Argininosuccinate Synthetase Gene Research Articles

Citrullinemia type I in Chinese children: Identification of two novel argininosuccinate synthetase gene mutations.

BackgroundIn this study, we evaluated the clinical characteristics, prognosis, and gene mutations of five children with citrullinemia type I (CTLN1) diagnosed in our department and identified two novel ASS1 gene mutations.MethodsWe examined the clinical characteristics, prognosis, and gene mutations of the five children through data collection, tandem mass spectrometry, and whole-exon sequencing. MutationTaster, regSNP-intron, and SWISS-MODEL were used for bioinformatic analysis to evaluate the two novel gene mutations. We analyzed differences in blood ammonia and citrulline levels based on clinical phenotypes. Finally, we reviewed the medical literature describing Chinese children with CTLN1.ResultsASS1 C773 + 6T > G and c.848 delA as well as c.952_953 del insTT and c.133G > A have not been previously reported in the Human Gene Mutation Database. Using MutationTaster and regSNP-intron, we predicted that these mutations affected protein function. The 3D structure obtained using SWISS-MODEL supported this prediction. Through comparative analysis showed that the ammonia level of the neonatal type was markedly higher than that of other types, whereas citrulline levels did not differ between groups.ConclusionWe identified two novel mutations that cause disease. The blood ammonia level of neonatal form citrullinemia was markedly higher than that of other types. The genotype-phenotype association in Chinese patients remains unclear and should be further evaluated in genetic studies of larger sample sizes.

The argininosuccinate synthetase can differentially regulate nitric oxide synthase in yellow catfish Pelteobagrus fulvidraco

Fish are at high risk of exposure to ammonia in aquaculture systems. When ammonia stress occurs, fish are more prone to disease outbreaks, but the mechanism is not very clear. The argininosuccinate synthetase (ASS) plays an important role in the regulation of urea synthesis and nitric oxide synthesis. We speculated that there must be some relationship between ASS expression and disease outbreak. In this study, ASS was cloned from the yellow catfish. The full-length cDNAs of ASS was 1558 bp, with open reading frames of 1236 bp. The mRNA expression of ASS gene was the highest in liver, kidney and brain. This study consists of two parts: 1) For ammonia challenge in vivo, yellow catfish (15.00 ± 1.50 g) were divided into control group, low ammonia group (1/10 96 h LC50), and high ammonia group (1/2 96 h LC50). The experiment continued for 192 h. The results showed that ammonia stress elevated serum ammonia content, and inhibited urea synthesis enzymes activities but up-regulated the expression levels of related genes except ARG, and induced arginine accumulation and nitric oxide synthase (nNOS and iNOS) different expression, and decreased resistance to Aeromonas hydrophage; 2) For ammonia challenge in vitro, the primary culture of liver cell was divided into four groups: control group, BPP group (Bj-BPP-10c was added as ASS activator), Amm group (96 h LC50), and Amm + BPP group. The experiment continued for 96 h. The results showed that the Bj-BPP-10c can inhibit nNOS activity and improve cell survival rate, and enhance iNOS activity and immune response (lysozyme, complement, respiratory burst, and phagocytic index) by activate ASS when ammonia stress occurred. Our results indicated that targeted regulation of ASS can improve iNOS activity, and enhance the immune response of yellow catfish under ammonia stress.

