| 1. |
- Ekström, Ulf, et al.
(författare)
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Expression of an LDL receptor allele with two different mutations (E256K and I402T)
- 2000
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Ingår i: Molecular Pathology. - 1366-8714. ; 53:1, s. 31-36
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Tidskriftsartikel (refereegranskat)abstract
- AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effects of the mutations on LDL receptor function. RESULTS: The two mutations were located on the same allele of the LDL receptor gene. All mutant constructs resulted in the production of a detectable protein in CHO cells. The cells expressing only the I402T mutation, or the combination of I402T and E256K mutations, were seriously affected in mediating uptake and degradation of LDL. Contrary to initial predictions, the cells expressing only the E256K mutation showed essentially the same binding, uptake, and degradation of 125I labelled LDL as cells transfected with normal LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation, and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients studied. CONCLUSIONS: Despite the information available on the structure-function relations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitution of E256K alone is a rare sequence variation, without a detectable phenotype modulating effect.
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| 2. |
- Johansson, L, et al.
(författare)
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A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro
- 2000
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Ingår i: Bone. - 1873-2763. ; 26:5, s. 451-459
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Tidskriftsartikel (refereegranskat)abstract
- Human cystatin C is a cysteine proteinase inhibitor belonging to the cystatin superfamily, which previously has been shown to inhibit bone resorption in bone organ culture. The aminoterminal segment, Arg8-Leu9-Val10-Gly11 (RLVG), of the single polypeptide chain of cystatin C constitutes an essential part of its inhibitory center. In the present study, the effect of benzyloxycarbonyl-Arg8-Leu9-Val10-Gly11-diazomethane (Z-RLVG-CHN2) on bone resorption in vitro was compared with the effects of cystatin C and calcitonin. Bone resorption was assessed by the release of 45Ca and 3H from mouse calvarial bones prelabeled with [45Ca]CaCl2 and [3H]-proline, respectively. Z-RLVG-CHN2 concentration-dependently inhibited the release of 45Ca and 3H in bones stimulated by parathyroid hormone (PTH), with half-maximal inhibition obtained at 1 μmol/L. The inhibitory actions of Z-RLVG-CHN2 and cystatin C were persistent, whereas action induced initially by calcitonin was lost with time. The inhibition caused by Z-RLVG-CHN2 and cystatin C on PTH-stimulated 45Ca release was observed after 6 h, whereas inhibition by calcitonin was seen already after 2 h. In contrast, the inhibitory effects of Z-RLVG-CHN2 and cystatin C, as well as that of calcitonin, on 3H release was seen already after 2 h. Z-RLVG-CHN2, in which the reactive carboxyterminal diazomethane was substituted by nonreactive groups [−OH, −NH2, or −N(CH3)2], resulted in peptidyl derivatives, which, in contrast to Z-RLVG-CHN2 and cystatin C, inhibited neither cysteine proteinases nor bone resorption. In contrast to wild-type cystatin C, recombinant human cystatin C with Gly substitutions for residues Arg8, Leu9, Val10, and Trp106, and with low or nonexistent affinity for cysteine proteinases, did not display any inhibitory effect on bone resorption. These data strongly indicate that Z-RLVG-CHN2 inhibits bone resorption in vitro by a mechanism that seems primarily to be due to an inhibition of bone matrix degradation via cysteine proteinases. The data also corroborate the hypothesis that cystatin C inhibits bone resorption by virtue of its cysteine proteinase inhibitory capacity.
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| 3. |
- John, R J, et al.
(författare)
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Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor
- 2000
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Ingår i: Clinical and Experimental Allergy. - : Wiley. - 1365-2222 .- 0954-7894. ; 30:6, s. 784-793
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. OBJECTIVE: To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. METHODS: The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. RESULTS: PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. CONCLUSION: PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.
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| 4. |
- Pavlova, A, et al.
(författare)
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Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration by release of loop anchoring through mutation of His110
- 2000
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Ingår i: FEBS Letters. - 1873-3468. ; 487:2, s. 156-156
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Tidskriftsartikel (refereegranskat)abstract
- Cystatins A and C were both shown to inhibit cathepsin B by a two-step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one-step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.
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