ABSTRACT
BACKGROUND: The autosomal recessive disorder N-acetylglutamate synthase (NAGS) deficiency is the rarest defect of the urea cycle, with an incidence of less than one in 2,000,000 live births. Hyperammonemic crises can be avoided in individuals with NAGS deficiency by the administration of carbamylglutamate (also known as carglumic acid), which activates carbamoyl phosphatase synthetase 1 (CPS1). The aim of this case series was to introduce additional cases of NAGS deficiency to the literature as well as to assess the role of nutrition management in conjunction with carbamylglutamate therapy across new and existing cases. METHODS: We conducted retrospective chart reviews of seven cases of NAGS deficiency in the US and Canada, focusing on presentation, diagnosis, medication management, nutrition management, and outcomes. RESULTS: Five new and two previously published cases were included. Presenting symptoms were consistent with previous reports. Diagnostic confirmation via molecular testing varied in protocol across cases, with consecutive single gene tests leading to long delays in diagnosis in some cases. All patients responded well to carbamylglutamate therapy, as indicated by normalization of plasma ammonia and citrulline, as well as urine orotic acid in patients with abnormal levels at baseline. Although protein restriction was not prescribed in any cases after carbamylglutamate initiation, two patients continued to self-restrict protein intake. One patient experienced two episodes of hyperammonemia that resulted in poor long-term outcomes. Both episodes occurred after a disruption in access to carbamylglutamate, once due to insurance prior authorization requirements and language barriers and once due to seizure activity limiting the family's ability to administer carbamylglutamate. CONCLUSIONS: Follow-up of patients with NAGS deficiency should include plans for illness and for disruption of carbamylglutamate access, including nutrition management strategies such as protein restriction. Carbamylglutamate can help patients with NAGS deficiency to liberalize their diets, but the maximum safe level of protein intake to prevent hyperammonemia is not yet known. Patients using this medication should still monitor their diet closely and be prepared for any disruptions in medication access, which might require immediate dietary adjustments or medical intervention to prevent hyperammonemia.
Subject(s)
Glutamates , Hyperammonemia , Urea Cycle Disorders, Inborn , Humans , Amino-Acid N-Acetyltransferase/genetics , Amino-Acid N-Acetyltransferase/metabolism , Hyperammonemia/drug therapy , Retrospective StudiesABSTRACT
BACKGROUND: L-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. N-acetylglutamate, synthesized from L-glutamate and acetyl-CoA, is a precursor of the L-arginine biosynthetic branch in microorganisms. The enzyme that produces N-acetylglutamate, N-acetylglutamate synthase, is allosterically inhibited by L-arginine. L-glutamate, as a central metabolite, provides carbon backbone for diverse biological compounds besides L-arginine. When glucose is the sole carbon source, the theoretical maximum carbon yield towards L-arginine is 96.7%, but the experimental highest yield was 51%. The gap of L-arginine yield indicates the regulation complexity of carbon flux and energy during the L-arginine biosynthesis. Besides endogenous biosynthesis, N-acetylglutamate, the key precursor of L-arginine, can be obtained by chemical acylation of L-glutamate with a high yield of 98%. To achieve high-yield production of L-arginine, we demonstrated a novel approach by directly feeding precursor N-acetylglutamate to engineered Escherichia coli. RESULTS: We reported a new approach for the high yield of L-arginine production in E. coli. Gene argA encoding N-acetylglutamate synthase was deleted to disable endogenous biosynthesis of N-acetylglutamate. The feasibility of external N-acetylglutamate towards L-arginine was verified via growth assay in argA- strain. To improve L-arginine production, astA encoding arginine N-succinyltransferase, speF encoding ornithine decarboxylase, speB encoding agmatinase, and argR encoding an arginine responsive repressor protein were disrupted. Based on overexpression of argDGI, argCBH operons, encoding enzymes of the L-arginine biosynthetic pathway, ~ 4 g/L L-arginine was produced in shake flask fermentation, resulting in a yield of 0.99 mol L-arginine/mol N-acetylglutamate. This strain was further engineered for the co-production of L-arginine and pyruvate by removing genes adhE, ldhA, poxB, pflB, and aceE, encoding enzymes involved in the conversion and degradation of pyruvate. The resulting strain was shown to produce 4 g/L L-arginine and 11.3 g/L pyruvate in shake flask fermentation. CONCLUSIONS: Here, we developed a novel approach to avoid the strict regulation of L-arginine on ArgA and overcome the metabolism complexity in the L-arginine biosynthesis pathway. We achieve a high yield of L-arginine production from N-acetylglutamate in E. coli. Co-production pyruvate and L-arginine was used as an example to increase the utilization of input carbon sources.