Suitability of nitisinone for alkaptonuria

In many, but not all, genetic metabolic conditions, biochemical markers are used in the diagnosis and monitoring of patients. However, these markers are not always relevant for the pathogenesis of the respective conditions. For example, in the classical urea cycle disorder citrullinaemia type 1, 1 Diez-Fernandez C Rüfenacht V Häberle J Mutations in the human argininosuccinate synthetase (ASS1) gene, impact on patients, common changes, and structural considerations. Hum Mutat. 2017; 38: 471-484 Crossref PubMed Scopus (30) Google Scholar the metabolite citrulline, an amino acid, is greatly elevated in the blood and urine of affected patients but has no known role in the pathogenesis of the condition (but rather the accompanying hyperammonaemia). In other conditions, an abnormal metabolite is crucial for developing disease; for example, the phenolic acid homogentisic acid is markedly elevated in the blood and urine in alkaptonuria, a disorder of phenylalanine and tyrosine degradation. 2 Phornphutkul C Introne WJ Perry MB et al. Natural history of alkaptonuria. N Engl J Med. 2002; 347: 2111-2121 Crossref PubMed Scopus (443) Google Scholar , 3 Ranganath LR Cox TF Natural history of alkaptonuria revisited: analyses based on scoring systems. J Inherit Metab Dis. 2011; 34: 1141-1151 Crossref PubMed Scopus (57) Google Scholar Importantly, this metabolite is not only elevated but also considered to be central to the many complications of alkaptonuria, which mainly occur in adulthood. Unfortunately, once these complications develop, the most prominent of which are damage to ochronotic tissue resulting in premature spondyloarthritis, tendon ruptures, cardiac valve disease, and nephropathy, they are irreversible. Efficacy and safety of once-daily nitisinone for patients with alkaptonuria (SONIA 2): an international, multicentre, open-label, randomised controlled trialNitisinone 10 mg daily was well tolerated and effective in reducing urinary excretion of HGA. Nitisinone decreased ochronosis and improved clinical signs, indicating a slower disease progression. Full-Text PDF

Mutations in the Human Argininosuccinate Synthetase (ASS1) Gene, Impact on Patients, Common Changes, and Structural Considerations.

Citrullinemia type 1 is an autosomal recessive urea cycle disorder caused by defects in the argininosuccinate synthetase (ASS) enzyme due to mutations in ASS1 gene. An impairment of ASS function can lead to a wide spectrum of phenotypes, from life-threatening neonatal hyperammonemia to a later onset with mild symptoms, and even some asymptomatic patients exhibiting an only biochemical phenotype. The disease is panethnic. In this update, we report 137 mutations (64 of which are novel), consisting of 89 missense mutations, 19 nonsense mutations, 17 mutations that affect splicing, and 12 deletions. The change p.Gly390Arg is by far the most common mutation and is widely spread throughout the world. Other frequent mutations (p.Arg157His, p.Trp179Arg, p.Val263Met, p.Arg304Trp, p.Gly324Ser, p.Gly362Val, and p.Arg363Trp), each found in at least 12 independent families, are mainly carried by patients from the Indian subcontinent, Turkey, Germany, and Japan. To better understand the disease, we collected clinical data of >360 patients, including all published information available. This information is related to the patients' genetic background, the conservation of the mutated residues and a structural rationalization of the effect of the most frequent mutations. In addition, we review ASS regulation, animal models, diagnostic strategies, newborn screening, and treatment options.

Expression profile and down-regulation of argininosuccinate synthetase in hepatocellular carcinoma in a transgenic mouse model

