Papain-like cysteine proteases

Properties of cathepsin L may be compared among papain-like cysteine cathepsins (36-39) for understanding its role in producing neuropeptides. Cathepsin L belongs to the CIA subfamily of Clan CA (36). Clan CA was formed based on recognition of the first cysteine protease papain. The crystal structure of papain shows two structural domains separated by an active-site cleft. The N-terminal domain is comprised of a-helices, and the C-terminal domain contains a -barrel. 

The alignment of them realized in the tetrapetide allows for a simultaneous inhibition of the proteolytic activity of trypsin-like serine proteases, papain-like cysteine proteases, and pepsin-like aspartyl proteases. Therefore, this unique compound represents a blueprint for the design of protease class-spanning inhibitors [85, 86]. The capability of (59) to inhibit proteases belonging to different classes for trypsin, cathepsin B, cathepsin L, and papain was reported (see Table 30.3). Miraziridine A [85] also inhibited cathepsin B with an IC50 value of 1.4 pg/mL. Aziridine-2,3-dicarboxylic acid (14) is a rare natural product, reported from a Streptomyces [36], and vArg has never before reported as a natural product. 

Cathepsin K (Cat K) is a member of the CA1 family of lysosomal cysteine proteases. This family is comprised of 11 human members (cathepsins B, C, F, H, K, L, O, S, V, W, Z) which share a common papain-like structural fold and a conserved active site Cys-Asn-His triad of residues [1-3]. These enzymes are synthesized as pre-pro-enzymes and are converted from the catalytically inactive zymogen into the active form in acidic lysosomal environment. In some cases, cathepsins are also secreted in the active form from cells. The sequence identity of... 

Phylogenetic analysis, based on the primary sequences of the catalytic domains of cysteine proteases, has demonstrated a number of different clades within the trematode non-cathepsin B-like papain superfamily (Fig. 18.6 Tort et al., 1999 Park et al., 2002 Kang et al., 2004). The parasite enzymes fall into two main... 

Domain II is composed of two subdomains (Ha and lib) and represents the catalytic core of the protease. A cys at position 115 (p,-calpain) or 105 (m-calpain), a His residue at position 272 ((x-calpain) or 262 (m-calpain) and an Asn residue at position 296 ( x-calpain) or 286 (m-calpain) form the catalytic triad characteristic of cysteine proteases such as papain or cathepsins B, L, or S. Domain II, however, shares only limited sequence homology with other cysteine proteases, and is likely to have evolved from a different ancestral gene. 

The cysteine proteases can be divided into three classes the papain-like, the caspases (and related enzymes), and the picorna viral cysteine proteases. The proposed catalytic mechanism for cysteine protease peptide cleavage is related to the serine protease mechanism but with a cysteine thiol acting as the nucleophile that attacks the scissile peptide bond carbonyl. 

A recent publication described the synthesis of a series of dipeptide nitriles and the evaluation of their interaction with various papain-like cysteine proteases. Different P2 side chains were explored within the series of 44 compounds and microwave-assisted Suzuki couplings were used in a limited part of the synthetic efforts. Two different biphenyl derivatives were synthesized from the corresponding aryl bromides, as shown in Scheme 25, using 30 min of microwave heating at 70 °C [87]. Especially the meta-biphenyl derivative 52 was shown to be a potent inhibitor of papain and the cathepsins L, S, and K. 

Scheme 25 Preparation of a papain-like cysteine protease inhibitor...

Van der Hoorn RA, Leeuwenburgh MA, Bogyo M, Joosten MH, Peck SC (2004) Activity profiling of papain-like cysteine proteases in plants. Plant Physiol 135 1170-1178... 

Biological Roles of Papain-like Cysteine Proteases. 64... 

The occurrence of proteins which inhibit papain-like cysteine proteases has been known at least since 1946, when Grob demonstrated that blood serum (G7) and... 

