Expression, purification, crystallization and preliminary X-ray diffraction analysis of the dihydroorotase domain of human CAD.

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Šresolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a=82.1, b=159.3, c=61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.

Ribosome occupancy of the yeast CPA1 upstream open reading frame termination codon modulates nonsense-mediated mRNA decay.

Saccharomyces cerevisiae CPA1 mRNA contains an upstream open reading frame (uORF) encoding the arginine attenuator peptide (AAP). Negative translational regulation of CPA1 occurs when the nascent AAP responds to arginine (Arg) by stalling ribosomes at the uORF termination codon. CPA1 expression is also controlled by nonsense-mediated mRNA decay (NMD). Using wild-type and decay-defective strains expressing CPA1-LUC, we determined how this uORF contributes to NMD control. Arg addition to media rapidly destabilized the CPA1 transcript in wild-type but not upf1delta cells. The wild-type uORF exerted translational control and induced NMD of CPA1-LUC; the mutated D13N uORF, which eliminates stalling and regulation, did not. Thus, regulation by NMD was not governed simply by ribosomes encountering the uORF terminator but appeared dependent on the AAP's ribosome-stalling ability. Improving the D13N uORF initiation context also promoted NMD. Hence, NMD appears to be triggered by increased ribosomal occupancy of the uORF termination codon.

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha.

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.

Role of RNA surveillance proteins Upf1/CpaR, Upf2 and Upf3 in the translational regulation of yeast CPA1 gene.

Gene CPA1, encoding one of the subunits of carbamoylphosphate synthetase (CPSase A) is subject to a translational control by arginine of which the essential element is a 25 amino acid peptide encoded by the CPA1 messenger. The peptide is the product of an open reading frame located upstream (uORF) of the coding phase of the gene, within a 250 nucleotide leader. In the past, a series of mutations impairing the repression of gene CPA1 by arginine had been selected in vivo. Most of the mutations were located in the CPA1 uORF, but mutations unlinked to the CPA1 gene were also isolated and mapped in a gene called CPAR. In this work, we show that the CPAR gene is identical to the UPF1 gene, encoding a protein responsible for the premature termination step of RNA surveillance by nonsense-mediated mRNA decay (NMD). Deletion of UPF1, or deletion of UPF2 and UPF3, the other genes involved in the NMD pathway, enhances the synthesis of CPSase A, whether arginine is present or not in the growth medium. The regulatory effect of the NMD protein complex is only observed when the uORF is present in the CPA1 messenger, indicating that the arginine-peptide repression mechanism and the RNA surveillance pathway are complementary mechanisms. Our results indicate that the NMD destabilizes the 5' end of the CPA1 message and this decay is strongly enhanced when arginine is present in the growth medium.

Enzymatic activities of Ura2 and Ura1 proteins (aspartate carbamoyltransferase and dihydro-orotate dehydrogenase) are present in both isolated membranes and cytoplasm of Saccharomyces cerevisiae.

Computational analysis predicted three potential hydrophobic transmembrane alpha-helices within the Ura2 multidomain protein of Saccharomyces cerevisiae, the C-terminal subdomain of which catalyses the second step of uridine-monophosphate biosynthesis by its L-aspartate carbamoyltransferase activity (EC 2.1.3.2). The fourth step of pyrimidine biosynthesis is catalysed by dihydro-orotate dehydrogenase (Ura1 protein; EC 1.3.99.11), which was similarly characterized as a peripheral membrane protein. Ex situ, the activities of the investigated enzymes were associated both with isolated yeast membranes, fractionated by differential centrifugation to remove intact nuclei, and with soluble cytoplasmic proteins.

CAD, a c-Myc target gene, is not deregulated in Burkitt's lymphoma cell lines.

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.

Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding.

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.

Opposite fluxes of glutamine and alanine in the splanchnic area are an efficient mechanism for nitrogen sparing in rats.

