

Figure 9

Binding of protein inhibitors. Stefin B superimposed on cathepsin L-p41 composite in positions ( a ) across and ( B ) along the active-site cleft of cathepsin L. ( a ) is shown in about the standard position ( Fig. 1 ) , whereas ( B ) is generated by an 90i?? rotary motion about the perpendicular axis. Superposition of the p41 fragment and stefin B is based on the 3-dimensional alliance of papain and cathepsin L structures in the papain-stefin B and cathepsin L-p41 fragment composites. Chain hints of the p41 fragment, stefin B and cathepsin L are shown in orange, ruddy and bluish, severally.

The cuneus form and the three-loop agreement of the p41 fragment edge to the active-site cleft of cathepsin L is evocative of the inhibitory border of cystatins and therefore show the first ascertained illustration of convergent development in the cysteine peptidase inhibitors. The interactions within the active-site cleft are non-specific. They are either hydrophobic or mediated via solvent molecules or affect hydrogen-bond interactions with groups which are conserved throughout the household of papain-like enzymes. This suggests that the p41 fragment, like the stefins, displaces the characteristic structural elements of exopeptidases from the active site, as revealed by the stefin A-cathepsin H composite ( Jenko, unpublished consequences ) . However, the different crease of the p41 fragment consequences in extra contacts with the extremely variable parts of the cringles at the top of the R-domain, which build the surface of the S2 and S1 ‘ substrate-binding sites. This enables the p41 fragment to organize specific interactions with its mark enzymes and at the same time prevents the attack of cathepsins S and B ( Guncar et al. , 1999 ) .

6. Prospectives

Presently, no drug targeted towards a lysosomal cysteine peptidase is in usage ; nevertheless, many are in development. The cognition and experience gathered in the field suggest that there are adequate leads to drive the research. The protein-inhibitor composite, nevertheless, suggest to interior decorators of low-molecular-weight inhibitors that there are still undiscovered countries on the surface of the enzymes. In peculiar, it may be worthwhile to research them in the instance of endopeptidases, which have fewer restraints within the active-site cleft than the exopeptidases.

The drug-design procedure is challenged besides from another point of position. Are the mature human enzymes truly the most appropriate marks for possible drugs? Labelled inhibitors aiming a papain-like cysteine peptidase from Trypanosoma cruzi indicate that such inhibitors may interact already with their zymogen signifier within the Golgi composite ( Engel et al. , 1998 ) .

Recognitions

The Slovenian Ministry of Education, Science and Sport and the ICGEB are appreciatively acknowledged for their fiscal support.

Biographic Information

Dusan Turk is a research associate at Jozef Stefan Institute, Ljubljana, Slovenia, heading the Structural Biology group in the Department of Biochemistry and Molecular Biology. He received his PhD grade at the Technical University, Munich in 1992 in the research lab of Professor Robert Huber at the Max-Planck Institute of Biochemistry, Martinsried, Germany, after finishing his BSc in chemical science and Masterss degree in the country of computational chemical science at Ljubljana University ( Chemical Institute ) . His postoctoral experience was divided between the research labs of Professors Robert Huber and Vito Turk at the Jozef Stefan Institute and he started a macromolecular crystallography research lab in the latter. His chief involvements are in the structural biological science of peptidases, preponderantly cysteine peptidases, and their control mechanisms, and in the development of computational methods for macromolecular crystal construction finding assembled in the computing machine plan MAIN.

Gregor Guncar obtained a grade in chemical science in 1995 and his doctor’s degree in 2000, both from the University of Ljubljana, Slovenia. He joined the Structural Biology group at Josef Stefan Institute after his BSc and worked in the field of cysteine peptidases and their inhibitors, of which he determined several constructions. He is now go oning his work in the field and is looking frontward to happening a nice postdoctoral place in the close hereafter.

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