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Argininosuccinate lyase (ASL) deficiency is an autosomal recessive disorder of the urea cycle with a diverse spectrum of clinical presentation that is detectable in newborn screening. We report an 8-year-old girl with ASL deficiency who was detected through newborn screening and was confirmed using biochemical and functional assay. She is compound heterozygous for a likely pathogenic variant NM_000048.4(ASL):c.283C>T (p.Arg95Cys) and a likely benign variant NM_000048.4(ASL): c.1319T>C (p.Leu440Pro). Functional characterisation of the likely benign genetic variant in ASL was performed. Genomic sequencing was performed on the index patient presenting with non-specific symptoms of poor feeding and lethargy and shown to have increased serum and urine argininosuccinic acid. Functional assay using HEK293T cell model was performed. ASL enzymatic activity was reduced for Leu440Pro. This study highlights the role of functional testing of a variant that may appear benign in a patient with a phenotype consistent with ASL deficiency, and reclassifies NM_000048.4(ASL): c.1319T>C (p.Leu440Pro) variant as likely pathogenic., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Cancer-specific TERT promoter mutations have been linked to the reactivation of epigenetically silenced TERT gene by creating de novo binding motifs for E-Twenty-Six transcription factors, especially GABPA. How these mutations switch on TERT from epigenetically repressed states to expressed states have not been defined. Here, we revealed that EGFR activation induces ERK1/2-dependent phosphorylation of argininosuccinate lyase (ASL) at Ser417 (S417), leading to interactions between ASL and GABPA at the mutant regions of TERT promoters. The ASL-generated fumarate inhibits KDM5C, leading to enhanced trimethylation of histone H3 Lys4 (H3K4me3), which in turn promotes the recruitment of c-Myc to TERT promoters for TERT expression. Expression of ASL S417A, which abrogates its binding with GABPA, results in reduced TERT expression, inhibited telomerase activity, shortened telomere length, and impaired brain tumor growth in mice. This study reveals an unrecognized mechanistic insight into epigenetically activation of mutant TERT promoters where GABPA-interacted ASL plays an instrumental role., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Key Message: This study revealed different catalytic efficiencies of cyanobacterial argininosuccinate lyases in non-nitrogen-fixing and nitrogen-fixing cyanobacteria, demonstrating that L-arginine inhibition of L-argininosuccinate lyase is conserved among enzymes of three cyanobacterial orders. Arginine is a nitrogen-rich amino acid that uses a nitrogen reservoir, and its biosynthesis is strictly controlled by feedback inhibition. Argininosuccinate lyase (EC 4.3.2.1) is the final enzyme in arginine biosynthesis that catalyzes the conversion of argininosuccinate to L-arginine and fumarate. Cyanobacteria synthesize intracellular cyanophycin, which is a nitrogen reservoir composed of aspartate and arginine. Arginine is an important source of nitrogen for cyanobacteria. We expressed and purified argininosuccinate lyases, ArgHs, from Synechocystis sp. PCC 6803, Nostoc sp. PCC 7120, and Arthrospira platensis NIES-39. The catalytic efficiency of the Nostoc sp. PCC 7120 ArgH was 2.8-fold higher than those of Synechocystis sp. PCC 6803 and Arthrospira platensis NIES-39. All three ArgHs were inhibited in the presence of arginine, and their inhibitory effects were lowered at pH 7.0, compared to those at pH 8.0. These results indicate that arginine inhibition of ArgH is widely conserved among the three cyanobacterial orders. The current results demonstrate the conserved regulation of enzymes in the cyanobacterial aspartase/fumarase superfamily., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Argininosuccinate lyase (ASL) is essential for the NO-dependent regulation of tyrosine hydroxylase (TH) and thus for catecholamine production. Using a conditional mouse model with loss of ASL in catecholamine neurons, we demonstrate that ASL is expressed in dopaminergic neurons in the substantia nigra pars compacta, including the ALDH1A1  +  subpopulation that is pivotal for the pathogenesis of Parkinson disease (PD). Neuronal loss of ASL results in catecholamine deficiency, in accumulation and formation of tyrosine aggregates, in elevation of α-synuclein, and phenotypically in motor and cognitive deficits. NO supplementation rescues the formation of aggregates as well as the motor deficiencies. Our data point to a potential metabolic link between accumulations of tyrosine and seeding of pathological aggregates in neurons as initiators for the pathological processes involved in neurodegeneration. Hence, interventions in tyrosine metabolism via regulation of NO levels may be therapeutic beneficial for the treatment of catecholamine-related neurodegenerative disorders., (© 2021. The Author(s).)

