| 51. |
- Balbin, Milagros, et al.
(författare)
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Structural and functional characterization of two allelic variants of human cystatin D sharing a characteristic inhibition spectrum against mammalian cysteine proteinases
- 1994
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 269:37, s. 23156-23162
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin D is a novel member of the cystatin superfamily of cysteine proteinase inhibitors present in saliva and tears. Two alleles of the cystatin D gene (CST5), encoding protein variants with either Cys or Arg as residue 26 in their 122-residue polypeptide chains, are present in the population. Expression of the two alleles was investigated by immunochemical analyses of the secreted cystatin D in saliva from individuals homozygous for each of the two alleles, with results demonstrating that both are expressed at similar levels. The inhibitory characteristics of the two cystatin D variants were studied, by determination of dissociation equilibrium constants (Ki) for their complexes with papain and with the mammalian cysteine proteinases, cathepsins B, H, L, and S. The results demonstrate that 1) cystatin D has a characteristic inhibition profile since it does not inhibit cathepsin B (Ki > 1 microM), and when compared to cystatin C and all other known cystatins it is a much poorer inhibitor of cathepsin L (mean Ki 25 nM) but binds cathepsin H and S relatively tightly (mean Ki values of 8.5 and 0.24 nM, respectively); and 2) the inhibitory activities of the two cystatin D variants are not significantly different, demonstrating that the presence of an extra cysteine residue in the cystatin D molecule affects neither the stability nor the functional activity of the inhibitor, thus explaining the widespread distribution of the Cys26-cystatin D encoding allele in the population. The inhibitory properties displayed by cystatin D suggest that it has a function in saliva as inhibitor of either endogenous or exogenous enzymes with cathepsin S- or H-like properties.
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| 52. |
- Bauer, S, et al.
(författare)
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Phenotype in a Swedish family with X-linked retinitis pigmentosa caused by a novel splice defect in the RPGR gene
- 1998
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Ingår i: Investigative Ophthalmology & Visual Science. - 1552-5783. ; 39:12, s. 2470-2474
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To assess the clinical phenotype in a Swedish family with X- linked retinitis pigmentosa (XLRP) resulting from a novel splice defect in the RPGR gene. METHODS: RPGR mutation analysis was performed in one family with XLRP, and several individuals from the family were examined clinically. RESULTS: The causative mutation in the family was demonstrated to be a single base-pair change at the splice donor site in intron 7 that resulted in skipping of the complete exon 7 in the mature RPGR transcript. The aberrant mRNA is predicted to produce an RPGR protein with an in-frame deletion of 53 amino acids, corresponding to an RCC1-homology repeat. Clinical studies that included ophthalmological examination and full-field electroretinography showed that this splice mutation resulted in a comparatively less severe form of RP. CONCLUSIONS: Correlation of a causative RPGR genotype with clinical findings in hemizygotes and carrier heterozygotes is an important step toward predictive diagnosis and should assist in the development of gene-based therapies in the future.
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| 53. |
- Berti, Paul J, et al.
(författare)
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Affinity purification and elimination of methionine oxidation in recombinant human cystatin C
- 1997
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 11:1, s. 111-118
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant human cystatin C (cC), a cysteine protease inhibitor, contained methionine sulfoxide [Met(O)] residues when expressed in Escherichia coli under aerobic conditions or upon allowing osmotic shock solutions from anaerobically grown cultures to warm to room temperature. Oxidation occurred in the periplasmic space or intracellularly during aerobic expression. Both Met14 and Met41 were subject to oxidation, as determined by NMR spectroscopy and mass spectrometry. Oxidation of Met110 was not observed. Growth under anaerobic conditions and modified purification procedures prevented oxidation. Through the use of a new form of affinity purification, cC was purified to > 99% in one step on E-64-papain-Sepharose (E-64 is 1-[N-[(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]amino]-4-g uanidinobutane), with elution with sodium trichloroacetate. The dissociation equilibrium constants (Kd) for the interaction of unoxidized cC, (Met(O)14)cC, and (Met(O)41)cC with S-(N-ethylsuccinimidyl)papain were experimentally identical: 1.8 (+/-0.2) x 10(-7), 1.6 (+/-0.2) x 10(-7), and 1.4 (+/-0.5) x 10(-7) M, respectively. This implies that the structure of the protease-binding region of mono-oxidized cC's was unchanged. The NMR observation of small, localized conformational changes was consistent with this. (Met(O)14)cC and (Met(O)14,Met(O)41)cC eluted earlier upon analytical affinity chromatography.
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| 54. |
- Bjarnadottir, M, et al.
(författare)
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Intracellular accumulation of the amyloidogenic L68Q variant of cystatin C in NIH/3T3 cells
- 1998
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Ingår i: Molecular Pathology. - 1366-8714. ; 51:6, s. 317-326
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Tidskriftsartikel (refereegranskat)abstract
- AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-- >Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.
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| 55. |
- Bjork, I, et al.
