Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.
J Mol Biol 1992 Oct 20
PMID:Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes. 143 93

Comparison of the mutagenicity of nine isomeric benzo(a)pyrenyl [B(a)P] phenols conjugated with either sulfate or glucuronide was carried out using strain Salmonella typhimurium TA98. Of the nine conjugates tested, only B(a)P-1-sulfate was mutagenic. Accordingly, the mutagenicity of B(a)P-1-sulfate was compared with that of B(a)P and 1-hydroxybenzo(a)pyrene [B(a)P-1-OH] in the presence and absence of rat lung S9 and Aroclor-induced liver S9 with and without an NADPH-generating system. B(a)P-1-sulfate was slightly mutagenic, whereas B(a)P and the 1-hydroxy derivative were nonmutagenic when S9 fractions and NADPH were omitted. Addition of induced liver S9 with NADPH caused mutagenicity with B(a) -1-OH greater than B(a)P greater than B(a)P-1-sulfate. B(a)P-1-sulfate was the only mutagenic species when lung S9 was added. This mutagenicity did not require NADPH. Sodium sulfite, an inhibitor of arylsulfatase, decreased the mutagenicity of B(a)P-1-sulfate. These data suggest that a unique mutagenic species is generated from B(a)P-1-sulfate via arylsulfatase in rat lung.
Environ Mol Mutagen 1992
PMID:Mutagenicity of benzo(a)pyrenyl-1-sulfate in the Ames test. 157 48

The Ca2+ uptake activity of rat cardiac sacroplasmic reticulum (CSR) in ventricular homogenates is highly unstable, and this instability probably accounts for the low specific activity of Ca2+ uptake in previously reported fractions of isolated rat CSR. The instability was observed at either 0 degrees or 37 degrees, but the Ca2+ uptake activity was relatively stable at 25 degrees. The decay of Ca2+ uptake activity at 0 degrees could not be prevented by either PMSF or leupeptin, but dithiothreitol exerted some protective effects. Sodium metabisulfite prevented decay of the Ca2+ uptake activity of homogenates kept on ice but not of homogenates kept at 37 degrees. We also found that release of the CSR from the cellular debris required homogenization in high KCl. This distinguishes rat CSR from canine CSR. Isolated CSR was produced by a combination of differential centrifugation and discontinuous sucrous gradient centrifugation. The average rate of the sustained oxalate-supported calcium uptake in the resulting CSR fraction was 0.36 mumol/min-mg in the absence of CSR calcium channel blockers and 0.67 mumol/min/mg in the presence of 10 microM ruthenium red. Thus, this preparation has the advantage of containing both the releasing and non-releasing fractions of the CSR. The Ca2(+)-ATPase rates averaged 1.07 mumol/min/mg and 0.88 mumol/min-mg in the absence and presence of ruthenium red, respectively. Although these rates are higher than previously reported rates, this CSR preparation should still be considered a 'crude' preparation. A major distinction between the rat CSR and dog CSR was the lower content of Ca2(+)-ATPase in rat CSR, as judged by SDS-PAGE. Preparations of CSR isolated by this method may be useful in evaluating alterations in CSR function.
Mol Cell Biochem 1990 Dec 03
PMID:Stabilization of rat cardiac sacroplasmic reticulum Ca2+ uptake activity and isolation of vesicles with improved calcium uptake activity. 214 64

Bisulfite mutagenesis techniques have been used to introduce single-point mutations within a region of the Rous sarcoma virus src gene defined by a BglI restriction endonuclease cleavage site. The mutants of Rous sarcoma virus that are produced by these techniques encode src proteins which contain single amino acid changes within a highly conserved amino acid sequence encompassing residues 430 to 433. DNA from the mutants CHpm26 ( Ala430 to Val), CHpm9 ( Pro431 to Ser), CHpm6 ( Glu432 to Lys), and CHpm65 ( Ala433 to Thr) each failed to transform chicken cells upon transfection, whereas DNA from CHpm59 (a third base alteration in the codon for Glu432 ) readily transformed chicken cells. Analysis of immune complexes containing the altered src proteins indicates that these proteins have decreased tyrosine protein kinase activity in vitro. In vivo labeling of cells infected with the mutant virus revealed diminished levels of the tyrosine-phosphorylated 34,000-molecular-weight protein. These data indicate that mutations within the sequence Ala430 - Pro431 - Glu432 - Ala433 lead to alterations in pp60src-specific tyrosine protein kinase activity and a concomitant loss of transforming potential of the mutant virus.
Mol Cell Biol 1984 May
PMID:Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity. 632 73

To identify amino acid residues of the D2 protein that are critical fo r functional photosystem II (PS II), sodium bisulfite was utilized for in vitro random mutagenesis of the psbDI gene from Synechocystis sp. PCC 6803. Sodium bisulfite reacts specifically with cytosine in single-stranded regions of DNA and does not attack double-stranded DNA. Using a hybrid plasmid that was single-stranded in the region to be mutagenized and that was double-stranded elsewhere, mutations were targeted to a specific psbDI region coding for the lumenal A-B loop of the D2 protein. Several mutants were isolated with a total of 15 different amino acid changes in the loop. The majority of these mutations did not result in a loss of photoautotrophic growth or in significantly altered PS II function. However, mutation of Glu-69 to Lys, Ser-79 to Phe, and Ser-88 to Phe were found to influence photosystem II activity; the importance of the latter two residues for proper PS II function was unexpected. Cells carrying the double mutation S79F/S88F in D2 did not grow photoautotrophically and had no functionally active PS II centers. The single mutant S79F was also incapable of photoautotrophic growth, but displayed reasonably stable oxygen evolution, while PS II function in the single mutant S88F appeared to be close to normal. Because of the more pronounced phenotype of the S79F/S88F strain as compared to the single mutants, both Ser residues appear to affect stable assembly and function of the PS II complex. The mechanism by which the S79F mutant loses photoautotrophic growth remains to be established. However, these results show the potential of targeted random mutagenesis to identify functionally important residues in selected regions of proteins.
Plant Mol Biol 1996 Jan
PMID:Random chemical mutagenesis of a specific psbDI region coding for a lumenal loop of the D2 protein of photosystem II in Synechocystis sp. PCC 6803. 861 49