Macrophages are indispensable immune cells tasked at eliminating intracellular pathogens. Mycobacterium tuberculosis ( Mtb ), one of the most virulent intracellular bacterial pathogens known to man, infects and resides within macrophages. While macrophages can be provoked by extracellular stimuli to inhibit and kill Mtb bacilli, these host defense mechanisms can be blocked by limiting nutritional metabolites, such as amino acids. The amino acid L-arginine has been well described to enhance immune function, especially in the context of driving macrophage nitric oxide (NO) production in mice. In this study, we aimed to establish the necessity of L-arginine on anti- Mtb macrophage function independent of NO. Utilizing an in vitro system, we identified that macrophages relied on NO for only half of their L-arginine-mediated host defenses and this L-arginine-mediated defense in the absence of NO was associated with enhanced macrophage numbers and viability. Additionally, we observed macrophage glycolysis to be driven by both L-arginine and mechanistic target of rapamycin (mTOR), and inhibition of glycolysis or mTOR reduced macrophage control of Mtb as well as macrophage number and viability in the presence of L-arginine. Our data underscore L-arginine as an essential nutrient for macrophage function, not only by fueling anti-mycobacterial NO production, but also as a central regulator of macrophage metabolism and additional host defense mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 McKell, Crowther, Schmidt, Robillard, Cantrell, Lehn, Janssen and Qualls.)

Short-term hypoxic states can influence the health and life activities of lowlanders who travel shortly to high altitudes, in transitory situations, such as surgical ischemia-reperfusion (to one or several organs), and in some sporting activities, such as parachuting and extreme skiing, mountain rescue teams, regular commercial flight crews, in which the subject may not even notice the hypoxia. NO is an integral part of the human physiological response to hypoxia. Until recently, the urea cycle (UC) was only considered as an important mechanism for neutralizing ammonia. We are the first to reveal an interrelation in hypoxic states between the activities of NO-synthase and UC enzymes in male rats' liver, kidney and brain. In the presented work, we have shown that during short-term intermittent hypobaric hypoxia (IHH) all enzymes of UC play an important role in the maintenance of NO quantity. The results allow thinking that kidney and brain argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) and liver ASS and ASL can be different isoenzymes. It is worth mentioning that the results have revealed new sides of l-arginine metabolism in a hypoxic state in male rats., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox" led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling., (Copyright © 2020. Published by Elsevier Inc.)

Argininosuccinic aciduria is an autosomal, recessive amino acid disorder that is caused by a deficiency of the argininosuccinate lyase enzyme. Citrulline is the most significant marker to detect this disorder. We used the High-performance liquid chromatography with fluorescence detection with 450 nm emission and 330 nm excitation wavelengths, 15 mmol/L potassium dihydrogen phosphate and 5 mmol/L dipotassium hydrogen phosphate as Mobile Phase A, and 50 mL water, 250 mL acetonitrile, and 200 mL methanol as Mobile Phase B in gradient mode with flow rate of 1.2 mL/min. The citrulline concentration was 22 µmol/L in healthy infants and 220 µmol/L in infants suffering from the disorder.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

The air-breathing magur catfish (Clarias magur) is a potential ureogenic teleost because of its functional ornithine-urea cycle (OUC), unlike typical freshwater teleosts. The ability to convert ammonia waste to urea was a significant step towards land-based life forms from aquatic predecessors. Here we investigated the molecular characterization of some OUC genes and the molecular basis of stimulation of ureogenesis via the OUC in magur catfish. The deduced amino acid sequences from the complete cDNA coding sequences of ornithine transcarbamyolase, argininosuccinate synthase, and argininosuccinate lyase indicated that phylogenetically magur catfish is very close to other ureogenic catfishes. Ammonia exposure led to a significant induction of major OUC genes and the gene products in hepatic and in certain non-hepatic tissues of magur catfish. Hence, it is reasonable to assume that the induction of ureogenesis in magur catfish under hyper-ammonia stress is mediated through the activation of OUC genes as an adaptational strategy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity., (© 2020 Wiley Periodicals, Inc.)

BACKGROUNDLiver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear.METHODSWe estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTest to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD.RESULTSWe demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, 2 markers of disease severity, were significantly (P < 0.001 and P = 0.001, respectively) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTest revealed increased echogenicity and liver stiffness, even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted before death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver-specific (ApoE) promoter.CONCLUSIONOur results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction.TRIAL REGISTRATIONThis study has been registered at ClinicalTrials.gov (NCT03721367, NCT00237315).FUNDINGFunding was provided by NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, and CPRIT.

