Altered spacing of promoter elements due to the dodecamer repeat expansion contributes to reduced expression of the cystatin B gene in EPM1.
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Progressive myoclonus epilepsy of the Unverricht-Lundborg type (EPM1; MIM 254800) is an autosomal recessive disorder characterized by seizures, myoclonus and progression to cerebellar ataxia. EPM1 arises due to mutations in the cystatin B (CSTB) gene which encodes a cysteine proteinase inhibitor. Only a minority of EPM1 alleles carry point mutations, while the majority contain large expansions of the dodecamer CCCCGCCCCGCG repeat which is present at two to three copies in normal individuals. The dodecamer repeat is located in the 5' flanking region of the CSTB gene, presumably in its promoter. The pathological repeat expansion results in a reduction in CSTB mRNA, which may be cell specific. To elucidate the mechanism of this reduction of gene expression, we have studied the putative CSTB promoter in vitro. A 3.8 kb fragment, containing the putative promoter with a 600 bp repeat expansion, showed a 2- to 4-fold reduction in luciferase activity compared with an identical fragment with a normal repeat; this reduction was observed only in certain cell types. Introduction of heterologous DNA fragments of 730 and 1000 bp into the normal promoter, instead of the repeat expansion, showed similarly reduced activity. Terminal deletions of the promoter implicate a putative AP-1 binding site, upstream of the repeat, in CSTB transcription activation. We propose that a novel mechanism of pathogenesis, the altering of the spacing of transcription factor binding sites from each other and/or the transcription initiation site due to repeat expansion, is among the causes of reduction in CSTB expression and thus EPM1. (+info)
Instability of the EPM1 minisatellite.
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Inherited mutations in the cystatin B gene ( CSTB ) are responsible for progressive myoclonus epilepsy type 1 (EPM1; MIM 254800). This autosomal recessive disease is characterized by variable progression to mental retardation, dementia and ataxia. The majority of EPM1 alleles identified to date contain expansions of a dodecamer repeat located upstream of the transcription start site of the CSTB gene. Normal alleles contain two or three copies of the repeat, whereas pathogenic alleles contain >40 repeats. We examined the meiotic stability of pathogenic, expanded EPM1 alleles from 17 EPM1 families by employing a fluorescence-based PCR-based genotyping assay capable of detecting single dodecamer repeat unit differences on an automated DNA sequencer. We followed 74 expanded allele transmissions to 30 affected individuals and 22 carriers. Thirty-five of 74 expanded allele transmissions demonstrated either contraction or expansion of the minisatellite, typically by a single repeat unit. Thus expanded alleles of the EPM1 minisatellite demonstrate a mutation rate of 47%, the highest yet observed for pathogenetic alleles of a human minisatellite. (+info)
Cysteine proteinase inhibitors stefin A, stefin B, and cystatin C in sera from patients with colorectal cancer: relation to prognosis.
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The levels of cysteine proteinase inhibitors stefin A, stefin B, and cystatin C were determined using ELISAs in sera obtained preoperatively from 345 patients with colorectal cancer and in control sera from 125 healthy blood donors. The levels of stefin A and cystatin C were found to be moderately increased in patient sera (1.4-fold and 1.6-fold, respectively; P < 0.0001), whereas the level of stefin B remained statistically unchanged when compared with controls. The medians were 4.3 ng/ml versus 3.2 ng/ml for stefin A, 1.2 ng/ml versus 1.7 ng/ml for stefin B, and 679 ng/ml versus 425 ng/ml for cystatin C. In patient sera, a weak correlation of cystatin C with age (r = 0.34; P < 0.001) and gender (P = 0.01) was found. Stefin A and cystatin C levels were independent of Dukes' stage, whereas stefin B correlated significantly with Dukes' stage, its level being the highest in stage D (P < 0.007). Stefin B and cystatin C correlated with survival, whereas stefin A was not a significant prognostic factor in this study. Using medians as cutoff values, patients with high levels of stefin B and patients with high levels of cystatin C exhibited a significantly higher risk of death than those with low levels of inhibitors (hazard ratio = 1.6; 95% confidence interval, 1.2-2.2; P = 0.002 for stefin B; hazard ratio = 1.3; 95% confidence interval, 1.0-1.8; P = 0.04 for cystatin C). Our results reveal a correlation between high levels of extracellular cysteine proteinase inhibitors and short survival in patients with colorectal cancer, and the data thus support previous studies suggesting a contributing role of protease inhibitors in the progression of cancer. (+info)
Structural basis of the endoproteinase-protein inhibitor interaction.
