UNIPROT:P01034 - FACTA Search

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Query: UNIPROT:P01034 (cystatin C)
3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin-peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.
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PMID:Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi. 880 25

The specific inhibitor of cysteine proteinases, cystatin C, was purified from ram rete testis fluid and the conditioned medium of Sertoli cells. This molecule associated with sheep liver cathepsin L at one of the fastest rates ever described for a proteinase/inhibitor interaction (1.75 +/- 0.20 x 10(8) M-1.s-1). But the association rate constant for the interaction of cathepsin L with alpha 2-macroglobulin, a non-specific inhibitor of proteinases, was also extremely high (8.8 +/- 0.75 x 10(6) M-1.s-1). Cathepsin L complexed with alpha 2-macroglobulin was protected from inhibition by type 2 and type 3 cystatins. The data indicate that cystatin C is the most potent inhibitor of cathepsin L in mammalian male genital tract fluids, whereas alpha 2-macroglobulin may act as a terminal acceptor of this enzyme. These inhibitors could therefore inhibit the activated form of procathepsin L which may appear during the complex process of spermatozoa production and maturation in the testis and epididymis.
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PMID:Interactions between ovine cathepsin L, cystatin C and alpha 2-macroglobulin. Potential role in the genital tract. 906 57

The implantation of the mouse embryo requires the controlled invasion of the uterine stroma by the embryonic trophoblast. This event is dependent, in part, on the secretion of matrix metalloproteinases and serine proteinases for the extracellular degradation of the uterine matrix. Proteinase activity is controlled by stromal decidualization and specific proteinase inhibitors. This work adds to our understanding of implantation and placentation by reporting the expression and function of another class of proteinases/inhibitors closely related to invasive cell behavior. We focused on the cysteine proteinases, cathepsins B and L, and their inhibitor cystatin C. Northern blots showed that trophoblast expressed cathepsin B throughout the invasive period (days 5.5-10.5). Both cathepsin B message and cathepsin L protein were localized to the mature, invasive trophoblast giant cells. Substrate gel electrophoresis showed an increase in giant cell cathepsin activity with enzyme profiles changing at the end of the invasive period. Northern and western blotting showed that cystatin C, the main inhibitor of cathepsins, was a major product of the decidualizing stroma. Message levels first increased in peripheral decidualizing cells, with the protein localizing close to the embryo during implantation (days 5.5-8.5). With the regression of the decidua beginning on day 9.5, a coordinated upregulation of both cathepsin B and cystatin C was observed implying a role for controlled cathepsin expression during apoptosis. E-64, a synthetic inhibitor of cathepsins B and L, was injected into pregnant females at the stage of blastocyst attachment (days 4.5-5.5). High doses resulted in the complete failure of implantation while lower doses resulted in stunted embryos and a reduced decidual reaction. These results suggested that cathepsins B and L are necessary for normal embryo development and uterine decidualization, and that decidua contributes to their control by a coordinated expression of cystatin C within the implantation site.
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PMID:The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation. 931 Mar 36

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.
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PMID:Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro. 943 34

We used site-directed mutagenesis to alter the specificity of human cystatin C, an inhibitor with a broad reactivity against cysteine proteinases. Nine cystatin C variants containing amino acid substitutions in the N-terminal (L9W, V10W, V10F and V10R) and/or the C-terminal (W106G) enzyme-binding regions were designed and produced in Escherichia coli. It was discovered that the inhibition profile of the cystatin could be altered by changing residues 9 and 10, which are proposed to bind in the S3 and S2 substrate-binding pockets respectively of the enzymes. All of the variants with substitutions in the N-terminal segment displayed decreased binding to cathepsins B and H, indicating that the S3 and S2 pockets of these enzymes cannot easily accommodate large aromatic residues. The introduction of a charged residue into S2 (variant V10R) created a more specific inhibitor to distinguish cathepsin B from cathepsin H. Cathepsin L showed a preference for larger aromatic residues in S2. In contrast, cathepsin S preferred phenylalanine to valine in S2, but bound less tightly to the V10W cystatin variant. The latter variant proved to be valuable for discriminating between cathepsin L and cathepsin S (Ki 2.4 and 190 pM respectively). The equilibrium dissociation constant of the complex between cathepsin L and variant L9W/W106G showed little difference in affinity from that of the cathepsin L complex with the singly substituted W106G variant. In contrast, the L9W/W106G variant displayed increased specificity for cathepsin S with a Ki of 10 pM. Our results clearly indicate differences in the specificity of interaction between the N-terminal region of cystatin C and cathepsins B, H, L and S, and that, although cystatin C has evolved to be a good inhibitor of all of the mammalian cysteine proteinases, more specific inhibitors of the individual enzymes can be engineered.
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PMID:Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases. 948 Aug 98