Patients with germline mutations in the urea-cycle enzyme argininosuccinate lyase (ASL) are at risk for developing neurobehavioral and cognitive deficits. We find that ASL is prominently expressed in the nucleus locus coeruleus (LC), the central source of norepinephrine. Using natural history data, we show that individuals with ASL deficiency are at risk for developing attention deficits. By generating LC-ASL-conditional knockout (cKO) mice, we further demonstrate altered response to stressful stimuli with increased seizure reactivity in LC-ASL-cKO mice. Depletion of ASL in LC neurons leads to reduced amount and activity of tyrosine hydroxylase (TH) and to decreased catecholamines synthesis, due to decreased nitric oxide (NO) signaling. NO donors normalize catecholamine levels in the LC, seizure sensitivity, and the stress response in LC-ASL-cKO mice. Our data emphasize ASL importance for the metabolic regulation of LC function with translational relevance for ASL deficiency (ASLD) patients as well as for LC-related pathologies., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Argininosuccinate lyase catalyses the reversible breakdown of argininosuccinate into arginine and fumarate and is known to form tetramers in its quaternary association. The absence of structures involving competent enzymes bound to substrate/products came in the way of the precise elucidation of the catalytic mechanism of this family of proteins. Crystal structures of the enzyme from Mycobacterium tuberculosis in an unliganded form and its complex with the substrate/products have now been determined at 2.2 and 2.7 Å, respectively. The refinement of the structure of the complex was bedevilled by the presence of a lattice translocation defect. The two tetramers in the apo-crystals and the one in the crystals of the liganded protein, have the same structure except for the movements associated with enzyme action. Each molecule consists of an N-domain, an M-domain, and a C-domain. The molecule consists of four binding sites, each made up of peptide stretches from three subunits. Three binding sites appear to be occupied by the ligand in the transition state, while the products occupy the fourth site. The structure exhibits the movement of a loop in the M-domain and parts of the C-domain. This is the first instance when the appropriate movements are observed in a complex with bound substrate/product. The detailed picture of the binding site, active site residues and the movements associated with catalysis thus obtained, enabled a revisit of the mechanism of action of the enzyme. © 2019 IUBMB Life, 71(5):643-652, 2019., (© 2019 International Union of Biochemistry and Molecular Biology.)

The development of CRISPR interference (CRISPRi) technology has dramatically increased the pace and the precision of target identification during platform strain development. In order to develop a simple, reliable, and dual-inducible CRISPRi system for the industrially relevant Corynebacterium glutamicum, we combined two different inducible repressor systems in a single plasmid to separately regulate the expression of dCas9 (anhydro-tetracycline-inducible) and a given single guide RNA (IPTG-inducible). The functionality of the resulting vector was demonstrated by targeting the l-arginine biosynthesis pathway in C. glutamicum. By co-expressing dCas9 and a specific single guide RNA targeting the 5'-region of the argininosuccinate lyase gene argH, the specific activity of the target enzyme was down-regulated and in a l-arginine production strain, l-arginine formation was shifted towards citrulline formation. The system was also employed for down-regulation of multiple genes by concatenating sgRNA sequences encoded on one plasmid. Simultaneous down-regulated expression of both argH and the phosphoglucose isomerase gene pgi proved the potential of the system for multiplex targeting. The system can be a promising tool for further pathway engineering in C. glutamicum. Cumulative effects on targeted genes can be rapidly evaluated avoiding tedious and time-consuming traditional gene knockout approaches., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Pathogenic variants in the argininosuccinate lyase ( ASL ) gene have been shown to cause argininosuccinate lyase deficiency (ASLD); therefore, sequencing analysis offers advantages for prenatal testing and counseling in families afflicted with this condition. Here, we performed a genetic analysis of an ASLD patient and his family with an aim to offer available information for clinical diagnosis. The research subjects were a 23-month-old patient with a high plasma level of citrulline and his unaffected parents. Whole-exome sequencing identified potential related ASL gene mutations in this trio. Enzymatic activity was detected spectrophotometrically by a coupled assay using arginase and measuring urea production. We identified a novel nonsynonymous mutation (c.206A>G, p.Lys69Arg) and a stop mutation (c.637C>T, p.Arg213∗) in ASL in a Chinese Han patient with ASLD. The enzymatic activity of a p.Lys69Arg ASL construct in human embryonic kidney 293T cells was significantly reduced compared to that of the wild-type construct, and no significant activity was observed for the p.Arg213∗ construct. Compound heterozygous p.Lys69Arg and p.Arg213∗ mutations that resulted in reduced ASL enzyme activity were found in a patient with ASLD. This finding expands the clinical spectrum of ASL pathogenic variants.

Currently available cell culture media may not reproduce the in vivo metabolic environment of tumors. To demonstrate this, we compared the effects of a new physiological medium, Plasmax, with commercial media. We prove that the disproportionate nutrient composition of commercial media imposes metabolic artifacts on cancer cells. Their supraphysiological concentrations of pyruvate stabilize hypoxia-inducible factor 1α in normoxia, thereby inducing a pseudohypoxic transcriptional program. In addition, their arginine concentrations reverse the urea cycle reaction catalyzed by argininosuccinate lyase, an effect not observed in vivo, and prevented by Plasmax in vitro. The capacity of cancer cells to form colonies in commercial media was impaired by lipid peroxidation and ferroptosis and was rescued by selenium present in Plasmax. Last, an untargeted metabolic comparison revealed that breast cancer spheroids grown in Plasmax approximate the metabolic profile of mammary tumors better. In conclusion, a physiological medium improves the metabolic fidelity and biological relevance of in vitro cancer models.

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.

Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ASS1 gene encodes argininosuccinate synthetase-1, a cytosolic enzyme with a critical role in the urea cycle. Mutations are found in all ASS1 exons and cause the autosomal recessive disorder citrullinemia. Using CRISPR/Cas9-editing, we established the WAe001-A-13 cell line, which was heterozygous for an ASS1 mutation, from the human embryonic stem cell line H1. The WAe001-A-13 cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype., (Copyright © 2017. Published by Elsevier B.V.)

Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

Arginine biosynthesis pathway is crucial to the survival and pathogenesis of Mycobacterium tuberculosis (Mtb). Arginine is a critical amino acid that contributes to the inflection of cellular immune responses during pathogenesis. Argininosuccinate lyase from Mtb (MtArgH), the last enzyme in the pathway, catalyzes the production of arginine from argininosuccinic acid. MtArgH is an essential enzyme for the growth and survival of M. tuberculosis. We biochemically characterized MtArgH and deciphered the role of a previously unexplored cysteine (Cys 441 ) residue at the C-terminal region of the protein. Chemical modification of Cys 441 completely abrogated the enzymatic activity suggesting its involvement in the catalytic mechanism. Replacement of Cys 441 to alanine showed a striking decrease in the enzymatic activity, while retaining the overall secondary to quaternary structure of the protein, hence corroborating the involvement of Cys 441 in the process of catalysis. Interestingly, replacement of Cys 441 to serine, showed significant increase in activity, as compared to the wild-type MtArgH. Inactivity of C 441 A and elevated activity of its conservative mutant (C 441 S) confirmed the participation of Cys 441 in the MtArgH activity. We also, observed that C 441 S mutant has higher thermal stability and maintains significant activity at high temperatures. This is in concordance with our observation that Cys 441 in Mtb is replaced by a serine in the ArgH from thermophilic microorganisms. Furthermore, we also propose a potential feedback mechanism, wherein the Cys 441 is covalently modified to S-(2-succinyl) cysteine (succination) by one of the products, fumarate, thereby inactivating MtArgH. These insights into the mechanism of MtArgH activity unravel novel regulations of arginine biosynthetic pathway in Mtb. © 2017 IUBMB Life, 69(11):896-907, 2017., (© 2017 International Union of Biochemistry and Molecular Biology.)

Current methodologies for the assessment of urea cycle (UC) enzymatic activity are insufficient to accurately evaluate this pathway in biological specimens where lower UC is expected. Liver cell lines, including HepaRG, have been described to have limited nitrogen fixation through the UC, limiting their applicability as biocomponents for Bioartificial Livers (BAL). This work aims to develop novel and sensitive analytical solutions using Mass Spectrometry-based methodology to measure the activity of four UC enzymes in human liver and HepaRG cells. Activity of carbamoyl-phosphate synthetase I (CPS I), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL) and arginase (ARG I and II) was determined on homogenates from normal human liver and HepaRG cells cultured in monolayer or in the AMC-BAL. Enzyme products were determined by stable-isotope dilution UPLC-MS/MS. Activity of CPS I, OTC and ARG I/II enzymes in HepaRG monolayer cultures was considerably lower than in human control livers albeit an increase was achieved in HepaRG-BAL cultures. Improved analytical assays developed for the study of UC enzyme activity, contributed to gain understanding of UC function in the HepaRG cell line. The decreased activity of CPS I suggests that it may be a potential rate-limiting factor underlying the low UC activity in this cell line., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cysteamine is a small aminothiol endogenously derived from coenzyme A degradation. For some decades, synthetic cysteamine has been employed for the treatment of cystinosis, and new uses of the drug continue to emerge. In this review, we discuss the role of cysteamine in cellular and extracellular homeostasis and focus on the potential use of aminothiols to reconstitute the function of proteins harboring arginine (Arg) to cysteine (Cys) mutations, via repair of the Cys residue into a moiety that introduces an amino group, as seen in basic amino acid residues Lys and Arg. Cysteamine has been utilized in vitro and ex vivo in four different genetic disorders, and thus provides "proof of principle" that aminothiols can modify Cys residues. Other aminothiols such as mercaptoethylguanidine (MEG) with closer structural resemblance to the guanidinium moiety of Arg are under examination for their predicted enhanced capacity to reconstitute loss of function. Although the use of aminothiols holds clinical potential, more studies are required to refine specificity and treatment design. The efficacy of aminothiols to target proteins may vary substantially depending on their specific extracellular and intracellular locations. Redox potential, pH, and specific aminothiol abundance in each physiological compartment are expected to influence the reactivity and turnover of cysteamine and analogous drugs. Upcoming research will require the use of suitable cell and animal models featuring Arg to Cys mutations. Since, in general, Arg to Cys changes comprise about 8% of missense mutations, repair of this specific mutation may provide promising avenues for many genetic diseases.

Nilaparvata lugens is a typical phloem feeder. Rice phloem is high in simple sugars and very low in essential amino acids. Nilaparvata lugens harbors an ascomycete Entomomyces delphacidicola that hypothetically biosynthesizes several amino acids to meet the nutrition requirement of the planthopper. Among these amino acids, here, we focused on arginine biosynthesis. A complete cDNA of an E. delphacidicola gene, arginine-succinate lyase, EdArg4, the last step in arginine biosynthesis, was obtained. RNAi-mediated suppression of EdArg4 reduced arginine content in the hemolymph, and decreased the expression of several arginine biosynthesis genes. Silencing of EdArg4 delayed nymphal development and led to nymphal lethality. About 20% of the EdArg4 RNAi surviving adults were deformed. The most obvious defect was wider and larger abdomen. The EdArg4 RNAi-treated planthoppers had thickened wings and enlarged antennae, legs, and anal tubes and a few adults did not normally emerge. Arginine deficiency in the EdArg4 RNAi planthoppers repressed nitric oxide signaling, determined at the transcriptional level. We infer that E. delphacidicola biosynthesizes essential arginine to compensate for nutrition deficiency in N. lugens., (© 2017 Wiley Periodicals, Inc.)

