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Query: UNIPROT:P06889 (Mol)
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Cells often acquire resistance to the antiproliferative agents methotrexate (MTX) or N-phosphonacetyl-L-aspartate (PALA) through amplification of genes encoding the target enzymes dihydrofolate reductase or carbamylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), respectively. We showed previously that Syrian hamster BHK cells resistant to selective concentrations of PALA (approximately 3 x ID50) arise at a rate of approximately 10(-4) per cell per generation and contain amplifications of the CAD gene as ladder-like structures on one of the two B9 chromosomes, where CAD is normally located. We now find that BHK cells resistant to high concentrations of PALA (approximately 15 x ID50) appear only after prior exposure to selective concentrations of PALA for approximately 72 h. Furthermore, in contrast to untreated cells, BHK cells pretreated with selective concentrations of MTX give colonies in high concentrations of PALA, and cells pretreated with selective concentrations of PALA give colonies in high concentrations of MTX or 5-fluorouracil. As judged by measuring numbers of cells and metaphase cell pairs, BHK cells do not arrest completely when starved for pyrimidine nucleotides by treatment with selective concentrations of PALA for up to 72 h. We propose that DNA damage, caused when cells fail to stop DNA synthesis promptly under conditions of dNTP starvation, stimulates amplification throughout the genome by mechanisms--such as bridge-breakage-fusion cycles--that are triggered by broken DNA. Amplified CAD genes were analyzed by fluorescence in situ hybridization both in cells where amplification was induced by PALA pretreatment and in cells in which the amplification occurred spontaneously, before selection with PALA. The ladder-like structures that result from bridge-breakage-fusion cycles were observed in both cases.
Mol Biol Cell 1996 Mar
PMID:Inefficient growth arrest in response to dNTP starvation stimulates gene amplification through bridge-breakage-fusion cycles. 886 64

Carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, which is encoded by the cad gene, is required for the first three rate-limiting steps of de novo pyrimidine biosynthesis. It has been previously demonstrated that cad transcription increases at the G1/S-phase boundary, as quiescent cells reenter the proliferative cell cycle. The growth-responsive element has been mapped to an E box at +65 in the hamster cad promoter. Using an in vivo UV cross-linking and immunoprecipitation assay, we show that Myc, Max, and upstream stimulatory factor (USF) bind to the chromosomal cad promoter. To determine whether binding of Myc-Max or USF is critical for cad growth regulation, we analyzed promoter constructs which contain mutations in the nucleotides flanking the E box. We demonstrate that altering nucleotides which flank the cad E box to sequences which decrease Myc-Max binding in vitro correlates with a loss of cad G1/S-phase transcriptional activation. This result supports the conclusion that binding of Myc-Max, but not USF, is essential for cad regulation. Our investigations demonstrate that the endogenous cad E box can be bound by more than one transcription factor, but growth-induced cad expression is achieved only by Myc.
Mol Cell Biol 1997 May
PMID:Myc versus USF: discrimination at the cad gene is determined by core promoter elements. 911 22

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.
J Mol Biol 1998 Aug 14
PMID:Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins. 969 53

A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis. Peculiarities in the trypanosomatid pyr6-5 gene product are discussed.
J Mol Biol 1999 Jan 08
PMID:Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi. 987 95

The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans. The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid. The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it. Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII. Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway. The 'silent' dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases.
Mol Microbiol 1999 Aug
PMID:Structural and transcriptional analysis of the pyrABCN, pyrD and pyrF genes in Aspergillus nidulans and the evolutionary origin of fungal dihydroorotases. 1041 50

Fur (ferric uptake regulation) binding fragments were isolated by in vitro binding of purified Fur protein with Sau3AI-digested genomic DNA fragments. The Fur-bound DNA fragments were filtered on nitrocellulose paper, isolated, cloned, and sequenced. The protein binding was confirmed by gel retardation assay for five DNA fragments. The sequence data were used to identify the genes by comparison with the GenBank data. The proposed Fur binding regions lie on or near the putative promoter regions of marAB (multiple antibiotic resistance), pyrC (dihydroorotase), mreB (mecillinam resistance) and an unidentified gene (ecouw93) near argI and in the middle of the treBC (trehalose permease enzyme II) coding region. The proposed Fur binding sites of the known iron regulating operators including the genes of this work are AAT(pyrimidine) and A(purine)TT. The two conserved sequences are 10 bases apart and palindromic to each other, which might suggest the classical pattern of protein binding toward one side of the DNA in contrast to the concept of the Fur protein wrapping around the DNA.
Mol Cells 1999 Oct 31
PMID:Isolation and identification of Fur binding genes in Escherichia coli. 1059 41

Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source. Previously, we screened several yeasts for their ability to catabolise pyrimidines. One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines. Here, a series of molecular techniques have been modified to clone pyrimidine catabolic genes, study their expression and purify the corresponding enzymes from this yeast. The pyd2-1 mutant, which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S. kluyveri genomic library. The complementing plasmid contained the full sequence of the PYD2 gene, which exhibited a high level of homology with mammalian DHPases and bacterial hydantoinases. The organisation of PYD2 showed a couple of specific features. The 542-codons open reading frame was interrupted by a 63 bp intron, which does not contain the Saccharomyces cerevisiae branch-point sequence, and the transcripts contained a long 5' untranslated leader with five or six AUG codons. The derived amino acid sequence showed similarities with dihydroorotases, allantoinases and uricases from various organisms. Surprisingly, the URA4 gene from S. cerevisiae, which encodes dihydroorotase, shows greater similarity to PYD2 and other catabolic enzymes than to dihydroorotases from several other non-fungal organisms. The S. kluyveri DHPase was purified to homogeneity and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product. The enzyme is a tetramer, likely consisting of similar if not identical subunits each with a molecular mass of 59 kDa. The S. kluyveri DHPase was capable of catalysing both dihydrouracil and dihydrothymine degradation, presumably by the same reaction mechanism as that described for mammalian DHPase. On the other hand, the regulation of the yeast PYD2 gene and DHPase seem to be different from that in other organisms. DHPase activity and Northern analysis demonstrated that PYD2 expression is inducible by dihydrouracil, though not by uracil. Apparently, dihydrouracil and DHPase represent an important regulatory checkpoint of the pyrimidine catabolic pathway in S. kluyveri.
J Mol Biol 2000 Jan 28
PMID:PYD2 encodes 5,6-dihydropyrimidine amidohydrolase, which participates in a novel fungal catabolic pathway. 1065 11