Human cystatins C and D share almost identical primary structures of two out of the three segments proposed to be of importance for enzyme interactions but have markedly different profiles for inhibition of the target cysteine peptidases, cathepsins B, H, L, and S. To investigate if the N-terminal binding regions of the inhibitors are responsible for the different inhibition profiles, and thereby confer biological selectivity, two hybrid cystatins were produced in Escherichia coli expression systems. In one hybrid, the N-terminal segment of cystatin C was placed on the framework of cystatin D, and the second was engineered with the N-terminal segment of cystatin D on the cystatin C scaffold. Truncated cystatin C and D variants, devoid of their N-terminal segments, were obtained by incubation with glycyl endopeptidase and isolated, in a second approach to assess the importance of the N-terminal binding regions for cystatin function and specificity. The affinities of the four cystatin variants for cathepsins B, H, L, and S were measured. By comparison with corresponding results for wild-type cystatins C and D, it was concluded (1) that both the N-terminal and framework part of the molecules significantly contribute to the observed differences in inhibitory activities of cystatins C and D and (2) that the N-terminal segment of cystatin C increases the inhibitory activity of cystatin D against cathepsin S and cathepsin L but results in decreased activity against cathepsin H. These differences in specificity were explained by the residues interacting with the S2 subsite of peptidases (Val- and Ala-10 in cystatin C and D, respectively). Also, removal of the N-terminal segment results in total loss of enzyme affinity for cystatin D but not for cystatin C. Therefore, structural differences in the framework parts, as well as in the N-terminal segments, are critical for both inhibitory specificity and potency. Homology modeling was used to identify residues likely responsible for the generally reduced inhibitory potency of cystatin D.
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PMID:Structural basis for different inhibitory specificities of human cystatins C and D. 952 28

The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.
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PMID:The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L. 958 70

Purified human central nervous system myelin contains an endogenous cysteine protease which degrades the 100-kDa myelin-associated glycoprotein into a slightly smaller 90-kDa derivative called dMAG, and which has been implicated in demyelinating diseases. The native proteolytic site in human MAG was determined in order to characterize this cysteine protease in humans further. This was accomplished by identifying the carboxy-terminus of purified dMAG. The results of these experiments, in conjunction with peptidolysis assays of myelin, demonstrated that the enzyme which proteolyses MAG is extracellular and has cathepsin L-like specificity. Furthermore, it was shown that this cathepsin L-like activity potentially was regulated by the endogenous extracellular inhibitor cystatin C.
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PMID:Characterization of myelin-associated glycoprotein (MAG) proteolysis in the human central nervous system. 969 Jul 44

A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nM, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.
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PMID:Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. 973 83

This study examined the role of cysteine proteinases and their inhibitor in the development of emphysema in comparison with neutrophil elastase (NE) complexed with alpha1-protease inhibitor (NE-alpha1-PI), which was previously demonstrated to be increased in bronchoalveolar lavage (BAL) fluid from subjects with subclinical emphysema. Eight nonsmokers and 31 current smokers with (n=17) and without (n=14) emphysema, as evidenced by lung computed tomographic scans, were studied. The concentrations of immunologically detected cathepsin L and cystatin C, but not cathepsin B, were significantly increased in BAL fluid from the smokers with emphysema compared with those without emphysema, although the activity of cathepsin L, measured using a synthetic substrate and cathepsin L, released from cultured alveolar macrophages at 24 h, did not show any significant difference between the two groups. When comparison was made only for the subjects aged <60 yrs, the difference between the two groups disappeared for cathepsin L, but remained for NE-alpha1-PI. There was no significant correlation between the level of cathepsin L and that of NE-alpha1-PI in BAL fluid from the subjects with emphysema. In conclusion, increased levels of cathepsin L and cystatin C were demonstrated in bronchoalveolar lavage fluid from subjects with subclinical emphysema. However, the roles of cathepsin L and neutrophil elastase in the development of emphysema may vary between subjects and between the young and the old.
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PMID:Cysteine proteinases and cystatin C in bronchoalveolar lavage fluid from subjects with subclinical emphysema. 986 93

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