Subject(s)
Escherichia coli , Glutamic Acid , Escherichia coli/metabolism , Amino-Acid N-Acetyltransferase/metabolism , Glutamic Acid/metabolism , Arginine , Pyruvates/metabolism , Carbon/metabolism , Metabolic Engineering/methodsABSTRACT
The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.
Subject(s)
Amino-Acid N-Acetyltransferase/genetics , Gene Transfer Techniques , Hyperammonemia/genetics , Urea Cycle Disorders, Inborn/genetics , Amino-Acid N-Acetyltransferase/metabolism , Animals , Arginine/metabolism , Arginine/pharmacology , Citrulline/metabolism , Citrulline/pharmacology , Dependovirus/genetics , Disease Models, Animal , Glutamates/metabolism , Glutamates/pharmacology , Humans , Hyperammonemia/metabolism , Hyperammonemia/pathology , Hyperammonemia/therapy , Mice , Mice, Knockout , Mutant Proteins/genetics , Urea/metabolism , Urea Cycle Disorders, Inborn/metabolism , Urea Cycle Disorders, Inborn/pathology , Urea Cycle Disorders, Inborn/therapyABSTRACT
Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.
Subject(s)
Amino-Acid N-Acetyltransferase , Carbamoyl-Phosphate Synthase (Ammonia) , Genes, Dominant , Mutation, Missense , Ornithine Carbamoyltransferase , Urea Cycle Disorders, Inborn , Amino Acid Substitution , Amino-Acid N-Acetyltransferase/chemistry , Amino-Acid N-Acetyltransferase/genetics , Amino-Acid N-Acetyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Ammonia)/chemistry , Carbamoyl-Phosphate Synthase (Ammonia)/genetics , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Female , Heterozygote , Homozygote , Humans , Male , Ornithine Carbamoyltransferase/chemistry , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Protein Domains , Urea Cycle Disorders, Inborn/enzymology , Urea Cycle Disorders, Inborn/geneticsABSTRACT
Three-dimensional full-atom model of the enzyme complex with acetyl-CoA and substrate was constructed on the basis of the primary sequence of amino acid residues of N-acetyl glutamate synthase. Bioinformatics approaches of computer modeling were applied, including multiple sequence alignment, prediction of co-evolutionary contacts, and ab initio folding. On the basis of the results of calculations by classical molecular dynamics and combined quantum and molecular mechanics (QM/MM) methods, the structure of the active site and the reaction mechanism of N-acetylglutamate formation are described. Agreement of the structures of the enzyme-product complexes obtained in computer modeling and in the X-ray studies validates the reliability of modeling predictions.
