| 1. |
- Åkesson, Per, et al.
(författare)
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Streptococcal inhibitor of complement-mediated lysis (SIC): an anti-inflammatory virulence determinant
- 2010
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Ingår i: Microbiology. - : Microbiology Society. - 1465-2080 .- 1350-0872. ; 156, s. 3660-3668
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Tidskriftsartikel (refereegranskat)abstract
- Since the late 1980s, a worldwide increase of severe Streptococcus pyogenes infections has been associated with strains of the M1 serotype, strains which all secrete the streptococcal inhibitor of complement-mediated lysis (SIC). Previous work has shown that SIC blocks complement-mediated haemolysis, inhibits the activity of antibacterial peptides and has affinity for the human plasma proteins clusterin and histidine-rich glycoprotein; the latter is a member of the cystatin protein family. The present work demonstrates that SIC binds to cystatin C, high-molecular-mass kininogen (HK) and low-molecular-mass kininogen, which are additional members of this protein family. The binding sites in HK are located in the cystatin-like domain D3 and the endothelial cell-binding domain D5. Immobilization of HK to cellular structures plays a central role in activation of the human contact system. SIC was found to inhibit the binding of HK to endothelial cells, and to reduce contact activation as measured by prolonged blood clotting time and impaired release of bradykinin. These results suggest that SIC modifies host defence systems, which may contribute to the virulence of S. pyogenes strains of the M1 serotype.
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| 2. |
- Ahuja, Sat pal, et al.
(författare)
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Glutathione S-transferase µ(GST) modifies activities of proteases and levels of cystatin C secreted by mouse retinal explants
- 2004
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Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 45, s. 352-352
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Konferensbidrag (refereegranskat)abstract
- Purpose: In one form of human autosomal recessive retinitis pigmentosa and in retinal degeneration (rd1) mouse, mutation occurs in the genes encoding ß subunit of rod photoreceptor cGMP phosphodiesterase. Therefore, rd1 mutant mouse is an appropriate model for human inherited retinal degeneration studies. Retinal explants are successfully cultured in serum free chemically defined R16 medium to evaluate effects of various rescue factors and retinal conditioned medium (RCM) for secreted molecules like proteases and their inhibitors. Cysteine protease inhibitor cystatin C has recently been identified in rodent neuroretina and RPE. RCM of explants treated with GST were analyzed for proteases and cystatin C to explain, in part, mode of action of GST in protection of degenerating retina. Methods: Postnatal day 2 (PN2) and PN7 control (wt) and rd1 were cultured with (10 ng / ml GST) and without GST in R16 medium, respectively, for 26 and 21 days in vitro (div). Retinal extracts (RE) and RCM were analyzed by fluorometry using casein green fluorescent labeled with BODIPY–FL (Molecular Probes) for total proteases; Z–Phe–Arg–NMec or Z–Arg–Arg–NMec for cysteine proteases and by ELISA for cystatin C, respectively, for levels and secretion of proteases and cystatin C. The protein content of RE was measured. Results: Protein content (µg) of RE from wt and rd1 retinal extracts respectively increased and decreased with age. Cystatin C (ng/ml RCM) content in wt and rd1 RE increased with age (was always higher in wt) up to PN14 and then decreased but was higher than that at PN2. Progressive secretion of cystatin C by PN2 explants was lower than that by PN7 explants; and that by rd1 PN2 and PN7 explants was initially lower up to in vitro age of PN19 and subsequently it was higher than that by wt explants. Secretion of total cystatin C by PN2 and PN7 wt and rd1 explants was similar and was increased by GST. During initial stage of culture total protease activity ({Delta} F / 100 µl RCM) in RCM of rd1 PN2 and PN7 explants was higher and was decreased in GST treated explants. Conclusions: Cystatin C content and secretion by wt RE is always higher and that of proteases is lower than that of rd1. Treatment with GST increases content of cystatin C and consequently decreases that of proteases especially cysteine proteases.
