Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.3.4.6 (urease)
 7,490 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the molecular basis of familial Type I hyperlipoproteinemia in two brothers of Turkish descent who had normal plasma apolipoprotein C-II levels and undetectable plasma post-heparin lipoprotein lipase (LPL) activity. We cloned the cDNAs of LPL mRNA from adipose tissue biopsies obtained from these individuals by the polymerase chain reaction and directional cloning into M13 vectors. Direct sequencing of pools of greater than 2000 cDNA clones indicates that their LPL mRNA contains two mutations: a missense mutation changing codon 156 from GAU to GGU predicting an Asp156----Gly substitution and a nonsense mutation changing the codon for Ser447 from UCA to UGA, a stop codon, predicting a truncated LPL protein that contains 446 instead of 448 amino acid residues. Both patients were homozygous for both mutations. Analysis of genomic DNAs of the patients and their family members by the polymerase chain reaction, restriction enzyme digestion (the GAT----GGT mutation abolishes a TaqI restriction site), and allele-specific oligonucleotide hybridization confirms that the patients were homozygous for these mutations at the chromosomal level, and the clinically unaffected parents and sibling were true obligate heterozygotes for both mutations. In order to examine the functional significance of the mutations in this family, we expressed wild type and mutant LPLs in vitro using a eukaryotic expression vector. Five types of LPL proteins were produced in COS cells by transient transfection: (i) wild type LPL, (ii) Asp156----Gly mutant, (iii) Ser447----Ter mutant, (iv) Gly448----Ter mutant, and (v) Asp156----Gly/Ser447----Ter double mutant. Both LPL immunoreactive mass and enzyme activity were determined in the culture media and intracellularly. Immunoreactive LPLs were produced in all cases. The mutant LPLs, Asp156----Gly and Asp156----Gly/Ser447----Ter, were devoid of enzyme activity, indicating that the Asp156----Gly mutation is the underlying defect for the LPL deficiency in the two patients. The two mutant LPLs missing a single residue (Gly448) or a dipeptide (Ser447-Gly448) from its carboxyl terminus had normal enzyme activity. Thus, despite its conservation among all mammalian LPLs examined to date, the carboxyl terminus of LPL is not essential for enzyme activity. We further screened 224 unrelated normal Caucasians for the Ser447----Ter mutation and found 36 individuals who were heterozygous and one individual who was homozygous for this mutation, indicating that it is a sequence polymorphism of no functional significance. Human LPL shows high homology to hepatic triglyceride lipase and pancreatic lipase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Catalytic triad residue mutation (Asp156----Gly) causing familial lipoprotein lipase deficiency. Co-inheritance with a nonsense mutation (Ser447----Ter) in a Turkish family. 190 78

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.
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PMID:Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis. 1092 73

We have studied the expression of the green fluorescent protein (GFP) gene to gain more understanding of the effects of additional nucleotide triplets (codons) downstream from the initiation codon on the translation of the GFP mRNA in CHO and Cos1 cells. A leader sequence of six consecutive identical codons (GUG, CUC, AGU or UCA) was introduced into a humanized GFP (hm gfp) gene downstream from the AUG to produce four GFP gene variants. Northern blot and RT-PCR analysis indicated that mRNA transcription from the GFP gene was not significantly affected by any of the additional sequences. However, immunoblotting and FACS analysis revealed that AGU and UCA GFP variants produced GFP at a mean level per cell 3.5-fold higher than the other two GFP variants and the hm gfp gene. [35S]-Methionine labeling and immunoprecipitation demonstrate that GFP synthesis was very active in UCA variant transfected-cells, but not in GUG variant and hm gfp transfected-cells. Moreover, proteasome inhibitor MG-132 treatment indicated that the GFPs encoded by each of the GFP variants and the hm gfp were equally stable, and this together with the comparable mRNA levels observed for each construct suggested that the different steady-state GFP concentrations observed reflected different translation efficiencies of the various GFP genes. In addition, the CUC GFP variant, when transiently transfected into CHO or COS-1 cells, did not produce any GFP expressing cells (fully green cells), and the GUG variant produced GFP expressing cells less than 10%, while AGU and UCA GFP variants up to 30-35% in a time course study from 8 to 36 h posttransfection. Analysis of the potential secondary structure of the GFP variant mRNAs especially in the translation initiation region suggested that the secondary structure of the GFP mRNAs was unlikely to explain the different translation efficiencies of the GFP variants. The present findings indicate that a change of the initiation context of the GFP gene by addition of extra coding sequence can alter the translation efficiency of GFP mRNA, providing a means of more efficient expression of GFP in eukaryotic cells.
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PMID:Effects of additional sequences directly downstream from the AUG on the expression of GFP gene. 1465 38

Host cell pathology induced by nuclear targeting of bacterial proteins has recently been identified as a pathogenic mechanism of bacteria. However, very few bacterial proteins were identified to target the nuclei of host cells. This study was designed to screen nuclear targeting proteins with nuclear localization signals (NLSs) in Helicobacter pylori using a combination of bioinformatic analysis and the Gateway recombinational cloning system. Forty-nine functional or hypothetical proteins were predicted to carry the putative NLSs among 1570 open reading frames (ORFs) of H. pylori 26695. Entire sets of 49 H. pylori ORFs were cloned for the generation of green fluorescent protein-tagged proteins using the Gateway recombinational cloning system. Twenty-six H. pylori proteins with the putative NLSs were found to target in the nuclei of COS-7 cells, whereas 23 were localized in the cytoplasm of host cells. Deletion of NLS sequences from four selected nuclear targeting proteins, urease subunit A, Omp18, secreted protein involved in flagellar motility, and response regulator, resulted in cytoplasmic localization of these mutant proteins. In conclusion, a combination of bioinformatic analysis and the Gateway cloning system was shown to be a useful tool for large-scale screening of nuclear targeting proteins with NLSs in H. pylori, which can be used to better understand the H. pylori-directed host cell pathology.
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PMID:Prediction and screening of nuclear targeting proteins with nuclear localization signals in Helicobacter pylori. 2307 23

Nuclear targeting of bacterial proteins and their pathological effects on host cells are an emerging pathogenic mechanism in bacteria. We have previously reported that urease subunit A (UreA) of Helicobacter pylori targets the nuclei of COS-7 cells through nuclear localization signals (NLSs). This study further investigated whether UreA of H. pylori targets the nuclei of gastric epithelial cells and then induces molecular and cellular changes in the host cells. H. pylori 26695 strain produced and secreted outer membrane vesicles (OMVs). UreA was translocated into gastric epithelial AGS cells through outer membrane vesicles (OMVs) and then targeted the nuclei of AGS cells. Nuclear targeting of rUreA did not induce host cell death, but resulted in morphological changes, such as cellular elongation, in AGS cells. In contrast, AGS cells treated with rUreA?NLS proteins did not show this morphological change. Next generation sequencing revealed that nuclear targeting of UreA differentially regulated 102 morphogenesis- related genes, of which 67 and 35 were up-regulated and down-regulated, respectively. Our results suggest that nuclear targeting of H. pylori UreA induces both molecular and cellular changes in gastric epithelial cells.
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PMID:Morphological changes in human gastric epithelial cells induced by nuclear targeting of Helicobacter pylori urease subunit A. 2602 73