Although the Myc family of transcription factors is upregulated in many human tumors, it is unclear which genes are targets for the deregulated Myc. Previous studies suggest that hamster and rat carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase Cad genes are regulated by c-Myc. In fact, of all putative target genes thought to be activated by c-Myc, only the Cad gene showed loss of growth regulation in rat cells nullizygous for c-Myc. However, it was unknown whether upregulation of CAD, which performs the first three rate-limiting steps of pyrimidine biosynthesis, contributes to c-Myc's role in human neoplasia. To explore this possibility, we cloned the human cad promoter. We found that c-Myc could bind to an E box in the human cad promoter in gel shift assays and that growth regulated transcription from the human cad promoter was dependent on this c-Myc binding site. However, the increased amount of c-Myc found in Burkitt's lymphoma cell lines did not lead to increased cad mRNA levels. Thus, we suggest that although c-Myc is clearly important for the normal transcriptional control of the cad promoter, it is unlikely that increased levels of CAD are important mediators of c-Myc-induced neoplasia. Therefore, an understanding of the mechanism by which overexpressed c-Myc contributes to the development of Burkitt's lymphoma requires the identification of additional c-Myc target genes.
Mol Carcinog 2000 Feb
PMID:CAD, a c-Myc target gene, is not deregulated in Burkitt's lymphoma cell lines. 1065 1

Carbamoyl phosphate synthetase II (CPSII) is part of carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Herein, we identify CAD as a substrate for caspase-3 degradation in both in vitro and in vivo models of apoptosis. Withdrawal of interleukin-3 or incubation with staurosporine (STS) or doxorubicin (Dox) resulted in proteolytic cleavage of CAD in a myeloid precursor cell line (32D) or in a cell line over-expressing CAD. The rapid decline in the CPSII activity paralleled the degradation of CAD and preceded the appearance of Annexin-V-stained apoptotic cells and DNA fragmentation. These events correlated closely with the activation of caspase-3 in these cells and were prevented by the cell-permeable caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone. Moreover, the incubation of purified CAD with recombinant caspase-3 in vitro generated CAD fragments that were similar to those obtained in vivo. Edman sequencing revealed that two of the major caspase-3 cleavage sites occurred at the sequences EAVD/G and VACD/G within the catalytic (B2) and allosteric (B3) domains of CAD, thus providing a potential mechanism for the rapid inactivation of CPSII during apoptosis. Consistent with this, an enhanced loss of the intracellular pyrimidines (UTP and CTP) was observed in response to STS or DOX-induced apoptosis. Therefore, these studies show that CAD is a novel target for caspase-dependent regulation during apoptosis and suggest that the selective inactivation of pyrimidine nucleotide synthesis accompanies the process of apoptosis.
Mol Pharmacol 2002 Mar
PMID:Caspase-dependent cleavage of carbamoyl phosphate synthetase II during apoptosis. 1185 37

Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida, a homohexameric enzyme with a molecular mass of 28.4 kDa per subunit, is a cyclic amidohydrolase catalysing the reversible conversion of creatinine to creatine. The enzyme plays a key role in the bacterial degradation of creatinine. The three-dimensional structure of creatininase from P.putida was determined and refined to 2.1A. The structure shows the six subunits arranged as a trimer of dimers and definitely disproves previous reports that the enzyme has an octameric quaternary structure. Each monomer consists of a central, four-stranded, parallel beta-sheet flanked by two alpha-helices on both sides of the beta-sheet. This topology is unique within the superfamily of amidohydrolases. Moreover, creatininase possesses a novel fold with no close structural relatives within the Protein Data Bank. Each creatininase monomer contains a binuclear zinc centre near the C termini of the beta-strands and the N termini of the main alpha-helices. These zinc ions indicate the location of the active site unambiguously. The active site is entirely buried and is not accessible from the solution without movement of parts of the protein. The two zinc ions are bridged by a water molecule and by an aspartate residue, which acts as a bidentate ligand. They differ from each other in the number and the spatial arrangement of their ligands. One of them is tetrahedrally and the other trigonal-bipyramidally ligated. Using two water molecules of the first coordination sphere as anchor points, a creatinine-water adduct resembling the transition state of the hydrolysation reaction was modelled into the active site. The resulting complex in combination with structural comparisons with other amidohydrolases enabled us to identify the most probable candidate for the catalytic base and to suggest a putative reaction mechanism. Surprisingly these structural comparisons revealed a similarity in the active-site arrangement between creatininase and the hydantoinase-like cyclic amidohydrolases that was unexpected, given the completely unrelated primary and tertiary structures. In particular, the zinc-bridging aspartate residue of creatininase is a spatially and functionally analogue to a carboxylated lysine residue found in dihydroorotase and the hydantoinases. Hence, creatininase and the hydantoinase-like cyclic amidohydrolases represent a further example of convergent evolution within the enzyme class of hydrolases.
J Mol Biol 2003 Sep 05
PMID:Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution. 1294 65


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