BackgroundArgininosuccinate synthetase (ASS) participates in urea and nitric oxide production and is a rate-limiting enzyme in arginine biosynthesis. Regulation of ASS expression appears complex and dynamic. In addition to transcriptional regulation, a novel post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. Moreover, many cancers, including hepatocellular carcinoma (HCC), have been found not to express ASS mRNA; therefore, they are auxotrophic for arginine. To study when and where ASS is expressed and whether post-transcriptional regulation is undermined in particular temporal and spatial expression and in pathological events such as HCC, we set up a transgenic mouse system with modified BAC (bacterial artificial chromosome) carrying the human ASS gene tagged with an EGFP reporter.ResultsWe established and characterized the transgenic mouse models based on the use of two BAC-based EGFP reporter cassettes: a transcription reporter and a transcription/post-transcription coupled reporter. Using such a transgenic mouse system, EGFP fluorescence pattern in E14.5 embryo was examined. Profiles of fluorescence and that of Ass RNA in in situ hybridization were found to be in good agreement in general, yet our system has the advantages of sensitivity and direct fluorescence visualization. By comparing expression patterns between mice carrying the transcription reporter and those carrying the transcription/post-transcription couple reporter, a post-transcriptional up-regulation of ASS was found around the ventricular zone/subventricular zone of E14.5 embryonic brain. In the EGFP fluorescence pattern and mRNA level in adult tissues, tissue-specific regulation was found to be mainly controlled at transcriptional initiation. Furthermore, strong EGFP expression was found in brain regions of olfactory bulb, septum, habenular nucleus and choroid plexus of the young transgenic mice. On the other hand, in crossing to hepatitis B virus X protein (HBx)-transgenic mice, the Tg (ASS-EGFP, HBx) double transgenic mice developed HCC in which ASS expression was down-regulated, as in clinical samples.ConclusionsThe BAC transgenic mouse model described is a valuable tool for studying ASS gene expression. Moreover, this mouse model is a close reproduction of clinical behavior of ASS in HCC and is useful in testing arginine-depleting agents and for studies of the role of ASS in tumorigenesis.

L-citrulline production by metabolically engineered Corynebacterium glutamicum from glucose and alternative carbon sources

L-citrulline plays an important role in human health and nutrition and is an intermediate of the L-arginine biosynthetic pathway. L-citrulline is a by-product of L-arginine production by Corynebacterium glutamicum. In this study, C. glutamicum was engineered for overproduction of L-citrulline as major product without L-arginine being produced as by-product. To this end, L-arginine biosynthesis was derepressed by deletion of the arginine repressor gene argR and conversion of L-citrulline towards L-arginine was avoided by deletion of the argininosuccinate synthetase gene argG. Moreover, to facilitate L-citrulline production the gene encoding a feedback resistant N-acetyl L-glutamate kinase argBfbr as well as the gene encoding L-ornithine carbamoylphosphate transferase argF were overexpressed. The resulting strain accumulated 44.1 ± 0.5 mM L-citrulline from glucose minimal medium with a yield of 0.38 ± 0.01 g⋅g−1 and a volumetric productivity of 0.32 ± 0.01 g⋅l−1⋅h−1. In addition, production of L-citrulline from the alternative carbon sources starch, xylose, and glucosamine could be demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-014-0085-0) contains supplementary material, which is available to authorized users.

Abstract 1435: Exploiting obligate arginine auxotrophy in tumor cells lacking arginino-succinate synthetase (ASS) expression to develop targeted molecular therapy for non-small cell lung cancer (NSCLC)