Arginine is a critical amino acid in specific cancer types including hepatocellular carcinoma (HCC) and melanoma. Novel molecular mechanisms and therapeutic targets in arginine metabolism-mediated cancer formation await further identification. Our laboratory has previously demonstrated that arginine metabolic enzyme argininosuccinate lyase (ASL) promoted HCC formation in part via maintenance of cyclin A2 protein expression and arginine production for channeling to nitric oxide synthase. In this study, we investigated the mechanism by which ASL regulates cyclin A2 expression. We found that ASL interacted with cyclin A2 in HCC cells and the localization of their interaction was in the cytoplasm. Mutation of essential residues for enzymatic activity of ASL did not affect the binding of ASL to cyclin A2. Moreover, the mutant ASL retained the ability to restore the decreased tumorigenicity caused by ASL shRNA. Furthermore, overexpression of ASL conferred resistance to arginine deprivation therapy. Finally, the important pathways and potential therapeutic targets in ASL-regulated HCC were identified by bioinformatics analyses with Metacore database and Connectivity Map database. Our analyses suggested that bisoprolol, celecoxib, and ipratropium bromide, are potential therapeutics for ASL-regulated HCC formation. Thus, ASL interacts with cyclin A2 in cytoplasm, and may promote HCC formation through this non-enzymatic function. Overexpression of ASL may be a contributing factor in drug resistance for arginine deprivation therapy.

Arginine and nitric oxide (NO) are important mediators of tumorigenesis in various types of cancer. Dysregulation of NO content by argininosuccinate lyase (ASL) has been previously demonstrated to inhibit the proliferation of liver and breast cancer cells. However, the function of ASL in colon cancer is not well defined. The present study aimed to determine the effect of ASL on colon cancer. Western blot analysis indicated that ASL expression was induced by endoplasmic reticulum stress in HCT116 and SW480 colon cancer cells. Additionally, the expression of ASL in colon cancer tissues was enhanced compared with that in the adjacent normal tissues, and the patients with colon cancer with higher ASL expression exhibited poorer survival rates. Transfection of ASL-targeting short hairpin RNA (shRNA) into HCT116 cells inhibited cell proliferation and decreased anchorage-independent growth in a soft agar assay. In addition, when injected subcutaneously into NOD/SCID mice, stable transfectant ASL-downregulated HCT116 cells exhibited decreased in vivo tumorigenic ability. Flow cytometric analysis of cell cycle progression indicated that ASL-targeting shRNA induced G2/M arrest, and western blot analysis showed that the inhibition of ASL was accompanied by cyclin A2 degradation. Furthermore, ASL-targeting shRNA resulted in increased autophagosomes and decreased NO levels. Inhibition of NO by the NO synthase inhibitor L-NMMA significantly reduced cell proliferation and colony formation. In summary, the results of the present study indicated that ASL-targeting shRNA-induced growth inhibition is associated with decreased cyclin A2 expression and NO content in colon cancer.

Introduction: Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins., Objectives: We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle., Methods: In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay., Results: Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue., Conclusion: Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.

Under high-energy diets, amino acid N is difficult to dispose of, as a consequence of the availability of alternative substrates. We found, recently, that WAT contains a complete functional urea cycle, we analyzed the possible overall changes in the WAT urea cycle (and other-related amino acid metabolism gene expressions) in rats subjected to a cafeteria diet. Adult female Wistar rats were fed control or simplified cafeteria diets. Samples of WAT sites: mesenteric, periovaric, retroperitoneal and subcutaneous, were used for the estimation of all urea cycle enzyme activities and gene expressions. Other key amino acid metabolism gene expressions, and lactate dehydrogenase were also measured. Subcutaneous WAT showed a differentiated amino acid metabolism profile, since its cumulative (whole site) activity for most enzymes was higher than the activities of the other sites studied. After one month of eating an energy-rich cafeteria diet, and in spite of doubling the size of WAT, the transforming capacity of most amino acid metabolism enzymes remained practically unchanged in the tissue. This was not only due to limited changes in the overall enzyme activity, but also a consequence of a relative decrease in the expression of the corresponding genes. Overall, the results of this study support the consideration of WAT as an organ, disperse but under uniform control. The metabolic peculiarities between its different sites, and their ability to adapt to different energy availability conditions only add to the variable nature of adipose tissue. We have presented additional evidence of the significant role of WAT in amino acid metabolism.