Subject(s)
Amino-Acid N-Acetyltransferase/chemistry , Amino-Acid N-Acetyltransferase/metabolism , Neisseria gonorrhoeae/enzymology , Catalysis , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Models, Molecular , Neisseria gonorrhoeae/chemistry , Neisseria gonorrhoeae/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Glucagon regulates the hepatic amino acid metabolism and increases ureagenesis. Ureagenesis is activated by N-acetylglutamate (NAG), formed via activation of N-acetylglutamate synthase (NAGS). With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we investigated whether glucagon receptor-mediated activation of ureagenesis is required in a situation where NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle in vivo. Female C57BL/6JRj mice treated with a glucagon receptor antagonist (GRA), glucagon receptor knockout (Gcgr-/-) mice, and wild-type (Gcgr+/+) littermates received an intraperitoneal injection of N-carbamoyl glutamate (Car; a stable variant of NAG), l-citrulline (Cit), Car and Cit (Car + Cit), or PBS. In separate experiments, Gcgr-/- and Gcgr+/+ mice were administered N-carbamoyl glutamate and l-citrulline (wCar + wCit) in the drinking water for 8 wk. Car, Cit, and Car + Cit significantly (P < 0.05) increased plasma urea concentrations, independently of pharmacological and genetic disruption of glucagon receptor signaling (P = 0.9). Car increased blood glucose concentrations equally in GRA- and vehicle-treated mice (P = 0.9), whereas the increase upon Car + Cit was impaired in GRA-treated mice (P = 0.008). Blood glucose concentrations remained unchanged in Gcgr-/- mice upon Car (P = 0.2) and Car + Cit (P = 0.9). Eight weeks administration of wCar + wCit did not change blood glucose (P > 0.2), plasma amino acid (P > 0.4), and urea concentrations (P > 0.3) or the area of glucagon-positive cells (P > 0.3) in Gcgr-/- and Gcgr+/+ mice. Our data suggest that glucagon-mediated activation of ureagenesis is not required when NAGS activity and/or NAG levels are sufficient to activate the first step of the urea cycle.NEW & NOTEWORTHY Hepatic ureagenesis is essential in amino acid metabolism and is importantly regulated by glucagon, but the exact mechanism is unclear. With the aim to identify the steps whereby glucagon both acutely and chronically regulates ureagenesis, we here show, contrary to our hypothesis, that glucagon receptor-mediated activation of ureagenesis is not required when N-acetylglutamate synthase activity and/or N-acetylglutamate levels are sufficient to activate the first step of the urea cycle in vivo.
Subject(s)
Citrulline/administration & dosage , Glucagon/metabolism , Glutamates/administration & dosage , Liver/drug effects , Receptors, Glucagon/deficiency , Receptors, Glucagon/metabolism , Urea/blood , Amino-Acid N-Acetyltransferase/metabolism , Animals , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Female , Glutamates/metabolism , Hormone Antagonists/administration & dosage , Liver/enzymology , Mice, Inbred C57BL , Mice, Knockout , Receptors, Glucagon/antagonists & inhibitors , Receptors, Glucagon/geneticsABSTRACT
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.
Subject(s)
Amino-Acid N-Acetyltransferase/genetics , Enhancer Elements, Genetic , Genetic Variation , Urea Cycle Disorders, Inborn/etiology , Amino-Acid N-Acetyltransferase/metabolism , Base Sequence , Child , Child, Preschool , Female , Humans , Hyperammonemia , Prognosis , Urea Cycle Disorders, Inborn/metabolism , Urea Cycle Disorders, Inborn/pathologyABSTRACT
N-acetylglutamate synthase deficiency (NAGSD) is an extremely rare urea cycle disorder (UCD) with few adult cases so far described. Diagnosis of late-onset presentations is difficult and delayed treatment may increase the risk of severe hyperammonemia. We describe a 52-year-old woman with recurrent headaches who experienced an acute onset of NAGSD. As very few papers focus on headaches in UCDs, we also report a literature review of types and pathophysiologic mechanisms of UCD-related headaches. In our case, headaches had been present since puberty (3-4 days a week) and were often accompanied by nausea, vomiting, or behavioural changes. Despite three previous episodes of altered consciousness, ammonia was measured for the first time at 52 years and levels were increased. Identification of the new homozygous c.344C>T (p.Ala115Val) NAGS variant allowed the definite diagnosis of NAGSD. Bioinformatic analysis suggested that an order/disorder alteration of the mutated form could affect the arginine-binding site, resulting in poor enzyme activation and late-onset presentation. After optimized treatment for NAGSD, ammonia and amino acid levels were constantly normal and prevented other headache bouts. The manuscript underlies that headache may be the presenting symptom of UCDs and provides clues for the rapid diagnosis and treatment of late-onset NAGSD.