(författare)
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Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes
- 1994
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Ingår i: Biochemical Journal. - 0264-6021. ; 299, s. 219-225
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Tidskriftsartikel (refereegranskat)abstract
- The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.
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| 56. |
- 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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| 57. |
- Björck, Lars, et al.
(författare)
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Bacterial growth inhibited by a synthetic inhibitor based upon the structure of a human proteinase inhibitor
- 1989
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 337:6205, s. 385-386
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Tidskriftsartikel (refereegranskat)abstract
- Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins1 and in the processing of prohormones and proenzymes2,3, but also in the penetration of normal human tissue by malignant cells4 and possibly microorganisms5, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids6. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C7 and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracy-cline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.
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| 58. |
- Björk, Ingemar, et al.
(författare)
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Probing the functional role of the N-terminal region of cystatins by equilibrium and kinetic studies of the binding of Gly-11 variants of recombinant human cystatin C to target proteinases
- 1995
-
Ingår i: Biochemical Journal. - 0264-6021. ; 306:2, s. 513-518
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between cystatin C variants, in which the evolutionarily conserved Gly-11 residue was substituted by Ala, Glu or Trp, and the cysteine proteinases, papain, ficin, actinidin and cathepsin B, was characterized. The substitutions reduced the affinity of binding in a manner consistent with the Gly residue of the wild-type inhibitor, allowing the N-terminal region to adopt a conformation that was optimal for interaction with target proteinases. Replacement of Gly-11 by Ala resulted in only a 5- to 100-fold reduction in binding affinity. Comparison with the affinities of wild-type cystatin C lacking the N-terminal region indicated that even this small structural change affects the conformation of this region sufficiently to largely abolish its interaction with the weakly binding proteinases, actinidin and cathepsin B. However, the substitution allows interactions of appreciable strength between the N-terminal region and the tightly binding enzymes, papain or ficin. Replacement of Gly-11 with the larger Glu and Trp residues substantially decreased the affinity of binding to all enzymes, from 10(3)- to 10(5)-fold. These substitutions further affect the conformation of the N-terminal region, so that interactions of this region with papain and ficin are also essentially eliminated. The decreased affinities of the three cystatin C variants for papain, ficin and actinidin were due exclusively to increased dissociation rate constants. In contrast, the decreased affinity between cathepsin B and the Ala-11 variant, the only one for which rate constants could be determined with this enzyme, was due almost entirely to a decreased association rate constant. This behaviour is analogous to that observed for forms of cystatin C lacking the N-terminal region and supports the conclusion that the mode of interaction of this region with target proteinases varies with the enzyme as a result of structural differences in the active-site region of the latter.
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| 59. |
- Bokarewa, Maria, 1963, et al.
(författare)
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Cystatin C binds serum amyloid A, downregulating its cytokine-generating properties
- 2007
-
Ingår i: Journal of Rheumatology. - 0315-162X. ; 34:6, s. 1293-1301
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Tidskriftsartikel (refereegranskat)abstract
- Objective. To assess the interaction between cystatin C (CysC) and serum amyloid A protein (SAA). Methods. Levels of CysC and SAA and antibodies against these proteins were assessed in the paired blood and synovial fluid (SF) samples of 90 patients with rheumatoid arthritis (RA). Age and sex matched individuals having normal iohexol clearance (n = 90) and SF following joint trauma (n = 40) were used as controls. In vitro experiments included assessment of interaction between CysC and SAA by ELISA and the influence of CysC on SAA functions. Results. A pilot screening for cystatins C, E, and F in blood and SF of patients with RA found CysC to be by far the predominant extracellular cystatin. Circulating CysC levels were significantly lower in patients with RA compared to the matched controls (0.81 +/- 0.03 vs 1.01 +/- 0.03 mg/l; p = 0.05). These low CysC levels could not be explained by the presence of anti-CysC antibodies in patients with RA. In contrast, concentrations of CysC that accumulated in the inflamed SF were significantly greater in patients with erosive RA (1.66 +/- 0.08 mg/l) compared to nonerosive RA (1.36 +/- 0.06 mg/l; p = 0.003) and controls (1.18 +/- 0.03 mg/l; p = 0.043). In vitro studies showed direct binding of CysC to SAA. CysC/SAA binding impaired proinflammatory effects of SAA, reducing its ability to trigger expression of proinflammatory cytokines. Conclusion. Our study shows a relative deficiency of circulating CysC during systemic inflammation in RA. Physical interaction between CysC and the acute-phase protein SAA (1) provides an explanation for CysC deficiency; and (2) suggests that CysC is regulating inflammatory responses. We hypothesize that decreased systemic CysC levels predispose to accelerated atherosclerosis and development of amyloidosis in patients with RA.
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| 60. |
- Brage, M, et al.
(författare)
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Different cysteine proteinases involved in bone resorption and osteoclast formation.
- 2005
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Ingår i: Calcified tissue international. - : Springer Science and Business Media LLC. - 0171-967X .- 1432-0827. ; 76:6, s. 439-47
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Tidskriftsartikel (refereegranskat)abstract
- 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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