Abstract Background. Malignant cells frequently exhibit dysregulation of nutrient/energy metabolism that can be exploited for development of novel targeted molecular therapy. Transcriptional repression of ASS, an enzyme essential for arginine production, in certain cancers and not normal cells makes these tumor cells selectively auxotrophic (dependent on external source) of this semi-essential amino acid and extremely susceptible to arginine depletion. One of the clinically applicable strategies to deplete extracellular arginine is systemic administration of the enzyme arginine di-iminase (ADI) conjugated with polyethylene glycol (PEG20) for optimal bioavailability. While well characterized in malignant pleural mesothelioma, melanoma and hepatocellular carcinoma, the status of ASS expression and sensitivity to ADI-PEG20 has not previously been investigated in NSCLC and this is the overarching objective of this study. Materials and Methods. ASS expression in 12 NSCLC cell lines and normal cells is determined by quantitative RT-PCR, western blots. ASS expression in lung cancers is quantified by immunohistochemistry (IHC) of tissue microarray of 72 NSCLC tumors. ADI-PEG20-mediated inhibition of cell viability, autophagy activation, induction of apoptosis and cell cycle arrest are assayed using MTT, fluorescence microscopy, AnnexinV/PI staining with flow cytometry respectively. Methylation of the promotor region of the ASS gene in ASS-negative cells is evaluated by methylation-specific PCR. Results. Six of 12 NSCLC cells have no or extremely low basal expression of ASS mRNA and protein and are, thus, exquisitely sensitive to ADI-PEG20 treatment (IC50 values: 45± 5 to 90±8 ng/ml (mean ± standard deviation, n=6)). ASS+ NSCLC cells and normal cells (primary or immortalized human fibroblasts) are totally resistant to ADI-PEG20. About 80% of NSCLC tumors do not express ASS by IHC using a well-validated anti-ASS antibody (Santa Cruz, clone H231). ADI-PEG20 treatment strongly activates autophagy with accumulation of autophagic vacuoles detected by fluorescence microscopy. Significant induction of apoptosis (15±3% to 55±6%, n=4) is noted in 5 NSCLC cells treated with ADI-PEG20 (100 or 200 ng/ml for 4 or 7 days) as well as cell cycle arrest. Two cell lines have methylated ASS promotor (one complete, one partial) as the cause of gene silencing. Conclusion. Up to 50% of NSCLC cell lines and 80% of NSCLC tumors lack ASS expression. ASS- NSCLC are selectively and significantly susceptible to ADI-PEG20-mediated arginine depletion in vitro. Induction of autophagy, apoptosis and cell cycle arrest collectively contribute ADI-PEG20-mediated cytotoxicity. As ADI-PEG20 is currently in phase II clinical trials for other carcinomas with encouraging results, it can be used to develop novel targeted molecular therapy for NSCLC tumors lacking ASS. Citation Format: Min You, Medhi Wangpaichitr, Jonathan D. Nguyen, Jennifer R. Chapman, Maureen Cioffi-Lavina, Niramol Savaraj, Dao M. Nguyen. Exploiting obligate arginine auxotrophy in tumor cells lacking arginino-succinate synthetase (ASS) expression to develop targeted molecular therapy for non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1435. doi:10.1158/1538-7445.AM2014-1435

A transgenic approach to study argininosuccinate synthetase gene expression.

BackgroundArgininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed.ResultsTwo lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene.ConclusionsWe demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene.

Concurrent triplication and uniparental isodisomy: evidence for microhomology-mediated break-induced replication model for genomic rearrangements

Whole-genome oligonucleotide single-nucleotide polymorphism (oligo-SNP) arrays enable simultaneous interrogation of copy number variations (CNVs), copy neutral regions of homozygosity (ROH) and uniparental disomy (UPD). Structural variation in the human genome contributes significantly to genetic variation, and often has deleterious effects leading to disease causation. Co-occurrence of CNV and regions of allelic homozygosity in tandem involving the same chromosomal arm are extremely rare. Replication-based mechanisms such as microhomology-mediated break-induced replication (MMBIR) are recent models predicted to induce structural rearrangements and gene dosage aberrations; however, supportive evidence in humans for one-ended DNA break repair coupled with MMBIR giving rise to interstitial copy number gains and distal loss of heterozygosity has not been documented. We report on the identification and characterization of two cases with interstitial triplication followed by uniparental isodisomy (isoUPD) for remainder of the chromosomal arm. Case 1 has a triplication at 9q21.11-q21.33 and segmental paternal isoUPD for 9q21.33-qter, and presented with citrullinemia with a homozygous mutation in the argininosuccinate synthetase gene (ASS1 at 9q34.1). Case 2 has a triplication at 22q12.1-q12.2 and segmental maternal isoUPD 22q12.2-qter, and presented with hearing loss, mild dysmorphic features and bilateral iris coloboma. Interstitial triplication coupled with distal segmental isoUPD is a novel finding that provides human evidence for one-ended DNA break and replication-mediated repair. Both copy number gains and isoUPD may contribute to the phenotype. Significantly, these cases represent the first detailed genomic analysis that provides support for a MMBIR mechanism inducing copy number gains and segmental isoUPD in tandem.