Corynebacterium crenatum SYPA5-5, an L-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits L-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by L-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (L-citrulline and L-ornithine) and decreased production of L-arginine. Genetic and enzymatic levels analysis involved in L-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from L-citrulline to L-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased L-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the L-arginine productivity improved about 63.6%. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM L-arginine with the productivity of 5.42 mM h(-1). These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward L-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for L-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct L-arginine high-producing C. crenatum.

The yeast ζ-crystallin (Zta1p) is a quinone oxidoreductase belonging to the ζ-crystallin family, with activity in the reduction of alkenal/alkenone compounds. Various biological functions have been ascribed to the members of this protein family, such as their ability to interact specifically with AU-rich sequences in mRNA, and thus they have been proposed to act as AU-rich element-binding proteins (AREBPs). In this study, we evaluated the specificity of Zta1p for RNA versus DNA by means of a novel nonisotopic method for the in vitro quantitative detection of protein · RNA complexes. Through comparative transcriptomic analysis, we found that the lack of Zta1p negatively affects the expression of a group of genes involved in amino acid biosynthesis, the argininosuccinate lyase (ARG4) gene being one of them. Here, we propose that Zta1p participates in the post-transcriptional regulation of ARG4 expression by increasing the ARG4 mRNA half-life. In addition, expression of the ζ-crystallin gene (ZTA1) is itself regulated by nutrient availability through the general amino acid control and target of rapamycin pathways. Our results shed new light on the ζ-crystallin family members from yeast to humans as stress response proteins with a bifunctional role in the detoxification of alkenal and alkenone compounds, and the regulation of gene expression., (© 2015 FEBS.)

A multi-vitamin auxotrophic Torulopsis glabrata strain, a pyruvate producer, was further engineered to produce fumaric acid. Using the genome-scale metabolic model iNX804 of T. glabrata, four fumaric acid biosynthetic pathways, involving the four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL), fumarylacetoacetase (FAA), and fumarase (FUM1), were found. Athough single overexpression of each of the four enzymes in the cytosol improved fumaric acid production, the highest fumaric acid titer (5.62 g L(-1) ) was obtained with strain T.G-ASL(H) -ADSL(L) by controlling the strength of ASL at a high level and ADSL at a low level. In order to further improve the production of fumaric acid, the SpMAE1 gene encoding the C4 -dicarboxylic acids transporter was overexpressed in strain T.G-ASL(H) -ADSL(L) -SpMAE1 and the final fumaric acid titer increased to 8.83 g L(-1) . This study provides a novel strategy for fumaric acid biosynthesis by utilizing the urea cycle and the purine nucleotide cycle to enhance the bridge between carbon metabolism and nitrogen metabolism., (© 2014 Wiley Periodicals, Inc.)

L-citrulline (Cit) is a co-product of NO synthesis and a direct L-arginine (Arg) precursor for de novo NO synthesis. Acute liver failure (ALF) is associated with increased nitric oxide (NO) and cyclic GMP (cGMP) synthesis in the brain, indirectly implicating a role for active transport of Cit. In the present study we characterized [(3)H]Cit uptake to the cortical brain slices obtained from control rats and rats with thioacetamide (TAA)-induced ALF ("TAA slices"). In both control and TAA slices the uptake was partially Na(+)-dependent and markedly inhibited by substrates of systems L and N, including L-glutamine (Gln), which accumulates in excess in brain during ALF. Cit uptake was not affected by Arg, the y(+)/y(+)L transport system substrate, nor by amino acids taken up by systems A, xc (-)or XAG. The Vmax of the uptake in TAA slices was ~60 % higher than in control slices. Chromatographic (HPLC) analysis revealed a ~30 % increase of Cit concentration in the cerebral cortical homogenates of TAA rats. The activity of argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), the two enzymes of Cit-NO cycle catalyzing synthesis of Arg, showed an increase in TAA rats, consistent with increased ASS and ASL protein expression, by ~30 and ~20 %, respectively. The increased Cit-NO cycle activity was paralleled by increased expression of mRNA coding for inducible nitric oxide synthase (iNOS). Taken together, the results suggest a role for Cit in the activation of cerebral NO synthesis during ALF.

Necrotizing enterocolitis (NEC), the most common neonatal gastrointestinal emergency, results in significant mortality and morbidity, yet its pathogenesis remains unclear. Argininosuccinate lyase (ASL) is the only enzyme in mammals that is capable of synthesizing arginine. Arginine has several homeostatic roles in the gut and its deficiency has been associated with NEC. Because enterocytes are the primary sites of arginine synthesis in neonatal mammals, we evaluated the consequences of disruption of arginine synthesis in the enterocytes on the pathogenesis of NEC. We devised a novel approach to study the role of enterocyte-derived ASL in NEC by generating and characterizing a mouse model with enterocyte-specific deletion of Asl (Asl(flox/flox); VillinCre(tg/+), or CKO). We hypothesized that the presence of ASL in a cell-specific manner in the enterocytes is protective in the pathogenesis of NEC. Loss of ASL in enterocytes resulted in an increased incidence of NEC that was associated with a proinflammatory state and increased enterocyte apoptosis. Knockdown of ASL in intestinal epithelial cell lines resulted in decreased migration in response to lipopolysaccharide. Our results show that enterocyte-derived ASL has a protective role in NEC., (Copyright © 2014 the American Physiological Society.)