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Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature. (+info)
Prognostic significance of cysteine proteinases cathepsins B and L and their endogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck.
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Cysteine proteinases cathepsins (Cats) B and L and their endogenous inhibitors stefins (Stefs) A and B are implicated in the processes of local and metastatic tumor spread. They were identified as potential prognosticators in various malignant diseases, particularly in breast cancer. The aim of the present study was to determine the concentrations of Cats B and L and Stefs A and B in the tumor and adjacent normal tissue samples collected from 49 patients (the present group) with squamous cell carcinoma of the head and neck (SCCHN), using quantitative immunosorbent assays (ELISA; KRKA d.d., Novo mesto, Slovenia). Their clinical significance was compared with that from a previous study (the reference group, 45 patients; Budihna et al., Biol. Chem. Hoppe-Seyler, 377: 385-390, 1996). The follow-up of patients from the latter report was updated for this purpose. In the present group, significantly higher concentrations of Cat B (P < 0.0001), Cat L (P < 0.0001) and Stef A (P = 0.006) were found in tumors compared with concentrations in their normal tissue counterparts. Cat concentrations in normal laryngeal tissue were significantly/marginally elevated compared with nonlaryngeal tissue (Cat B, P = 0.02; Cat L, P = 0.06). The tumor concentration of Cat L was found to correlate with pT classification (P = 0.005) and tumor-node-metastasis stage (P = 0.05), whereas the concentrations of Stefs A and B correlated with pN classification (P = 0.007 and P = 0.03, respectively) and tumor-node-metastasis stage of the disease (P = 0.02 and P = 0.03, respectively). There was no statistically significant difference between low and high Cat B or Cat L groups, regarding either disease-free survival or disease-specific survival, using a minimum P approach to determine cutoff concentrations. The risk of disease recurrence and SCCHN-related death was significantly higher in patients with low Stef A (P = 0.0006 and P = 0.0005, respectively) and Stef B (P = 0.0009 and P = 0.0007, respectively) tumors, compared with those with high-Stef A and Stef B tumors. These results remained significant even after Ps were adjusted for a possible bias in the estimated effect on survival. The survival analysis in the reference group also confirmed these findings (Stef A: P = 0.0009 and P = 0.002, respectively; Stef B: P = 0.03 and P = 0.009, respectively). To avoid any possible bias arising from the differences between the laboratories that performed the biochemical analysis, the concentrations of both Stefs in the present group and in the reference group were standardized and coupled together to form a uniform group. In univariate survival analysis, standardized values of Stef A and Stef B correlated inversely with the rate of relapse (P = 0.0000) and mortality rate (P = 0.0000). Multivariate regression analysis showed that the standardized value of Stef A is the strongest independent prognostic factor for both disease-free survival and disease-specific survival. These findings show the specific role of Cats B and L and Stefs A and B in the invasive behavior of SCCHN. Furthermore, Stef A proved to be a reliable prognosticator of the risk of relapse and death in patients with this type of cancer. (+info)
Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.
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The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation. (+info)
Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases.
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Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B. (+info)
Cystatin B-deficient mice have increased expression of apoptosis and glial activation genes.
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Loss-of-function mutations in the cystatin B (Cstb) gene cause a neurological disorder known as Unverricht-Lundborg disease (EPM1) in human patients. Mice that lack Cstb provide a mammalian model for EPM1 by displaying progressive ataxia and myoclonic seizures. We analyzed RNAs from brains of Cstb-deficient mice by using modified differential display, oligonucleotide microarray hybridization and quantitative reverse transcriptase polymerase chain reaction to examine the molecular consequences of the lack of Cstb. We identified seven genes that have consistently increased transcript levels in neurological tissues from the knockout mice. These genes are cathepsin S, C1q B-chain of complement (C1qB), beta2-microglobulin, glial fibrillary acidic protein (Gfap), apolipoprotein D, fibronectin 1 and metallothionein II, which are expected to be involved in increased proteolysis, apoptosis and glial activation. The molecular changes in Cstb-deficient mice are consistent with the pathology found in the mouse model and may provide clues towards the identification of therapeutic points of intervention for EPM1 patients. (+info)