Argininosuccinate synthetase gene is silenced by CpG methylation in children with phenylketonuria

ObjectivesThe concentration of tyrosine and the ratio of branch-amino acid to the aromatic amino acid in phenylketonuria (PKU) patients are much lower than that of normal people, which reveal that PKU patients have amino acid metabolism disorder. The aim of the present study was to investigate the arginine level in blood, the expression of argininosuccinate synthetase (ASS), the rate-limiting enzyme in arginine synthesis pathway, and the methylation of ASS in patients with PKU. Design and MethodsTwenty-five children with PKU and 65 healthy controls were investigated in this study. Blood concentration of arginine was analyzed by automatic amino acid analyzer. The methylation of ASS gene promoter was evaluated by using methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) methods, and the mRNA level of ASS was evaluated by semi-quantitative RT-PCR. ResultsBlood concentration of arginine in PKU patients without dietary control was 0.017±0.009mmol/L while in normal persons was 0.129±0.007mmol/L, which is statistically significant (P<0.001). The promoter of ASS was methylated in PKU (15/15, 100%) but not in normal persons (0/15). The mRNA level of ASS in PKU patients was lower than that of normal people, which was well correlated with its methylation status. ConclusionsThe silencing of ASS due to aberrant promoter CpG methylation may be an important mechanism for arginine biosynthesis disorders in PKU. High levels of phenylalanine and low levels of arginine are common characteristics in PKU patients. These findings would extend the current understanding of arginine, ASS in the development of PKU disease.

Citrullinemia type 1: Genetic diagnosis and prenatal diagnosis in subsequent pregnancy

Citrullinemia type 1 was diagnosed by tandem mass spectrometry in a full term male neonate who presented with an acute catastrophic collapse on the 3rd day of life. Both parents were identified to be carriers for the exon 15 p Gly390Arg mutation in the argininosuccinate synthetase gene located at chromosome 9q34.1. Chorionic villus sampling and prenatal genetic testing in the subsequent pregnancy revealed an affected fetus resulting in termination of pregnancy.

Hepatic Adaptation Compensates Inactivation of Intestinal Arginine Biosynthesis in Suckling Mice

Suckling mammals, including mice, differ from adults in the abundant expression of enzymes that synthesize arginine from citrulline in their enterocytes. To investigate the importance of the small-intestinal arginine synthesis for whole-body arginine production in suckling mice, we floxed exon 13 of the argininosuccinate synthetase (Ass) gene, which codes for a key enzyme in arginine biosynthesis, and specifically and completely ablated Ass in enterocytes by crossing Ass fl and Villin-Cre mice. Unexpectedly, Ass fl/fl /VilCre tg/- mice showed no developmental impairments. Amino-acid fluxes across the intestine, liver, and kidneys were calculated after determining the blood flow in the portal vein, and hepatic and renal arteries (86%, 14%, and 33%, respectively, of the transhepatic blood flow in 14-day-old mice). Relative to control mice, citrulline production in the splanchnic region of Ass fl/fl /VilCre tg/- mice doubled, while arginine production was abolished. Furthermore, the net production of arginine and most other amino acids in the liver of suckling control mice declined to naught or even changed to consumption in Ass fl/fl /VilCre tg/- mice, and had, thus, become remarkably similar to that of post-weaning wild-type mice, which no longer express arginine-biosynthesizing enzymes in their small intestine. The adaptive changes in liver function were accompanied by an increased expression of genes involved in arginine metabolism (Asl, Got1, Gpt2, Glud1, Arg1, and Arg2) and transport (Slc25a13, Slc25a15, and Slc3a2), whereas no such changes were found in the intestine. Our findings suggest that the genetic premature deletion of arginine synthesis in enterocytes causes a premature induction of the post-weaning pattern of amino-acid metabolism in the liver.