Fig. 1. Amino acid sequence and schematic structure of human cystatin C. The shaded area marks the inhibitory site for papain-like cysteine proteases, which does not overlap with the inhibitory site for mammalian legumains comprising, inter alia, the Asn39 residue. The arrow indicates the Leu68 residue, which is replaced with a Gin residue in the cerebral hemorrhage producing cystatin C variant. The asterisk marks the Pro3 residue, which is partly hydroxylated.

In addition to being an inhibitor of papain-like cysteine proteases, cystatin C has recently been shown be an efficient inhibitor of some of the cysteine proteases of another family of cysteine proteases, called the peptidase family C13, with human legumain as a typical enzyme (C6). Human legumain has, like cathepsin S, been proposed to be involved in the class n MHC presentation of antigens (M3). It has also been shown that the cystatin C inhibitory site for mammalian legumain does not overlap with the cystatin C inhibitory site for papain-like cysteine proteases (Fig. 1) and that the same cystatin C molecule therefore is able to simultaneously inhibit one cysteine protease of each type (A 10). 

Cysteine proteases are so called because of a critical cysteine involved (together with an adjacent histidine) in the catalytic mechanism. Cysteine proteases include papain-related proteases, calpain-related proteases and the caspases. Papain-like cysteine proteases include the plant enzymes actinidin, aleurain, bromelain, caricain, chymopapain, ficin and papain and the lysosomal cathepsins B, C, H, K, L and S. Cathepsin C is multimeric (MW -200,000), but the other papain-related proteases are monomeric with MWs of about 20,000-35,000. While cathepsin C is a dipeptidyl aminopeptidase, the other enzymes are endopeptidases. Cathepsin B is an endopeptidase and a dipeptidyl carboxypeptidase. Cathepsin H is an endopeptidase and an aminopeptidase. In higher animals, cathepsin B generates peptides from antigens for presentation to T cells by the major histocompatibility... 

Turk D, Guncar G, Podobnik M, Turk B. Revised definition of substrate binding sites of papain-like cysteine proteases. Biol. 55. Chem. 1998 379 137-147. 

The cystatins, which are a superfamily of proteins that inhibit papain-like cysteine proteases, are a classic example of these inhibitors. The cystatins (Fig. 3) insert a wedge-hke face of the inhibitor that consists of the protein N-terminus and two hairpin loops into the V-shaped active site of a cysteine protease. The N-terminal residues bind in the S3-S1 pockets in a substrate-like manner, but the peptide then turns away from the catalytic residues and out of the active site. The two hairpin loops bind to the prime side of the active site, which provides most of the binding energy for the interaction. Thus, both the prime and the nonprime sides of the active site are occupied, but no interactions are actually made with the catalytic machinery of the enzyme (23). 

For these thermodynamically controlled syntheses a variety of enzymes may be used, e.g. serine proteases like chymotrypsin and trypsin, cysteine proteases (thiol proteases) like papain, aspartate proteases like pepsin and metalloproteases like thermolysin. ... 

The first protease-catalyzed reaction in ILs was the Z-aspartame synthesis (Scheme 10.7) from carbobenzoxy-L-aspartate and L-phenylalanine methyl ester catalyzed by thermolysin in [BMIM] [PF ] [ 14]. Subtilisin is a serine protease responsible for the conversion of A -acyl amino acid ester to the corresponding amino acid derivatives. Zhao et al. [90] have used subtilisin in water with 15% [EtPy][CF3COO] as cosolvent to hydrolytically convert a series of A -acyl amino acid esters often with higher enantioselectivity than with organic cosolvent like acetonitrile (Scheme 10.8, Table 10.2). They specifically achieved l-serine and L-4-chlorophenylalanine with an enantiomeric access (ee) of-90% and -35% product yield which was not possible with acetonitrile as a cosolvent [90]. Another example is hydrolysis of A-unprotected amino acid ester in the presence of a cysteine protease known as papain. Liu et al. 

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