Glutamine release by the liver constitutes a process of nitrogen salvage through the recycling of a part of the nitrogen, which prevents irreversible nitrogen losses as urea. The aim of this work was to study the nitrogen cycling in the splanchnic bed under different nutritional conditions: fed state, postabsorptive state (16 h food deprivation) or prolonged starvation (24 or 40 h). Rats were adapted to a 15% casein diet for 15 d and then sampled. The digestive, hepatic and splanchnic balances of glucose, lactate, ketone bodies, urea and amino acids were determined. There was a net release of lactate and alanine by the digestive tract, due to the high rate of glycolysis and glutaminolysis. During prolonged starvation, ketone bodies became major energy fuel for the intestine. In fed rats, there was a net uptake of most amino acids by the liver, except for glutamine and glutamate. Urea, glutamine and glutamate released represented 33, 24 and 6% of total nitrogen taken up by the liver, respectively. In postabsorptive rats, compared with fed rats, there was a significant reduction of ureagenesis, and glutamine became the major form of nitrogen released by the liver. In fact, nitrogen cycling in the form of glutamine or glutamate in the liver may be interpreted as a nitrogen salvage process, rather than as an acid-base control process. In the splanchnic area, in parallel with a highly active cycling of glucose as lactate, there exists a nitrogen cycling involving opposite fluxes of glutamine and alanine.

The arg2 gene of Trichoderma virens: cloning and development of a homologous transformation system.

The arg2 gene which encodes the small subunit of carbamoyl phosphate synthetase for Trichoderma virens has been cloned and used to develop a homologous transformation system. A genomic clone containing the arg2 gene was isolated from a cosmid library of T. virens based on complementation of an arginine auxotrophic mutant of this fungus. The predicted amino acid sequence of the arg2 gene shows 56-82% identity with homologous polypeptides from other fungi. It also contains an upstream open reading frame which encodes 24 amino acids. As is observed with other gene sequences encoding this polypeptide in filamentous fungi, the N-terminus of the predicted polypeptide showed characteristic features of a mitochondrial signal sequence. The arg2 gene was used for genetic transformation of T. virens in frequencies of up to 370 transformants/microgram of DNA. Heat-shock treatment of T. virens protoplasts increased the transformation frequency by fivefold, but more than 85% of the transformants were abortive. Both single-copy, homologous integration events and ectopic, non-homologous integration events were detected by Southern analyses of genomic DNA from transformed strains.

Carbamyl-phosphate synthetase domain of the yeast multifunctional protein Ura2 is necessary for aspartate transcarbamylase inhibition by UTP.

In Saccharomyces cerevisiae, the first two reactions of pyrimidine biosynthesis are catalyzed by the multifunctional protein Ura2 carrying both carbamyl-phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) enzyme activities. In order to study how UTP regulates both of these activities mutant strains were constructed: one strain which expressed the Ura2 protein fused to the green fluorescent protein, and two strains expressed truncated Ura2 proteins. These strains exhibited a phenotype associated with a modified regulation of the pyrimidine pathway. Results presented in this report provide arguments in favor of a single UTP binding site located on the CPSase domain, and support a model in which ATCase activity is inhibited by UTP only when it can interact with the CPSase domain.

Requirement for the carboxyl-terminal domain of Saccharomyces cerevisiae carbamoyl-phosphate synthetase.

The arginine-specific carbamoyl phosphate synthetase of Saccharomyces cerevisiae is a heterodimeric enzyme, with a 45-kDa CPA1 subunit binding and cleaving glutamine, and a 124-kDa CPA2 subunit accepting the ammonia moiety cleaved from glutamine, binding all of the remaining substrates and carrying out all of the other catalytic events. CPA2 is composed of two apparently duplicated amino acid sequences involved in binding the two ATP molecules needed for carbamoyl phosphate synthesis and a carboxyl-terminal domain which appears to be less tightly folded than the remainder of the protein. Using deletion mutagenesis, we have established that essentially all of the carboxyl-terminal domain of CPA2 is required for catalytic function and that even small truncations lead to significant changes in the CPA2 conformation. In addition, we have demonstrated that the C-terminal region of CPA2 can be expressed as an autonomously folded unit which is stabilized by specific interactions with the remainder of CPA2. We also made the unexpected finding that, even when ammonia is used as the substrate and there is no catalytic role for CPA1, interaction with CPA1 led to an increase in the Vmax of CPA2 in crude extracts.

Molecular cloning of a human cDNA encoding a trifunctional enzyme of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine synthesis.

A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.

Overexpression of cyclin D1 enhances gene amplification.

Defects in cell cycle control and increased genomic instability, including gene amplification, often occur during cancer development. Cyclin D1 plays a pivotal role in G1, and this gene is frequently amplified and overexpressed in several types of human cancer. This study demonstrates that ectopic overexpression of cyclin D1 in a rat liver epithelial cell line markedly increased the yield of cells containing amplified copies of the CAD gene. This effect was associated with a loss of G1-S checkpoint control, although the cyclin D1-overexpressing cells had a normal p53 gene. The capacity of cyclin D1 to enhance gene amplification may contribute to the process of genomic instability during tumor development.