Abstract 3652: Pegylated arginine deiminase (ADI-PEG20) as a potential therapeutic agent for rarer variants of bladder cancer that are deficient for argininosuccinate synthetase

Abstract Background: A previous study had identified microsatellite instability in the argininosuccinate synthetase (ASS1) gene locus in urothelial cancers (UCC), although the significance of this finding was unclear. ASS1 plays a critical role in the biosynthesis of arginine from citrulline and a deficiency of ASS1 leads to arginine auxotrophy. This can be further exacerbated by administration of the pegylated form of arginine deiminase (ADI-PEG20), which has been shown to carry low toxicity in phase I/II clinical trials. Thus, reduced levels of ASS1 can serve as an indicator for ADI-PEG20-responsive tumors. We therefore evaluated the expression of ASS1 in bladder cancer variants to explore the possibility of expanding therapeutic avenues for this population. Design: Formalin fixed, paraffin embedded samples of normal urothelium (n=19), conventional UCC (n=148), and other variants including the micropapillary variant of UCC (n=17), small cell carcinoma (n=19), squamous cell carcinoma (n=39) and adenocarcinoma (n=19), were used in the construction of tissue microarrays (TMAs). TMAs were immunostained for ASS1 and scored based on the intensity of stain (0-3+; high staining was considered 2-3+). The results were then correlated with various parameters such as age at diagnosis, sex, race and pathologic staging. Also, these results were correlated with ASS1 expression as determined by western blotting in multiple bladder cancer derived cell-lines. Results: Compared to normal urothelium where 68.4% of the cases showed high expression of ASS, conventional UCCs demonstrated lower levels of staining (41% of the non-metastatic subset; n=61 and 52.8% of the metastatic subset; n=87). Surprisingly, the paired lymph node metastases from the latter group showed high staining in only 31.5% (n=38) of the patients. High levels of ASS1 immunoreactivity were maintained in the micropapillary variant of UCC (76.4% of cases) and pure bladder adenocarcinoma (84.2%). In contrast, both pure small cell and squamous cell carcinoma of the bladder demonstrated marked reduction in ASS1 expression: only 26.3% of small cell carcinoma specimens and 10.2% of the squamous cell carcinoma specimens had high expression of ASS1, suggesting these two subtypes of bladder cancer could potentially respond to ADI-PEG20 therapy. We have also identified 6 bladder cancer cell-lines (including one derived from a squamous cell carcinoma) that are deficient for ASS1, and a primary cell-line which has high levels of ASS1 expression. These can serve as excellent tools for further in vitro studies. Conclusion: ADI-PEG20 represents a potentially exciting new therapeutic agent for metastatic conventional UCC, small cell carcinoma and squamous cell carcinoma. This is especially relevant as limited therapies are currently available for these less common forms of bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3652. doi:1538-7445.AM2012-3652

Abstract 4096: TRAIL enhances cytotoxicity of arginine depletion therapy in argininosuccinate synthetase-negative melanoma cells through interruption of autophagy via activation of caspases