Objective: To detect potential mutations of Y9ASS1, ASL and SLC25A13 genes in four patients manifesting citrullinemia., Methods: Genomic DNA was extracted from peripheral blood leukocytes. Exons and their flanking sequences of the three genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing., Results: Based on DNA sequence analysis, one case was diagnosed with argininosuccinate synthetase deficiency, and the mutation type (ASS1 gene) was c.236C>T (p.S79F) + c.431C>G (p.P144R). Two cases were diagnosed with argininosuccinic aciduria (ASL gene), and their gene mutations were c.434A>G (p.D145G) + c.1366C>T (p.R456W) and c.331C>T (p.R111W) + IVS8+2insT, respectively. A thirteen months boy who carried a heterozygous 851del4 mutation (SLC25A13 gene) was diagnosed with citrullinemia adult-onset type II., Conclusion: Through analysis of relevant pathogenic genes, four patients have been diagnosed.

L-Arginine (L-Arg), a conditional essential amino acid in adults, has been shown to enhance pregnancy outcome. Argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl) are the key enzyme for L-Arginine (L-Arg) biosynthesis. Based our microarray analysis, Ass1 expression is upregulated significantly at implantation site on day 5 of pregnancy compared to that at inter-implantation site. However, the expression, regulation and function of Ass1 during early pregnancy remain unknown. Here we found that Ass1 is highly expressed in mouse decidua and uterine stromal cells undergoing decidualization, and Asl is weakly expressed in mouse decidua and uterine stromal cells undergoing decidualization. α-Methyl-DL-aspartic acid (MDLA), a specific inhibitor for Ass1, can significantly increase the rate of embryonic reabsorption. Under in vitro induced decidualization, MDLA clearly inhibits the expression of decidual/trophoblast prolactin-related protein (Dtprp), a marker for decidualization in mice. Only Ass1 expression is induced by cAMP through PKA/p-Creb signaling pathway. Results from our cell culture models further indicates that the high level of L-Arg enhances stromal proliferation, while enzymatic activity or Ass1 expression level is essential to determine the magnitude of both mouse and human decidualization. Interestingly, L-Arg at high concentration down-regulates Ass1 and Asl expression by negative feedback to maintain L-Arg homeostasis. These findings highlight that cAMP-induced Ass1 expression is important in controlling the magnitude of decidualization through regulating L-Arg level., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Plant roots play an important role in the uptake of water and nutrients, structural support and environmental sensing, but the molecular mechanisms involved in root development are poorly understood in rice (Oryza sativa), which is characterized by a dense fibrous root system. Here we report a rice mutant (red1 for root elongation defect 1) with short roots. Morphological and physiological analyses showed that the mutant had a shorter length from the quiescent center (QC) to the starting point of the elongation zone but a similar cell size and number of lateral and crown roots compared with the wild type. Furthermore, the mutant had similar radial structure and nutrient uptake patterns to the wild type. Map-based cloning revealed that the mutant phenotype was caused by a point mutation of a gene encoding an argininosuccinate lyase (ASL), catalyzing the last step of arginine biosynthesis. The OsASL1 gene has two distinct transcripts, OsASL1.1 and OsASL1.2, which result from different transcription start sites, but only OsASL1.1 was able to complement the mutant phenotype. OsASL1.1 was expressed in both the roots and shoots. The protein encoded by OsASL1.1 showed ASL activity in yeast. OsALS1.1 was localized to the plastid. The short root of the mutant was rescued by exogenous addition of arginine, but not by other amino acids. These results indicate that arginine produced by ASL is required for normal root elongation in rice., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Brucellosis caused by Brucella species is a zoonotic disease with a serious impact on public health and the livestock industry. To better understand the pathogenesis of the disease, in vivo-induced antigen technology (IVIAT) was used to investigate the in vivo-induced antigens of Brucella abortus in this study. A genomic expression library of B. abortus was constructed and screened using pooled bovine B. abortus-positive sera by IVIAT. In total, 33 antigens were identified. Five antigens were further expressed and tested for their seroreactivity against 33 individual bovine B. abortus-positive sera by Western blot analysis. The results showed a highest positive rate of 32/33 for argininosuccinate lyase (ASL), indicating that ASL may be used as a candidate marker for serodiagnosis of brucellosis. Furthermore, an asl gene-deleted mutant strain S2308ΔASL was constructed, and the intracellular survival and replication of the mutant strain in RAW264.7 cells were investigated. Interestingly, the numbers of bacteria recovered from cells infected with mutant strain S2308ΔASL were similar at all time points observed from 0 h to 96 h post-infection, suggesting the asl gene plays an important role in the bacterial replication in RAW264.7 cells. Real-time quantitative PCR (qPCR) analysis showed that the mRNA levels in S2308ΔASL were decreased for BvrR, BvrS and virB5 when compared with those in S2308 (P<0.05). Our results not only expand the knowledge of Brucella intracellular replication but also expand the list of candidates for serodiagnostic markers of brucellosis.