O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.
Mol Cell Biol 1997 Sep
PMID:Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene. 927 36

Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.
Mol Cell Biol 2000 Feb
PMID:Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression. 1062 41

The introduction of intracytoplasmic sperm injection (ICSI) has raised concern about safety in terms of a possible increase in the incidence of major congenital malformations, chromosomal aberrations or developmental problems. The possible influence of genetic imprinting on an ICSI procedure has not yet been investigated. We therefore studied the DNA-methylation status at a defined region in chromosome 15q11-q13 in 92 children born after an ICSI procedure. Imprinting defects in this region are associated with neurogenetic disorders, e.g. Angelman syndrome (AS) and Prader-Willi syndrome (PWS). Blood samples were taken directly after birth and stored at -80 degrees C. Genomic DNA purification was performed from 3-7 ml EDTA-blood. Sodium bisulphite treatment was carried out in order to distinguish methylated from unmethylated DNA by transferring the unmethylated nucleic acid cytosine into uracil and leaving the methylated cytosine unchanged. Subsequently, a methylation-specific polymerase chain reaction (M-PCR) was performed. In all 92 children (83 from ICSI with ejaculated spermatozoa and nine from ICSI with non-ejaculated spermatozoa), a regular DNA-methylation pattern was found in the PWS/AS region. In none of the children were clinical symptoms of PWS or AS present. In conclusion, the results of this study do not indicate a higher risk of DNA-methylation defects in children born after ICSI.
Mol Hum Reprod 2000 Nov
PMID:Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. 1104 69

Chromatin immunoprecipitation assays were employed to assess the kinetics of transcription factor assembly and histone modifications that occur during gamma interferon (IFN-gamma) induction of CIITA gene expression. CIITA is the master regulator of major histocompatibility complex class II transcription. Promoter IV (PIV), the major IFN-gamma responsive promoter for CIITA expression, requires both STAT1 and IFN regulatory factor 1 (IRF-1) for induction by IFN-gamma. STAT1 binding to PIV was detected first and was accompanied by a modest acetylation of histones H3 and H4 that were associated with the region. Despite these changes, which occurred within 30 min of IFN-gamma treatment, CIITA mRNA was not detected until IRF-1 protein was synthesized and bound to its site, a process that required >120 min. In contrast to these events, fetal trophoblast-like cell lines, which are refractory to CIITA induction by IFN-gamma, failed to assemble the above factors or modify their chromatin, suggesting that accessibility to the promoter is blocked. Bisulfite sequencing of PIV showed strong hypermethylation of PIV, providing a link between methylation, chromatin structure, and factor binding. Together, this analysis provides a kinetic view of the activation of the CIITA gene in response to IFN-gamma and shows that regulatory factor assembly, chromatin modification, and gene expression proceed in discrete steps.
Mol Cell Biol 2002 Jul
PMID:Kinetics of a gamma interferon response: expression and assembly of CIITA promoter IV and inhibition by methylation. 1205 85

Several studies have shown that E-cadherin expression is lost during malignant transformation. We hypothesized that CpG methylation in the promoter region may inactivate the expression of the E-cadherin gene in human bladder cancer. Normal and bladder cancer samples from 51 patients were compared in terms of E-cadherin gene expression and methylation status by immunohistochemistry, methylation-specific polymerase chain reaction (MSP), and bisulfite genome-sequencing techniques. Ten different CpG sites (nt 863, 865, 873, 879, 887, 892, 901, 918, 920, and 940) in the promoter region were studied. Thirty-five of 51 (69%) bladder cancer samples lacked E-cadherin expression, whereas only six of 51 (12%) normal bladder samples lacked E-cadherin immunoreactivity. MSP analysis of bladder cancer samples suggested that 43 of 51 (84%) showed methylation of the promoter region, whereas only 12 of 51 (24%) normal bladder samples showed hypermethylation. Sodium bisulfite genome-sequencing analysis revealed that of 10 CpG sites, two sites (nt 892 and nt 940) showed 100% methylation in all the cancer samples analyzed. Other CpG sites were partially methylated (47-91%). Normal tissue showed only 12% methylation (range, 1-33%) on various CpG sites. Also supporting these data, E-cadherin-negative bladder cancer cell lines restored expression of the E-cadherin gene after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The present study showed that CpG hypermethylation was an important mechanism of E-cadherin gene inactivation in bladder cancer and also that specific CpG sites consistently presented higher methylation levels than others. These findings may provide a better strategy for the diagnosis and management of bladder cancer.
Mol Carcinog 2002 Aug
PMID:CpG hypermethylation of promoter region and inactivation of E-cadherin gene in human bladder cancer. 1220 70


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