Abstract Melanomas which do not express argininosuccinate synthetase (ASS) cannot make arginine from citrulline; thus arginine depletion caused by pegylated arginine deiminase (ADI-PEG20, provided by Polaris Inc.) results in growth inhibition and eventually leads to cell death. However, autophagy does take place during arginine deprivation which prevents cell death. During this period, certain melanoma cells can turn on the ASS gene and hence become resistant to this form of treatment. In order to increase the effectiveness of ADI-PEG20 therapy, we have previously found that combining tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with ADI-PEG20 can accelerate cell death (apoptosis) process and hence greatly enhance cytotoxicity (Biochem. Biophys. Res. Commun. 2010; 394(3):760-6). We have found that the addition of TRAIL increases formation of tBid. In this study, we found that with the combination treatment, autophagy-related protein Atg5 and Beclin-1 are cleaved which suggests that autophagic process is interrupted and apoptosis can take place. In order to investigate whether caspase activation plays a role in Atg5 and Beclin-1 cleavage, we have used specific caspase inhibitors (all at 25 nM) to address this question. A375 (ASS non-inducible) and A2058 (ASS inducible) cell lines were used for this study. About 70-80 % cell death occurs at 24 h after combination treatment, while only 10-20% cell death occurs when treated with TRAIL alone. Almost no cell death is apparent when treated with arginine-free medium alone at this time point. Adding caspase-8 (Z-IETD-FMK), caspase-9 (Z-LEHD-FMK) or caspase-10 (Z-AEVD-FMK) single inhibitor can almost completely rescue cell death on combination treatment while caspase-6 inhibitor (Z-VEID-FMK) has less rescuing effect (about 10-30% cell death). Inhibitors for caspase-3 (Z-DEVD-FMK) or 3/7 (Ac-DNLD-CHO) have least effect (more than 50% cell death). These data confirm that the enhancement effect of the combination needs the participation of both the extrinsic and intrinsic pathways of apoptosis. The cleavage of Beclin-1 and Atg5 are also least affected when caspase-3 or 3/7 inhibitor is applied, but blocked or reduced when single inhibitor to caspase-6, 8, 10 or 9 is added. There is little difference between the ASS inducible or non-inducible cell lines. This finding suggests that the combination treatment turns off the autophagic process during arginine deprivation, possibly through caspase activation and allows apoptotic process to take place. Overall, our findings indicate that autophagy is a pro-survival mechanism in melanoma cells under nutritional stress, and interfering with this pathway may achieve greater efficacy with ADI-PEG20 treatment. (Supported by NIH grant 1R01CA109578) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4096. doi:10.1158/1538-7445.AM2011-4096

A Novel Argininosuccinate Synthetase Gene Mutation in a Korean Family with Type I Citrullinemia

A Novel Argininosuccinate Synthetase Gene Mutation in a Korean Family with Type I Citrullinemia

Citrullinemia Type I, Classical Variant. Identification of ASS∼p.G390R (c.1168G > A) mutation in families of a limited geographic area of Argentina: A possible population cluster

Objective Citrullinemia type I (CTLN1) is an urea cycle defect caused by mutations in the argininosuccinate synthetase gene. We report the first identification in Argentina of patients with CTLN1 in a limited geographic area. Design and methods Molecular analysis in patient/relatives included PCR, sequencing and restriction enzyme assay. Results The studied families showed the same mutation: ASS∼p.G390R, associated with the early-onset/severe phenotype. Conclusion We postulate a possible population cluster. A program to know the carrier frequency in that population is in progress.

Mutations and polymorphisms in the human argininosuccinate synthetase (ASS1) gene

Citrullinemia type I is an autosomal recessive disorder that is caused by a deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS1). Deficiency of ASS1 shows various clinical manifestations encompassing severely affected patients with fatal neonatal hyperammonemia as well as asymptomatic individuals with only a biochemical phenotype. This is a comprehensive report of all 87 mutations found to date in the ASS1 gene on chromosome 9q34.1. A large proportion of the mutations (n=27) are described here for the first time. Mutations are distributed throughout exons 3 to 15, most of them being identified in exons 5, 12, 13, and 14. The mutation G390R in exon 15 is the single most common mutation in patients with the classical phenotype. Certain mutations clearly link to specific clinical courses but the clinical phenotype cannot be anticipated in all patients. This update presents a survey of the correlation between mutations in the ASS1 gene and the respective clinical courses as described so far. It also sheds light on the geographic incidence of the mutations. Enzymatic studies have been done in bacterial and human cell systems. However, the prognostic value of genetic aberrations with respect to their effect on protein function and clinical manifestation remains uncertain.