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| 3. |
- Bjork, Ingemar, et al.
(författare)
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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
- 1996
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 35:33, s. 10720-10726
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Tidskriftsartikel (refereegranskat)abstract
- The single Trp of human cystatin C, Trp-106, is located in the second hairpin loop of the proteinase binding surface. Substitution of this residue by Gly markedly altered the spectroscopic changes accompanying papain binding and reduced the affinity for papain, actinidin, and cathepsins B and H by 300-900-fold. The decrease in affinity indicated that the side chain of Trp-106 contributes a similar free energy, -14 to -17 kJ·mol-1, to the binding to all four cysteine proteinases, corresponding to about 20-30% of the total binding energy. Replacement of Trp-106 by Phe led to a smaller (30-120-fold) decrease in affinity for the four enzymes than Gly substitution. The binding energy of the Phe residue corresponded to 20-45% of that of Trp, showing that a phenyl group can only partly substitute for the indole ring. The reduced affinities of the cystatin C Trp-106 variants for all proteinases studied were due almost exclusively to increased dissociation rate constants. The second hairpin loop thus contributes to the binding primarily by keeping cystatin C anchored to the proteinase once the complex has been formed. This role is partly in contrast to that of the N-terminal region, which increases the affinity of cystatin C for cathepsin B by increasing the association rate constant. Removal of the N-terminal region of the Trp-106Gly variant by proteolytic cleavage substantially weakened the binding to papain and cathepsin B. The resulting affinity indicated that the first hairpin loop (the "QVVAG-region"), which is the only region of the proteinase binding surface remaining intact in the truncated variant, contributes 40-60% of the total free energy of binding of cystatin C to both proteinases.
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| 4. |
- Hall, Anders, et al.
(författare)
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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
- 1995
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 270:10, s. 5115-5121
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Tidskriftsartikel (refereegranskat)abstract
- The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.
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| 5. |
- Huh, C, et al.
(författare)
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Decreased metastatic spread in mice homozygous for a null allele of the cystatin C protease inhibitor gene
- 1999
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Ingår i: Molecular Pathology. - 1366-8714. ; 52:6, s. 332-340
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: Increased or altered activities of cysteine proteases have been implicated in serious human disorders such as cancer, rheumatoid arthritis, sepsis, and osteoporosis. To improve the current knowledge of the regulatory role of a major mammalian cysteine protease inhibitor, cystatin C, in such disease processes, a cystatin C deficient mouse was generated and characterized. METHODS: The mouse cystatin C gene was inactivated by insertion of a bacterial neo gene through homologous recombination in 129/Sv embryonic stem cells. Embryonic stem cell clones were injected into C57BL/6J blastocysts followed by injection of the blastocysts into pseudopregnant female mice. F1 offspring with agouti coat colour after mating of chimaeric males with C57BL/6J females were examined by DNA analysis, and mice carrying the targeted mutation were intercrossed to obtain homozygous cystatin C deficient (CysC-/-) mice. To study the role of cysteine proteases and their inhibitors in metastasis, the spread of B16-F10 melanoma cells in CysC-/- and wild-type mice was compared. Analysis of the formation of remote metastases was carried out by intravenous injection of beta-galactosidase transfected B16-F10 cells and subsequent determination of cancer cell colonies in the lungs. RESULTS: Cystatin C deficient mice were fertile and showed no gross pathological abnormality up to 6 months of age. Compared with wild-type mice, seven times fewer large metastatic colonies were counted by means of a dissecting microscope in CysC-/- mice two weeks after tail vein injection of B16-F10 cells. At all of eight time points from 15 minutes to two weeks after intravenous injection of tumour cells, the CysC-/- mice had significantly fewer lung metastases. The observed differences were smaller when beta-galactosidase transfected cells were used to allow counting of small colonies. Subcutaneous and intracerebral tumour growth was not different in the CysC-/- mice. CONCLUSIONS: Cystatin C concentrations in vivo might influence metastasis in some tissues. The decreased metastatic spread of B16-F10 cells in CysC-/- mice is the result of both reduced seeding and reduced growth of tumour cells in their lungs.