Subject(s)
Urea Cycle Disorders, Inborn/diagnosis , Age of Onset , Amino-Acid N-Acetyltransferase/metabolism , Biomarkers , Brain/diagnostic imaging , Brain/pathology , Electroencephalography , Female , Glutamates/therapeutic use , Humans , Middle Aged , Symptom Assessment , Treatment Outcome , Urea Cycle Disorders, Inborn/metabolism , Urea Cycle Disorders, Inborn/pathology , Urea Cycle Disorders, Inborn/therapyABSTRACT
Toxin-antitoxin (TA) loci are prevalent in bacterial genomes. They are suggested to play a central role in dormancy and persister states. Under normal growth conditions, TA toxins are neutralized by their cognate antitoxins, and under stress conditions, toxins are freed and inhibit essential cellular processes using a variety of mechanisms. Here we characterize ataR-ataT, a novel TA system, from enterohemorrhagic Escherichia coli. We show that the toxin AtaT is a GNAT family enzyme that transfers an acetyl group from acetyl coenzyme A to the amine group of the methionyl aminoacyl moiety of initiator tRNA. AtaT specifically modifies Met-tRNAfMet, but no other aminoacyl-tRNAs, including the elongator Met-tRNAMet. We demonstrate that once acetylated, AcMet-tRNAfMet fails to interact with initiation factor-2 (IF2), resulting in disruption of the translation initiation complex. This work reveals a new mechanism of translation inhibition and confirms Met-tRNAfMet as a prime target to efficiently block cell growth.
Subject(s)
Amino-Acid N-Acetyltransferase/metabolism , Escherichia coli , Gene Expression Regulation/genetics , RNA, Transfer, Met/metabolism , Acetylation , Electrophoresis, Gel, Two-Dimensional , Models, Biological , Protein BiosynthesisABSTRACT
The biosynthesis of the azabicyclic ring system of the azinomycin family of antitumor agents represents the "crown jewel" of the pathway and is a complex process involving at least 14 enzymatic steps. This study reports on the first biosynthetic step, the inroads, in the construction of the novel aziridino [1,2-a]pyrrolidine, azabicyclic core, allowing us to support a new mechanism for azabicycle formation.
Subject(s)
Aldehyde Oxidoreductases/metabolism , Amino-Acid N-Acetyltransferase/metabolism , Antineoplastic Agents, Alkylating/metabolism , Azabicyclo Compounds/metabolism , Bacterial Proteins/metabolism , Drug Design , Phosphotransferases (Carboxyl Group Acceptor)/metabolism , Pyrrolidines/metabolism , Acetyl Coenzyme A/metabolism , Acetylation , Aldehyde Oxidoreductases/genetics , Amino-Acid N-Acetyltransferase/genetics , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/pharmacology , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Bacterial Proteins/genetics , Biocatalysis , Dipeptides/chemistry , Dipeptides/metabolism , Dipeptides/pharmacology , Gene Knockout Techniques , Glutamic Acid/metabolism , Intercellular Signaling Peptides and Proteins , Molecular Structure , Mutation , Naphthalenes/chemistry , Naphthalenes/metabolism , Naphthalenes/pharmacology , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Phosphotransferases (Carboxyl Group Acceptor)/genetics , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Recombinant Proteins/metabolism , Streptomyces/enzymology , Streptomyces/metabolism , Substrate SpecificityABSTRACT
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.
Subject(s)
Alphaproteobacteria/enzymology , Amino-Acid N-Acetyltransferase/chemistry , Arginine/chemistry , Bacterial Proteins/chemistry , Protein Multimerization , Xanthomonas campestris/enzymology , Amino-Acid N-Acetyltransferase/metabolism , Animals , Arginine/metabolism , Bacterial Proteins/metabolism , Protein Structure, QuaternaryABSTRACT
BACKGROUND: N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. METHODS: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. RESULTS: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice. CONCLUSION: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.