Participation of the purine repressor in control of the carbamoylphosphate synthetase operon in Salmonella typhimurium.

Previous work has shown that the carAB operon of Salmonella typhimurium is transcribed from tandem promoters, P1 and P2, that are negatively controlled by pyrimidines and arginine, respectively. The results reported here show that purines also negatively control expression of carAB and that this effect is absent in a purR::Tn10 derivative. Primer-extension experiments established that the purine effect is exerted at P1, thus redefining this promoter as sensitive to both purines and pyrimidines. The results of gel-retardation experiments as well as DNase I and premethylation footprintings indicate that the purine repressor interacts with a PUR box 85 bp upstream of P1. Modification of this PUR box by site-directed mutagenesis abolishes the repression by purines in a carA::lacZ fusion, confirming that this box functions in vivo in purine control of carAB expression.

Organization of carbamoyl-phosphate synthase genes in Neisseria gonorrhoeae includes a large, variable intergenic sequence which is also present in other Neisseria species.

The carbamoyl-phosphate synthase (CPS) enzyme in prokaryotes is a heterodimer, encoded by genes commonly called carA and carB. In most prokaryotes examined, these genes are separated by up to 24 bp and are cotranscribed. In Pseudomonas aeruginosa, carA and carB are also co-transcribed, but are separated by 682 bp. We have determined the complete DNA sequence of the carA and carB genes of Neisseria gonorrhoeae strain CH811. carA (1125 bp) and carB (3237 bp) are similar in size and sequence to other prokaryotic CPS genes, however they are separated by an intervening sequence of 3290 bp which has no similarity to the intervening sequence between other CPS genes; furthermore, putative transcription terminators are found downstream of both carA and carB. Several neisserial repetitive sequences were identified within the 9 kb sequenced, as well as novel 120 and 150 bp repeats (designated RS6 and RS7, respectively) which were found within the intervening sequence between carA and carB. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was not unusual, the sequence between carA and carB was amplified by PCR from 30 isolates of N. gonorrhoeae. The intervening sequence was found to vary in size, from approximately 2.2 to 3.7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS enzyme. A similar large, variably sized intervening sequence was also found between the carA and carB genes of 12 isolates of N. meningitidis and 18 commensal Neisseria isolates comprising nine species. This unexpected organization of the CPS genes in N. gonorrhoeae is therefore widespread throughout the genus Neisseria.

Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles.

In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.

Selection of N-(phosphonacetyl)-L-aspartate resistant Chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.

In mammalian cells selected in culture for resistance to PALA the CAD gene is amplified and these cells are a widely used model system to study gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD gene.

Vascular branching pattern and zonation of gene expression in the mammalian liver. A comparative study in rat, mouse, cynomolgus monkey, and pig.

BACKGROUND: A significant part of the liver volume consists of regions in which hepatocytes are in close contact with large branches of the afferent (portal vein) or efferent (hepatic vein) vessels. As most studies have addressed zonation of gene expression around the parenchymal branches of the portal and hepatic vein only, the patterns of gene expression in hepatocytes surrounding larger vessels are largely unknown. METHODS: For that reason, we studied the patterns of expression of the mRNAs and proteins of the pericentral marker enzymes glutamine synthase, ornithine aminotransferase, and glutamate dehydrogenase and the periportal marker enzymes phosphoenolpyruvate carboxykinase and carbamoylphosphate synthase in the rat liver, in relation to the branching pattern of the afferent and efferent hepatic veins with immuno and hybridocytochemical techniques. These patterns of expression were compared with those seen in mouse, monkey, and pig liver. RESULTS: The distribution patterns of the genes studied appear to reflect the "intensity" of the pericentral and periportal environment, glutamine synthase and phosphoenolypyruvate carboxykinase requiring the most pronounced environment, respectively. The patterns of gene expression around the large branches of the portal and hepatic vein were found to be related to the parenchymal branches in the neighbourhood of these large blood vessels. Only the cells of the limiting plate retain their periportal and pericentral phenotype for those marker enzymes that do not require a pronounced periportal or pericentral environment to be expressed. GS-negative areas in the pericentral limiting plate appear to correlate with a local absence of draining central veins, and become more frequent and extensive around the larger branches of the hepatic vein. CONCLUSIONS: The similarity of the observed patterns of gene expression of the genes studied in mouse, rat, monkey, pig, and man suggests that they reflect a general feature of gene expression in the mammalian liver. A comparison of mouse, rat, pig, and human liver suggests that the presence of glutamine synthase-negative areas reflects the branching order of the efferent hepatic blood vessel.