Cysteine protease cystatins

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). 

The N-terminal sequence of one peptide from the 35 kDa zone of H-gal-GP showed some homology to cathepsin B-like cysteine proteases. Molecular cloning has also identified a thrombospondin homologue associated with the diffusely staining region between zones A and B, a galectin associated with zone D (Newlands et al., 1999) and a low molecular weight (approximately 13 kDa) cysteine protease inhibitor, cystatin. 

Stefin or cystatin (cysteine protease inhibitor) Homo sapiens 95 (dimer form) 358-363... 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Arabidopsis clialiana (mouse-ear cress) (Brassicaceae) PRL1 PRL2 (proprotein 23 kDa) Cysteine protease inhibitor protein (cystatin) homologues [147]... 

Saccharum officinarum (sugar cane) (Poaceae) Sugar cane cystatins Scl- Sc25 (Phytocystatins) Cysteine proteases [184]... 

The Cystatin Superfamily of Inhibitors of Papain-like Cysteine Proteases. . 64... 

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.

The distribution in body fluids of the different cystatins is remarkably different (Fig. 3). For example, while cystatin C is present in appreciable amounts in all investigated body fluids, cystatins S, SN, and SA are virtually confined to saliva, tears, and seminal plasma (Al). Cystatin D is present only in saliva and tear fluid (Al, F3). In some body compartments, e.g., spinal fluid, cystatin C represents more than 90% of the total molar concentration of cysteine protease... 

Equilibrium Constants for Dissociation of Complexes between Human Cystatins and Cysteine Proteases Ki (nM)... 

CIO. Colle, A., Tavera, C., Laurent, P., Leung-Tack, J., and Girolami, J. P., Direct radioimmunoassay of rat cystatin C Increased urinary excretion of this cysteine proteases inhibitor during chromate nephropathy. J. Immunoassay 11(2), 199-214 (1990). 

Cystatin refers to a diverse family of protein cysteine protease inhibitors. There are three general types of cystatins Type 1 (stefens), which are primarily found in the cytoplasm but can appear in extracellular fluids Type 2, which are secreted and found in most extracellular fluids and Type 3, which are multidomain protease inhibitors containing carbohydrates and that include the kininogens. Cystatin 3 is used to measure renal function in clinical chemistry. See Barrett, A.J., The cystatins a diverse superfamily of cysteine peptidase inhibitors, Biomed. Biochim. Acta 45,1363-1374,1986 Katunuma, N., Mechanisms and regulation of lysosomal proteolysis, Revis. Biol. Cellular 20, 35-61, 1989 Gauthier, F., Lalmanach, G., Moeau, T. et al., Cystatin mimicry by synthetic peptides, Biol Chem. Hoppe Seyler 373, 465-470, 1992 Bobek, L.A. and Levine,... 

Cystatin A papain family cysteine proteases Competitive broad specificity picomolar to low nanomolar (19)... 

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). 

TIMPs inhibit matrix metaiioproteases (MMPs) via a two-step mechanism in a manner somewhat similar to that of cystatins (Fig. 3). While the N-terminal residues of cystatins bind to the nonprime side of cysteine proteases, TIMPs N-termini bind in the P1-P3 pockets of the protease, coordinate the catalytic... 

Although cystatins and aspins do not interact directly with the catalytic residues of cysteine proteases, many protease inhibitors, such as cytotoxic T-lymphocyte antigen 2-a and the cathepsin propeptides, do interact with the catalytic machinery... 

Because of the problems with changes in creatinine production and secretion, other endogenous compounds have been evaluated in an effort to provide a more accurate estimation of GFR. Perhaps the most promising is cystatin C, a low molecular weight protein that is a member of the cystatin super family of cysteine protease inhibitors [112]. Cystatin C is produced by all nucleated cells and its rate of production is relatively constant, being unaltered by inflammatory conditions... 

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