Data Mining Approaches for Understanding of Regulation of Expression of the Urea Cycle Genes

Urea cycle converts ammonia, a waste product of protein catabolism and a neurotoxin, into non-toxic urea. Urea cycle disorders are a group of rare genetic diseases that have protein-restricted diet as a common treatment modality. Expression of urea cycle genes is regulated in concert by the dietary protein intake, but the mechanism of this regulation is not well understood. Data mining of databases such as ENCODE and Cistrome can be used to gain new information about regulatory elements, transcription factors, and epigenetic mechanisms that regulate expression of urea cycle genes. This can lead to better understanding of the common mechanism, which regulates urea cycle genes, and can generate testable hypotheses about regulation of gene expression and new treatments for urea cycle disorders

A novel bifunctional N-acetylglutamate synthase-kinase from Xanthomonas campestris that is closely related to mammalian N-acetylglutamate synthase

BACKGROUND: In microorganisms and plants, the first two reactions of arginine biosynthesis are catalyzed by N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK). In mammals, NAGS produces an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle, and no functional NAGK homolog has been found. Unlike the other urea cycle enzymes, whose bacterial counterparts could be readily identified by their sequence conservation with arginine biosynthetic enzymes, mammalian NAGS gene was very divergent, making it the last urea cycle gene to be discovered. Limited sequence similarity between E. coli NAGS and fungal NAGK suggests that bacterial and eukaryotic NAGS, and fungal NAGK arose from the fusion of genes encoding an ancestral NAGK (argB) and an acetyltransferase. However, mammalian NAGS no longer retains any NAGK catalytic activity. RESULTS: We identified a novel bifunctional N-acetylglutamate synthase and kinase (NAGS-K) in the Xanthomonadales order of gamma-proteobacteria that appears to resemble this postulated primordial fusion protein. Phylogenetic analysis indicated that xanthomonad NAGS-K is more closely related to mammalian NAGS than to other bacterial NAGS. We cloned the NAGS-K gene from Xanthomonas campestis, and characterized the recombinant NAGS-K protein. Mammalian NAGS and its bacterial homolog have similar affinities for substrates acetyl coenzyme A and glutamate as well as for their allosteric regulator arginine. CONCLUSION: The close phylogenetic relationship and similar biochemical properties of xanthomonad NAGS-K and mammalian NAGS suggest that we have identified a close relative to the bacterial antecedent of mammalian NAGS and that the enzyme from X. campestris could become a good model for mammalian NAGS in structural, biochemical and biophysical studies

Pkhd1

Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver (cyli), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd

N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-acetylglutamate Synthase Enhancer Region

N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD

Transcriptional Regulation of N-Acetylglutamate Synthase

The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes

Ljubica Caldovic's Quick Files

The Quick Files feature was discontinued and it’s files were migrated into this Project on March 11, 2022. The file URL’s will still resolve properly, and the Quick Files logs are available in the Project’s Recent Activity

Sources and Fates of Carbamyl Phosphate: A Labile Energy-Rich Molecule with Multiple Facets.

Carbamyl phosphate (CP) is well-known as an essential intermediate of pyrimidine and arginine/urea biosynthesis. Chemically, CP can be easily synthesized from dihydrogen phosphate and cyanate. Enzymatically, CP can be synthesized using three different classes of enzymes: (1) ATP-grasp fold protein based carbamyl phosphate synthetase (CPS); (2) Amino-acid kinase fold carbamate kinase (CK)-like CPS (anabolic CK or aCK); and (3) Catabolic transcarbamylase. The first class of CPS can be further divided into three different types of CPS as CPS I, CPS II, and CPS III depending on the usage of ammonium or glutamine as its nitrogen source, and whethe

Sources and Fates of Carbamyl Phosphate: A Labile Energy-Rich Molecule with Multiple Facets