Subject(s)
Amino-Acid N-Acetyltransferase/genetics , Carbamoyl-Phosphate Synthase (Ammonia)/genetics , Hyperammonemia/diagnosis , Urea Cycle Disorders, Inborn/diagnosis , Acetyl Coenzyme A/metabolism , Amino-Acid N-Acetyltransferase/metabolism , Animals , Carbamoyl-Phosphate Synthase (Ammonia)/deficiency , Humans , Hyperammonemia/genetics , Hyperammonemia/metabolism , Hyperammonemia/physiopathology , Liver/enzymology , Mice , Mice, Knockout , Tandem Mass Spectrometry , Urea Cycle Disorders, Inborn/genetics , Urea Cycle Disorders, Inborn/metabolism , Urea Cycle Disorders, Inborn/physiopathologyABSTRACT
N-acetyl-L-glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for â¼1/3 of mutations. Missense mutations frequency is inhomogeneous along NAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C-terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain-mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinant Pseudomonas aeruginosa NAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD-associated mutations found. The disease-causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation.
Subject(s)
Amino-Acid N-Acetyltransferase/genetics , Mutation, Missense , Urea Cycle Disorders, Inborn/genetics , Amino-Acid N-Acetyltransferase/chemistry , Amino-Acid N-Acetyltransferase/metabolism , Genetic Predisposition to Disease , Humans , Models, Molecular , Protein Binding , Protein Domains , Protein StabilityABSTRACT
Objetivos. Analizar la implicación clínica del aminoácido N-acetil-L-aspartato (NAA) y el péptido N-acetil-aspartil-glutamato (NAAG) en relación con su valoración diagnóstica y pronóstica mediante espectroscopia de resonancia magnética. Realizar una revisión del metabolismo del NAA y del NAAG, considerando su estructura química y fisiología, en relación con las variaciones de su concentración y en correlación con la clínica. Desarrollo. La revisión se divide en dos partes: en una se comprobó que el único sitio de síntesis del NAA es la mitocondria neuronal, y del NAAG, el citoplasma neuronal; la segunda parte aborda las técnicas de resonancia magnética y, particularmente, la espectroscopia. Se analizan diversas patologías en busca de criterios que posibiliten obtener pautas diagnósticas y pronósticas. Conclusiones. El estudio del aminoácido más abundante del sistema nervioso central (NAA) junto con un producto de su metabolismo, el NAAG, permite en patologías de diversos orígenes su diagnóstico y seguimiento y facilita la obtención de datos de densidad de la población celular y vitalidad de ésta, de manera que se accede, además, al estado funcional de las sinapsis (AU)
Aims. To analyse the clinical involvement of the amino acid N-acetyl-L-aspartate (NAA) and the peptide N-acetyl-aspartylglutamate (NAAG) regarding their diagnostic and prognostic value by means of magnetic resonance spectroscopy. To conduct a review of the metabolism of NAA and NAAG, bearing in mind their chemical structure and physiology, in terms of the variations in their concentration and the correlation with the clinical features. Development. The review is divided into two parts: in one it was found that the only site where NAA synthesis takes place is in the neuronal mitochondria, while the second part addresses magnetic resonance and, especially, spectroscopic techniques. An array of pathologies were analysed in search of criteria that allow diagnostic and prognostic guidelines. Conclusions. The study of the most abundant amino acid in the central nervous system (NAA) together with a product of its metabolism, NAAG, allows the diagnosis and follow-up of a variety of pathologies. At the same time, it makes it easier to obtain data about the density of the cell population and its vitality, thus also providing access to the functional status of the synapses (AU)
Subject(s)
Humans , Male , Female , Magnetic Resonance Spectroscopy/instrumentation , Magnetic Resonance Spectroscopy/methods , Magnetic Resonance Spectroscopy/therapeutic use , Amino-Acid N-Acetyltransferase/analysis , Amino-Acid N-Acetyltransferase/metabolism , Amino-Acid N-Acetyltransferase/therapeutic use , Aspartate Aminotransferases/analysis , Aspartate Aminotransferases/metabolism , Aspartate Aminotransferases/therapeutic use , Prognosis , Symptom Assessment/instrumentation , Symptom Assessment/methods , Symptom AssessmentABSTRACT