Identification of a liver-specific cAMP response element in the human argininosuccinate synthetase gene

Argininosuccinate synthetase (ASS), a key enzyme in the urea cycle, participates in many metabolic processes including arginine biosynthesis and the citrulline–nitric oxide (NO) cycle. Factors like diets, hormones and pro-inflammatory stimuli are known to regulate ASS gene expression primarily at the transcription level. However, little is known about the cis-elements for transcriptional regulation of the ASS gene. In this study, we employed DNase I hypersensitive sites mapping to identify potential regulatory sites of the gene and revealed a site located at 10 kb upstream of the transcription start site which is responsible for liver-specific cAMP induction. Furthermore, a cAMP response element (CRE) highly conserved among mammals was identified and was experimentally verified. Our results show that liver-specific enhancement of ASS gene expression is mediated in part by the cAMP signaling pathway through a distal CRE site.

Glutamine and interleukin‐1β interact at the level of Sp1 and nuclear factor‐κB to regulate argininosuccinate synthetase gene expression

We previously demonstrated that the expression of the argininosuccinate synthetase (ASS) gene, a key step in nitric oxide production, is stimulated either by interleukin-1beta[Brasse-Lagnel et al. (2005) Biochimie 87, 403-9] or by glutamine in Caco-2 cells [Brasse-Lagnel et al. (2003) J. Biol. Chem. 278, 52504-10], through the activation of transcription factors nuclear factor-kappaB and Sp1, respectively. In these cells, the fact that glutamine stimulated the expression of a gene induced by pro-inflammatory factors appeared paradoxical as the amino acid is known to exert anti-inflammatory properties in intestinal cells. We therefore investigated the effect of simultaneous addition of both glutamine and interleukin-1beta on ASS gene expression in Caco-2 cells. In the presence of both compounds for 4 h, the increases in ASS activity, protein amount and mRNA level were almost totally inhibited, implying a reciprocal inhibition between the amino acid and the cytokine. The inhibition was exerted at the level of the transcription factors Sp1 and nuclear-kappaB: (a) interleukin-1beta inhibited the glutamine-stimulated DNA-binding of Sp1, which might be related to a decrease of its glutamine-induced O-glycosylation, and (b) glutamine induced per se a decrease in the amount of nuclear p65 protein without affecting the stimulating effect of interleukin-1beta on nuclear factor-kappaB, which might be related to the metabolism of glutamine into glutamate. The present results constitute the first demonstration of a reciprocal inhibition between the effects of an amino acid and a cytokine on gene expression, and provide a molecular basis for the protective role of glutamine against inflammation in the intestine.

Characterization of Late-Onset Citrullinemia 1 in a Korean Patient: Confirmation by Argininosuccinate Synthetase Gene Mutation Analysis

A 16-month old boy was referred to our hospital for evaluation of recurrent generalized tonic clonic seizures. Metabolic evaluation revealed significant hyperammonemia (1,112 microg/dl). Amino acid/acylcarnitine screening using tandem mass spectrometry showed markedly increased plasma levels of citrulline (1,350 microM/l) with undetectable levels of arginine and arginosuccinic acid. Urinary excretion of citrulline was markedly increased (38,617 microM/g creatinine). Brain MRI findings showed diffuse high-signal intensity lesions, that involved gray and white matter in both frontal lobes and insula with edematous changes; these findings were consistent with the acute stage of citrullinemia (CTLN). Mutation analysis of the argininosuccinate synthetase (ASS) gene, in this patient, showed a Gly324Ser mutation in exon 13, and a 67-bp duplication mutation in exon 15 (c.1128-6_1188dup67). The patient was confirmed as having late-onset CTLN1 and treated with anticonvulsants, lactulose enema, protein restricted diet and arginine. Here we describe a case of late-onset CTLN1 in a patient by biochemical analyses and ASS gene mutation confirmation. This is the first report of a Korean patient with late-onset CTLN1 confirmed by ASS gene mutation identification.