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

Effect of arginine on oligomerization and stability of N-acetylglutamate synthase.

N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-acetylglutamate synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS

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

Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs

Glutathione peroxidase 3 is a potential biomarker for konzo.

Konzo is a neglected paralytic neurological disease associated with food (cassava) poisoning that affects the worlds poorest children and women of childbearing ages across regions of sub-Saharan Africa. Despite understanding the dietary factors that lead to konzo, the molecular markers and mechanisms that trigger this disease remain unknown. To identify potential protein biomarkers associated with a disease status, plasma was collected from two independent Congolese cohorts, a discovery cohort (n = 60) and validation cohort (n = 204), sampled 10 years apart and subjected to multiple high-throughput assays. We identified that Glutathione Peroxidase 3 (GPx3), a critical plasma-based antioxidant enzyme, was the sole protein examined that was both significantly and differentially abundant between affected and non-affected participants in both cohorts, with large reductions observed in those affected with konzo. Our findings raise the notion that reductions in key antioxidant mechanisms may be the biological risk factor for the development of konzo, particularly those mediated through pathways involving the glutathione peroxidase family

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

Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses

A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified

Fatty Acid Binding Protein 1 Is Related with Development of Aspirin-Exacerbated Respiratory Disease

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) refers to the development of bronchoconstriction in asthmatics following the ingestion of aspirin. Although alterations in eicosanoid metabolites play a role in AERD, other immune or inflammatory mechanisms may be involved. We aimed to identify proteins that were differentially expressed in nasal polyps between patients with AERD and aspirin-tolerant asthma (ATA). METHODOLOGY/PRINCIPAL FINDINGS: Two-dimensional electrophoresis was adopted for differential display proteomics. Proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS). Western blotting and immunohistochemical staining were performed to compare the amount of fatty acid-binding protein 1 (FABP1) in the nasal polyps of patients with AERD and ATA. Fifteen proteins were significantly up- (seven spots) or down-regulated in the nasal polyps of patients with AERD (n = 5) compared to those with ATA (n = 8). LC-MS revealed an increase in seven proteins expression and a decrease in eight proteins expression in patients with AERD compared to those with ATA (P = 0.003-0.045). FABP1-expression based on immunoblotting and immunohistochemical analysis was significantly higher in the nasal polyps of patients with AERD compared to that in patients with ATA. FABP1 was observed in epithelial, eosinophils, macrophages, and the smooth-muscle cells of blood vessels in the polyps. CONCLUSIONS/SIGNIFICANCE: Our results indicate that alterations in 15 proteins, including FABP1, may be related to the development of AERD