The small GTP-binding protein Ran is involved in the regulation of essential cellular processes in interphase but also in mitotic cells: Ran controls the nucleocytoplasmic transport of proteins and RNA, it regulates mitotic spindle formation and nuclear envelope assembly. Deregulations in Ran dependent processes were implicated in the development of severe diseases such as cancer and neurodegenerative disorders. To understand how Ran-function is regulated is therefore of highest importance. Recently, several lysine-acetylation sites in Ran were identified by quantitative mass-spectrometry, some being located in highly important regions such as the P-loop, switch I, switch II and the G5/SAK motif. We recently reported that lysine-acetylation regulates nearly all aspects of Ran-function such as RCC1 catalyzed nucleotide exchange, intrinsic nucleotide hydrolysis, its interaction with NTF2 and the formation of import- and export-complexes. As a hint for its biological importance, we identified Ran-specific lysine-deacetylases (KDACs) and -acetyltransferases (KATs). Also for other small GTPases such as Ras, Rho, Cdc42, and for many effectors and regulators thereof, lysine-acetylation sites were discovered. However, the functional impact of lysine-acetylation as a regulator of protein function has only been marginally investigated so far. We will discuss recent findings of lysine-acetylation as a novel modification to regulate Ras-protein signaling.
Subject(s)
Signal Transduction , ran GTP-Binding Protein/metabolism , ras Proteins/metabolism , Acetylation , Amino Acid Motifs , Amino-Acid N-Acetyltransferase/genetics , Amino-Acid N-Acetyltransferase/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Lysine/genetics , Lysine/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , ran GTP-Binding Protein/genetics , ras Proteins/geneticsABSTRACT
Folate metabolism is central to cell proliferation and a target of commonly used cancer chemotherapeutics. In particular, the mitochondrial folate-coupled metabolism is thought to be important for proliferating cancer cells. The enzyme MTHFD2 in this pathway is highly expressed in human tumors and broadly required for survival of cancer cells. Although the enzymatic activity of the MTHFD2 protein is well understood, little is known about its larger role in cancer cell biology. We here report that MTHFD2 is co-expressed with two distinct gene sets, representing amino acid metabolism and cell proliferation, respectively. Consistent with a role for MTHFD2 in cell proliferation, MTHFD2 expression was repressed in cells rendered quiescent by deprivation of growth signals (serum) and rapidly re-induced by serum stimulation. Overexpression of MTHFD2 alone was sufficient to promote cell proliferation independent of its dehydrogenase activity, even during growth restriction. In addition to its known mitochondrial localization, we found MTHFD2 to have a nuclear localization and co-localize with DNA replication sites. These findings suggest a previously unknown role for MTHFD2 in cancer cell proliferation, adding to its known function in mitochondrial folate metabolism.
Subject(s)
Amino-Acid N-Acetyltransferase/metabolism , Cell Nucleus/enzymology , Folic Acid/metabolism , Methylenetetrahydrofolate Dehydrogenase (NADP)/metabolism , Neoplasms, Experimental/enzymology , Neoplasms, Experimental/pathology , Animals , Cell Line, Tumor , Cell Proliferation , HeLa Cells , Humans , Mice , Mitochondria/metabolism , Nuclear Proteins/metabolism , Rats , Species SpecificityABSTRACT
BACKGROUND: Borna disease virus (BDV) is a neurotropic RNA virus persistently infecting mammalian hosts including humans. Lysine acetylation (Kac) is a key protein post-translational modification (PTM). The unexpectedly broad regulatory scope of Kac let us to profile the entire acetylome upon BDV infection. METHODS: The acetylome was profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. RESULTS: We identified and quantified 791 Kac sites in 473 Kac proteins in human BDV Hu-H1-infected and non-infected oligodendroglial (OL) cells. Bioinformatic analysis revealed that BDV infection alters the acetylation of metabolic proteins, membrane-associated proteins and transmembrane transporter activity, and affects the acetylation of several lysine acetyltransferases (KAT). CONCLUSIONS: Upon BDV persistence the OL acetylome is manipulated towards higher energy and transporter levels necessary for shuttling BDV proteins to and from nuclear replication sites.