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| 6. |
- Håkansson, Katarina, et al.
(författare)
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Mouse and rat cystatin C: Escherichia coli production, characterization and tissue distribution
- 1996
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Ingår i: Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology. - : Elsevier BV. - 1879-1107 .- 1096-4959. ; 114:3, s. 303-311
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant mouse (Mus musculus) and rat (Rattus norvegicus) cystatin C were produced by expression in Escherichia coli, isolated and functionally characterized. The mouse and rat inhibitors were both fully active in titrations of papain. Determination of equilibrium constants for dissociation (Ki) for their complexes with the target proteinase, cathepsin B, produced values not largely different from that for human cystatin C (Ki 0.07-0.13 nM). Rabbit antisera against mouse and rat cystatin C were produced and used for improved affinity purification of the recombinant inhibitors. Affinity purified immunoglobulins isolated from the antiserum against mouse cystatin C were used for construction of a sensitive enzyme-linked immunosorbent assay. The assay was used to demonstrate a high degree of immunological cross-reactivity between mouse and rat cystatin C and could be used for cystatin C quantification in mouse and rat tissue homogenates. All tissues analyzed contained cystatin C, with a relative content very similar to that of human tissues. For all species, brain tissue contained the highest cystatin C amounts and liver the lowest, whereas kidney, spleen and muscle tissues were intermediate in content. In the mouse, a notable high cystatin C content in parotid gland tissue was observed. The high degree of similarity in distribution pattern and functional properties for mouse, rat and human cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary cystatin C amyloid angiopathy.
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| 7. |
- Pirttila, T J, et al.
(författare)
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Cystatin C modulates neurodegeneration and neurogenesis following status epilepticus in mouse
- 2005
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Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 20:2, s. 241-253
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Tidskriftsartikel (refereegranskat)abstract
- Brain damaging insults cause alterations in neuronal networks that trigger epileptogenesis, and eventually lead to the appearance of spontaneous seizures. The present experiments were designed to study the cellular expression and functions of a cysteine proteinase inhibitor, cystatin C, whose gene expression is previously shown to be upregulated in the rat hippocampus during status epilepticus (SE)induced epileptogenesis. The present data showed that the expression of cystatin C protein increased in the mouse hippocampus 7 days following SE and localized mainly to astrocytes and microglia. Acute neuronal death in the hippocampus at 24 h after SE was reduced in cystatin C-/- mice. Also, the basal level of neurogenesis in the subgranular layer of dentate gyros was decreased in cystatin C-/- mice compared to wildtype littermates. Interestingly, migration of newly born neurons within the granule cell layer was attenuated in cystatin C-/- mice. These data demonstrate that cystatin C has a role in neuronal death and neurogenesis during SE-induced network reorganization.
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| 8. |
- Wasselius, Johan, et al.
(författare)
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Cystatin C in the anterior segment of rat and mouse eyes.
- 2004
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Ingår i: Acta Ophthalmologica Scandinavica. - : Wiley. - 1395-3907 .- 1600-0420. ; 82:1, s. 68-75
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Cystatin C is a mammalian cysteine protease inhibitor. This study describes the localization of cystatin C in the anterior segment of normal rat and mouse eyes. Cysteine proteases play an important role in protein degradation (e.g. of photoreceptor outer segments in the retinal pigment epithelium) and the balance between these proteases and their specific inhibitors is therefore of great interest. Methods: Cells containing cystatin C were identified by immunohistochemistry and quantified by ELISA. Messenger RNA levels were analysed by quantitative real-time polymerase chain reaction. Results: Cystatin C is present at biologically significant levels in the corneal epithelium, endothelium and stromal keratinocytes, lens epithelium, epithelial cells in the ciliary processes, aqueous humour and iris stromal cells. In the rat anterior segment, the highest cystatin C concentrations were found in the ciliary epithelium. Conclusions: Cystatin C is present in several cell types and is probably locally produced. The inhibitor is likely to be an important regulator of cysteine proteases in the retinal pigment epithelium, ciliary epithelium, aqueous humour, lens epithelium and in the corneal endothelium and epithelium.
