Article

N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy

Research Center for Genetic Medicine, Children's National Medical Center, The George Washington University, Washington, DC, USA.
Human Mutation (Impact Factor: 5.14). 10/2011; 32(10):1153-60. DOI: 10.1002/humu.21553
Source: PubMed

ABSTRACT

N-acetylglutamate synthase (NAGS) catalyzes the conversion of glutamate and acetyl-CoA to NAG, the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAGS gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (c.-3064C>A), affecting a highly conserved nucleotide within the hepatic nuclear factor 1 (HNF-1) binding site, was not found in single nucleotide polymorphism databases and in a screen of 1,086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia. Inc.

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    • "Our experiments and other studies [74] confirm the role of HNF-1 in NAGS expression. HNF-1 is essential for stimulation of NAGS expression by its enhancer. "
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    ABSTRACT: 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.
    Full-text · Article · Feb 2012 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
    No preview · Article · Jan 2012
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    ABSTRACT: Abstract. Urea cycle disorders (UCD) may be due to defects of any of its six enzymes or two transporters. The diagnosis of the three mitochondrial enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase I (CPS1) and ornithine transcarbamylase (OTC) depends on either molecular mutation analysis or measurement of enzyme activity for definitive diagnosis, whereas the diagnosis of deficiencies of the three cytosolic enzymes argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase I (ARG1) is usually straightforward, based on marker metabolites. Enzyme assays for all UCD have been used since the first description, for disease confirmation and in some instances even for prenatal diagnosis. The genetic bases of the UCD have only been unraveled from the 1980s; the last gene cloned being the NAGS gene in 2002. In this review we discuss the enzymatic assays for all urea cycle enzymes from a historical perspective, their potential and drawbacks, and the current role of enzymatic analysis in UCD in general.
    Full-text · Article · Jan 2014 · Journal of Pediatric Biochemistry
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