Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P01034 (cystatin C)
 3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The utility of biomolecular interaction analysis-mass spectrometry (BIA/MS) in screening for protein-protein interactions was explored in this work. Experiments were performed in which proteins served as ligands for screening of possible interactions with other proteins from human plasma and urine. The proteins utilized were beta-2-microglobulin, cystatin C (cysC), retinol binding protein (RBP), transthyretin (TTR), alpha-1-microglobulin, C-reactive protein, transferrin and papain. The immobilization of functionally active proteins was confirmed via interactions with antibodies to the corresponding proteins. Various dilutions of human urine and plasma were injected over the protein-derivatized surfaces. It was observed that the urine injections generally yielded smaller SPR responses than those observed after the plasma injections. The BIA/MS experiments did not reveal novel protein-protein interactions, although several established interactions (such as those between RBP and TTR, and cysC and papain) were validated. Few protein ligand deficiencies (such as truncations) leading to false negative and false positive BIA/MS results were also discovered.
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PMID:Delineating protein-protein interactions via biomolecular interaction analysis-mass spectrometry. 1255 33

The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a K(i) of 25 nM. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.
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PMID:The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2. 1258 66

A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.
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PMID:Cystatin C in milk basic protein (MBP) and its inhibitory effect on bone resorption in vitro. 1259 44

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.
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PMID:Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases. 1450 83

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.
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PMID:Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system. 1469 Jun 87

Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P(5)-P(1) part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'(1)-P'(2) dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'(1)-S'(2) sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P(4) is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P(2) causes an increase of the substrate preference towards this activity.
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PMID:Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B. 1509 28

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.
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PMID:Domain swapping in N-truncated human cystatin C. 1531 69

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.
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PMID:Inhibitory properties of cystatin F and its localization in U937 promonocyte cells. 1575 68

Cysteine proteinases, especially cathepsin K, play an important role in osteoclastic degradation of bone matrix proteins and the process can, consequently, be significantly inhibited by cysteine proteinase inhibitors. We have recently reported that cystatin C and other cysteine proteinase inhibitors also reduce osteoclast formation. However, it is not known which cysteine proteinase(s) are involved in osteoclast differentiation. In the present study, we compared the relative potencies of cystatins C and D as inhibitors of bone resorption in cultured mouse calvariae, osteoclastogenesis in mouse bone marrow cultures, and cathepsin K activity. Inhibition of cathepsin K activity was assessed by determining equilibrium constants for inhibitor complexes in fluorogenic substrate assays. The data demonstrate that whereas human cystatins C and D are equipotent as inhibitors of bone resorption, cystatin D is 10-fold less potent as an inhibitor of osteoclastogenesis and 200-fold less potent as an inhibitor of cathepsin K activity. A recombinant human cystatin C variant with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106 did not inhibit bone resorption, had 1,000-fold decreased inhibitory effect on cathepsin K activity compared to wildtype cystatin C, but was equipotent with wildtype cystatin C as an inhibitor of osteoclastogenesis. It is concluded that (i) different cysteine proteinases are likely to be involved in bone resorption and osteoclast formation, (ii) cathepsin K may not be an exclusive target enzyme in any of the two systems, and (iii) the enzyme(s) involved in osteoclastogenesis might not be a typical papain-like cysteine proteinase.
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PMID:Different cysteine proteinases involved in bone resorption and osteoclast formation. 1590 14

Tumor cell invasion and metastasis are associated with degradation of components of the extracellular matrix by different proteinases. Among those, papain-like cysteine proteases, such as cathepsin B, seem to play an important role, as they are associated with poor clinical outcome in different cancers. In this study, we tested whether cystatin C, a natural extracellular inhibitor of papain-like cysteine proteases, can inhibit metastasis when overexpressed at the tumor-host interface. Local overexpression of cystatin C in liver and lungs of CD1 nu/nu mice was achieved by gene transfer with a novel adenoviral construct, which also led to the presence of 60 ng/mL of cystatin C in the serum. Three days after gene transfer, these mice were challenged by i.v. inoculation of lacZ-tagged human fibrosarcoma cells (HT1080lacZ-K15), leading to the formation of experimental lung and liver metastases. In this model, formation of experimental metastatic foci correlated with expression of cathepsin B in lungs, whereas there was no correlation with metastasis to the liver. In mice overexpressing cystatin C, the number of lung metastases was significantly reduced by 92%, as compared with mice receiving control adenovirus. The efficacy of extravasation of HT1080lacZ-K15 cells into the liver was not affected, indicating the independence of this process from the activity of cysteine-cathepsins. The present report is the first evidence of successful reduction of metastasis by inhibition of cysteine-cathepsins by cystatin C overexpression in the host microenvironment. Furthermore, organ-specific protease expression during tumor-host cell interactions could affect the success of antiproteolytic intervention against metastasis.
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PMID:Reduction of experimental human fibrosarcoma lung metastasis in mice by adenovirus-mediated cystatin C overexpression in the host. 1620 25


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