Subject(s)
Amino-Acid N-Acetyltransferase/metabolism , Borna disease virus/physiology , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Oligodendroglia/metabolism , Protein Processing, Post-Translational , Proteome/metabolism , Acetylation , Amino Acid Sequence , Amino-Acid N-Acetyltransferase/genetics , Carrier Proteins/genetics , Cell Line , Computational Biology , Energy Metabolism , Fetus , Host-Pathogen Interactions , Humans , Isotope Labeling , Lysine/metabolism , Membrane Proteins/genetics , Molecular Sequence Annotation , Molecular Sequence Data , Oligodendroglia/virology , Protein Interaction Mapping , Proteome/genetics , Virus ReplicationABSTRACT
The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between palmitoyl coenzyme A (palmitoyl-CoA) and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular histone acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling.
Subject(s)
Acyl Coenzyme A/metabolism , Amino-Acid N-Acetyltransferase/metabolism , Histone Acetyltransferases/metabolism , Lysine/metabolism , Acetylation , Acyl Coenzyme A/biosynthesis , Acyl Coenzyme A/chemistry , Amino-Acid N-Acetyltransferase/chemistry , HEK293 Cells , Histone Acetyltransferases/chemistry , Humans , Kinetics , Lysine/chemistry , Models, Chemical , Palmitoyl Coenzyme A/chemistry , Palmitoyl Coenzyme A/metabolism , ProteomicsABSTRACT
N-acetylglutamate synthase (NAGS) catalyzes the production of N-acetylglutamate (NAG) from acetyl-CoA and L-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups.
Subject(s)
Amino-Acid N-Acetyltransferase/chemistry , Amino Acid Sequence , Amino-Acid N-Acetyltransferase/metabolism , Animals , Bacteria/enzymology , Catalytic Domain , Humans , Molecular Sequence DataABSTRACT
Even though protein initiator methionine excision (NME) and N-terminal acetylation (NTA) have been relatively well investigated in eukaryotic proteomes, few studies were dedicated to these modifications in bacteria up to now. In this work, we investigated, for the first time, the N-terminal proteome of the bacterium Pseudomonas aeruginosa PA14 by studying the NME and NTA processes using proteomic approaches. For NME, most of proteins had their initiator Met cleaved (63%) and the nature of the penultimate residue seems to be essential for this cleavage. Concerning NTA, two methods were applied (protein fractionation and peptide enrichment). This allowed us to identify 117 Nα-acetylated proteins, among them 113 have not yet been described as modified in bacteria. Most often, the non-acetylated form was over-represented compared to the acetylated form, arguing that this latter was a minor part of the total abundance of a given protein. Furthermore, some proteins with acetylated initiator methionine were observed. The present work significantly enlarges the number of N-terminally modified proteins in bacteria and confirms that these modifications are a general and fundamental process, not only restricted to eukaryotes. BIOLOGICAL SIGNIFICANCE: Protein modifications in prokaryotes have been detected more recently than in eukaryotes. Methionine cleavage and N-terminal acetylation are two common protein N-terminal modifications. Despite their importance in bacterial processes, they are less investigated. The characterization of N-terminal acetylation in bacteria is a challenge because no antibody exists and it is a less frequent modification than in eukaryotes. We used proteomic approaches (enrichment, fractionation, nanoLC-MS/MS, and bioinformatic analyses) to investigate the N-terminal methionine excision and to profile the N-terminal acetylome of P. aeruginosa strain PA14. From our results, around 60% of the proteins had their iMet cleaved. In total, 117 proteins were identified constituting the largest dataset in prokaryotes. Among them, proteins kept their initiator methionine and were acetylated. These results may facilitate the design of experiments to better understand the role of acetylation at the protein N-terminus of P. aeruginosa PA14.