Carbamyl phosphate (CP) is well-known as an essential intermediate of pyrimidine and arginine/urea biosynthesis. Chemically, CP can be easily synthesized from dihydrogen phosphate and cyanate. Enzymatically, CP can be synthesized using three different classes of enzymes: (1) ATP-grasp fold protein based carbamyl phosphate synthetase (CPS); (2) Amino-acid kinase fold carbamate kinase (CK)-like CPS (anabolic CK or aCK); and (3) Catabolic transcarbamylase. The first class of CPS can be further divided into three different types of CPS as CPS I, CPS II, and CPS III depending on the usage of ammonium or glutamine as its nitrogen source, and whether N-acetyl-glutamate is its essential co-factor. CP can donate its carbamyl group to the amino nitrogen of many important molecules including the most well-known ornithine and aspartate in the arginine/urea and pyrimidine biosynthetic pathways. CP can also donate its carbamyl group to the hydroxyl oxygen of a variety of molecules, particularly in many antibiotic biosynthetic pathways. Transfer of the carbamyl group to the nitrogen group is catalyzed by the anabolic transcarbamylase using a direct attack mechanism, while transfer of the carbamyl group to the oxygen group is catalyzed by a different class of enzymes, CmcH/NodU CTase, using a different mechanism involving a three-step reaction, decomposition of CP to carbamate and phosphate, transfer of the carbamyl group from carbamate to ATP to form carbamyladenylate and pyrophosphate, and transfer of the carbamyl group from carbamyladenylate to the oxygen group of the substrate. CP is also involved in transferring its phosphate group to ADP to generate ATP in the fermentation of many microorganisms. The reaction is catalyzed by carbamate kinase, which may be termed as catabolic CK (cCK) in order to distinguish it from CP generating CK. CP is a thermally labile molecule, easily decomposed into phosphate and cyanate, or phosphate and carbamate depending on the pH of the solution, or the presence of enzyme. Biological systems have developed several mechanisms including channeling between enzymes, increased affinity of CP to enzymes, and keeping CP in a specific conformation to protect CP from decomposition. CP is highly important for our health as both a lack of, or decreased, CP production and CP accumulation results in many disease conditions

<i>NAGS</i>, <i>CPS1</i>, and <i>SLC25A13</i> (Citrin) at the Crossroads of Arginine and Pyrimidines Metabolism in Tumor Cells

Urea cycle enzymes and transporters collectively convert ammonia into urea in the liver. Aberrant overexpression of carbamylphosphate synthetase 1 (CPS1) and SLC25A13 (citrin) genes has been associated with faster proliferation of tumor cells due to metabolic reprogramming that increases the activity of the CAD complex and pyrimidine biosynthesis. N-acetylglutamate (NAG), produced by NAG synthase (NAGS), is an essential activator of CPS1. Although NAGS is expressed in lung cancer derived cell lines, expression of the NAGS gene and its product was not evaluated in tumors with aberrant expression of CPS1 and citrin. We used data mining approaches to identify tumor types that exhibit aberrant overexpression of NAGS, CPS1, and citrin genes, and evaluated factors that may contribute to increased expression of the three genes and their products in tumors. Median expression of NAGS, CPS1, and citrin mRNA was higher in glioblastoma multiforme (GBM), glioma, and stomach adenocarcinoma (STAD) samples compared to the matched normal tissue. Median expression of CPS1 and citrin mRNA was higher in the lung adenocarcinoma (LUAD) sample while expression of NAGS mRNA did not differ. High NAGS expression was associated with an unfavorable outcome in patients with glioblastoma and GBM. Low NAGS expression was associated with an unfavorable outcome in patients with LUAD. Patterns of DNase hypersensitive sites and histone modifications in the upstream regulatory regions of NAGS, CPS1, and citrin genes were similar in liver tissue, lung tissue, and A549 lung adenocarcinoma cells despite different expression levels of the three genes in the liver and lung. Citrin gene copy numbers correlated with its mRNA expression in glioblastoma, GBM, LUAD, and STAD samples. There was little overlap between NAGS, CPS1, and citrin sequence variants found in patients with respective deficiencies, tumor samples, and individuals without known rare genetic diseases. The correlation between NAGS, CPS1, and citrin mRNA expression in the individual glioblastoma, GBM, LUAD, and STAD samples was very weak. These results suggest that the increased cytoplasmic supply of either carbamylphosphate, produced by CPS1, or aspartate may be sufficient to promote tumorigenesis, as well as the need for an alternative explanation of CPS1 activity in the absence of NAGS expression and NAG