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| 9. |
- Wasselius, Johan, et al.
(författare)
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Cystatin C uptake in the eye.
- 2005
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Ingår i: Graefe's Archive for Clinical and Experimental Ophthalmology. - : Springer Science and Business Media LLC. - 1435-702X .- 0721-832X. ; 243:6, s. 583-592
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Tidskriftsartikel (refereegranskat)abstract
- Background: As a secreted protein, cystatin C is assumed to play its role in the extracellular compartment, where it can inhibit virtually all cysteine proteases of families C1 ( cathepsin B, L, S) and C13 ( mammalian legumain-related proteases). Since many of its potential target enzymes in the eye reside in intracellular compartments, we sought evidence for a cellular uptake of the inhibitor in ocular tissues. Methods: Fluorescence-labeled human cystatin C was injected intravitreally into normal rat eyes. Ocular tissues were subsequently examined using ELISA, fluorescence microscopy, and immunohistochemistry. Cystatin C uptake was additionally studied in an in vitro retina model. Results: Cystatin C administered intravitreally in vivo is taken up into cells of the corneal endothelium and epithelium, the epithelial cells lining the ciliary processes, and into cells in the neuroretina mostly ganglion cells) and the retinal pigment epithelium. The uptake is demonstrable also in vitro and was, in the neuroretina, found to be a high-affinity system, inhibited by cooling the specimens or by adding the microfilament polymerization inhibitor, cytochalasin D, to the medium. Conclusions: There is an active, temperature-dependent uptake system for cystatin C into several cell types in the cornea, ciliary body, and retina. The cell types that take up cystatin C are generally the same that contain endogenous cystatin C, suggesting that much or all cystatin C seen intracellularly in the normal eye may have been taken up from the surrounding extracellular space. The uptake indicates that the inhibitor may exert biological functions in intracellular compartments. It is also possible that this uptake system may regulate the extracellular levels of cystatin C in the eye.
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| 10. |
- Wasselius, Johan, et al.
(författare)
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Identification and localization of retinal cystatin C
- 2001
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Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 42:8, s. 1901-1906
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. Cystatin C is a mammalian cysteine protease inhibitor, synthesized in various amounts by many kinds of cells and appearing in most body fluids. There are reports that it may be synthesized in the mammalian retina and that a cysteine protease inhibitor may influence the degradation of photoreceptor outer segment proteins. In the current study cystatin C was identified, quantitated, and localized in mouse, rat, and human retinas. METHODS. Enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), DNA sequencing, Western blot analysis, and immunohistochemistry have been used on mouse, rat, and human retinas (pigment epithelium included). RESULTS. Cystatin C is present in high concentrations in the normal adult rat retina, as it is throughout its postnatal development. Its concentration increases to a peak at the time when rat pups open their eyes and then remains at a high level. It is mainly localized to the pigment epithelium, but also to some few neurons of varying types in the inner retina. Cystatin C is similarly expressed in normal mouse and human retinas. CONCLUSIONS. Cystatin C was identified and the localization described in the retinas of rat, mouse, and human using several techniques. Cystatin C is known to efficiently inactivate certain cysteine proteases. One of them, cathepsin S, is present in the retinal pigment epithelium and affects the proteolytic processing by cathepsin D of diurnally shed photoreceptor outer segments. Hypothetically, it appears possible that retinal cystatin C, given its localization to the pigment epithelium and its ability to inhibit cathepsin S, could be involved in the regulation of photoreceptor degradation.
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