Cystatin binding

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... 

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. 

Estimation of the glomerular filtration rate (GFR) is considered a sensitive index of functional nephron mass (Newman and Price 1999). Point measurements of the plasma levels of several endogenous small molecules (urea, creatinine, 2-(a-mannopyranosyl)-L-tryptophan) or small (less than 66 kDa) proteins (cystatin C (y-trace), prostaglandin D synthase (15-trace protein), ai-microglobulin, p2-microglobulin, and retinol binding protein) have been used to assess GFR in many species. 

B18. Bjork, I., Brieditis, I., Raub-Segall, E., Pol, E., Hakansson, K., and Abrahamson, M., The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases. Biochemistry 35(33),... 

H6. Hall, A., Hakansson, K., Mason, R. W., Grubb, A., and Abrahamson, M., Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases. J. Biol. Chem. 270(10), 5115-5121 (1995). 

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). 

Zn + ion, and exclude a catalytic water molecule from the active site. Meanwhile a second loop of the TIMP binds in both the P3 and the P2 pockets, and it binds to the N-terminus of the MMP. Despite the similarities in mechanistic architecture between TIMPs and cystatins (hairpin loops and N-terminal residues in substrate binding pockets), TIMPs interfere with the catalytic machinery of MMPs by chelating the catalytic Zn + (5). 

The ascaris pepsin inhibitor-3 is an aspin, which is a family of inhibitors of aspartic proteases that protect worms from host gastric enzymes. Like the cystatins, the aspins are competitive inhibitors that bind in the substrate-binding subsites, but they do not have an amide bond that is available for nucleophilic attack. They gain most of their inhibitory activity by inserting their 3 N-terminal residues in the ST-S3 subsites of the protease (6) (Fig. 3). 

The basis of bacterial adhesion is given by the acquired pelhcle formation on tooth surfaces. This pelhcle is a thin ( 0.5-l pm) layer of several sahvary proteins with calciumhydroxide-binding properties. The most important such proteins are salivary amylase, cystatins (S, SA, and SN type), histatin (HRPl), mucine (MGl), acidic PRPs, statherin, and immunoglobulins (slgA) (4, 7). 

A. Ritonja, A. D. Rowan, D. J. Buttle, N. D. Rawlings, V. Turk, and A. J. Barrett. Stem bromelain amino acid sequence and implications for weak binding of cystatin. FEBS Lett. 247 419 (1989). 

Cystatins, a human superfamily of cysteine peptidase inhibitors. They are tight-binding reversible inhibitors of many cysteine proteases, and are not capable of inhibiting other proteases. Members of this superfamily contain at least two intrachain disulfide bonds and an a-helical structure over a distance of about 100 aa. This superfamily comprises (i) Family 1 (intracellular cystatins) cystatin A and cystatin B (ii) Family 2 (extracellular and/or transcellular cystatins) cystatin G, cystatin D, cystatin E, cystatin F, cystatin G, cystatin S, cystatin SA and cystatin SN and (iii) Family 3 (intravascular cystatins) LMW-kininogen and HMW-kininogen [A. J. Barret, Trends Biol. Sci. 1987, 12, 193 S. Nagpal et al., J. Invest. Dermatol. 1997, 109, 91 M. Zanatti,... 

Cystatin C L-type fatty add-binding protein (L-FABP)... 

The GFR can be estimated by clearance measurements of endogenous or exogenous small molecules (urea, creatinine, 2-MPT, inulin, cystatin C, iohexol, or iodixanol). An ideal marker of GFR should be primarily excreted by the kidneys, freely filtered by the glomerulus, and neither secreted nor reabsorbed by the tubule. It should also be supplied to the plasma at a constant rate and exhibit no or minimal protein binding. If these criteria are met, such as for inulin, the GFR is equivalent to the renal/urinary clearance of the substance, as defined earlier in this chapter. 

Native ovoflavoprotein (49 kDa, pf=5.1) has, as does ovomucoid, certain antinutritional effects, as it inhibits serine proteases (trypsin, chymotrypsin and also microbial proteases) and has antiviral activity. Ovomacroglobulin (ovostatin) is an inhibitor of serine, cysteine, thiol and metalloproteases and shows antimicrobial activity. Some antinutritional effects are also seen in the basic glycoprotein avidin in raw egg white (relative molecular weight of the monomer is 15.6 kDa). It contains four identical subunits (pf = 9.5), each of which binds one molecule of biotin to give an unavailable complex. However, the denatured avidin, for example in hard-boiled eggs, does not interact with biotin. The interaction of riboflavin with flavoprotein (32 kDa, pf = 4.0) has, on the contrary, a positive influence on vitamin stability. Cystatin acts as cysteine protease inhibitor, and shows antimicrobial, antitumor and immunomodulating activities. 

Juszczyk P., Paraschiv G., Szymanska A., Kolodziejczyk A.S., Rodziewicz-Motowidlo S., Grzonka Z., Przybylski M. (2009) Binding epitopes and interaction structure of the neuroprotective protease inhibitor cystatin C with beta-amyloid revealed by proteolytic excision mass spectrometry and molecular docking simulation. J Med Chem, 52 2420-8. 

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