Plant roots play an important role in uptake of water and nutrients, support of above-ground part and environmental sensing, but the molecular mechanisms underlying the root development are poorly understood in rice. We found that a gene (OsASL1) encoding argininosuccinate lyase is involved in normal root development of rice. OsASL1 cleaves argininosuccinate to arginine and fumarate reversibly, the last step in the arginine biosynthetic pathway. Here, we further characterized OsASL1 in terms of expression pattern, subcellular localization, and arginine effect on the root growth. A detailed expression analysis revealed that 2 transcripts of OsASL1, OsASL1.1 and OsASL1.2, showed different expression patterns; OsASL1.1 was expressed in most organs throughout the whole growth period, whereas OsASL1.2 was mainly expressed in the roots. In contrast to plastid-localized OsASL1.1, OsASL1.2 was localized to the cytosol and nucleus. The short-root phenotype of the mutant was not rescued by exogenous addition of the sodium nitroprusside, a nitric oxide donor, but rescued by an appropriate concentration of Arg. Our results indicate that the subcellular localization was determined by the N terminus of OsASL1 and that appropriate concentration of Arg is required for normal root elongation in rice.

The last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from Mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary X-ray studies have been carried out on the crystals. The His-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kDa crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group P2(1)2(1)2(1). Molecular-replacement calculations and self-rotation calculations confirmed the space group and the tetrameric nature of the molecule.

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Arginine biosynthesis and nitric oxide (NO) production are important for cancer homeostasis. Degradation of arginine may be used to inhibit liver tumors with low argininosuccinate synthetase (ASS) expression. In this report, we investigated an alternative therapeutic approach by targeting argininosuccinate lyase (ASL). ASL is transcriptionally induced by endoplasmic reticulum stress and is overexpressed in some human liver tumors. Knockdown of ASL expression by short hairpin RNA (shRNA) in three liver cancer cell lines, ML-1, HuH-7, and HepG2, decreased colony formation in vitro and tumor growth in vivo. Furthermore, lentiviral infection of ASL shRNA inhibited tumor growth in a therapeutic animal tumor model. Analysis of ASL shRNA on the cell-cycle progression revealed a G2-M delay. Among cell-cycle regulatory molecules, cyclin A2 expression was reduced. Reintroduction of exogenous cyclin A2 restored the cell growth in ASL-knockdown cells. Autophagy was observed in the cells treated with ASL shRNA, as shown by an increase in LC3-II levels and autophagosome formation. The total cellular arginine level was not altered significantly. Inhibition of autophagy further attenuated cell growth, suggesting that autophagy induced by ASL shRNA plays a feedback prosurvival function. Knockdown of ASL reduced NO content, and addition of NO donor partially recovered the growth inhibition by ASL shRNA. In summary, downregulation of ASL attenuated tumor growth and the inhibition was mainly mediated by a decrease of cyclin A2 and NO., (©2013 AACR.)

The objective of this study was to examine the effects of 6 days of emersion on nitrogen metabolism and excretion in the Chinese soft-shelled turtle, Pelodiscus sinensis. Despite having a soft shell with a cutaneous surface that is known to be water permeable, P. sinensis lost only ~2% of body mass and was able to maintain its hematocrit and plasma osmolality, [Na(+)] and [Cl(-)] during 6 days of emersion. During emersion, it ameliorated water loss by reducing urine output, which led to a reduction (by 29-76%) in ammonia excretion. In comparison, there was a more prominent reduction (by 82-99%) in urea excretion during emersion due to a lack of water to flush the buccopharyngeal epithelium, which is known to be the major route of urea excretion. Consequently, emersion resulted in an apparent shift from ureotely to ammonotely in P. sinensis. Although urea concentration increased in several tissues, the excess urea accumulated could only account for 13-22% of the deficit in urea excretion. Hence, it can be concluded that a decrease (~80%) in urea synthesis occurred in P. sinensis during the 6 days of emersion. Indeed, emersion led to significant decreases in the activity of some ornithine-urea cycle enzymes (argininosuccinate synthetase/argininosuccinate lyase and arginase) from the liver of P. sinensis. As a decrease in urea synthesis occurred without the accumulation of ammonia and total free amino acids, it can be deduced that ammonia production through amino acid catabolism was suppressed with a proportional reduction in proteolysis in P. sinensis during emersion. Indeed, calculated results revealed that there could be a prominent decrease (~88%) in ammonia production in turtles after 6 days of emersion. In summary, despite being ureogenic and ureotelic in water, P. sinensis adopted a reduction in ammonia production, instead of increased urea synthesis, as the major strategy to ameliorate ammonia toxicity and problems associated with dehydration during terrestrial exposure.

Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.

Argininosuccinate lyase (ASL) is an important enzyme in the hepatic urea cycle, and catalyzes the reversible reaction of argininosuccinate to arginine and fumarate. Its expression is significantly reduced in some hepatocellular carcinomas (HCC). In this study, we aimed to investigate the correlation of reduced ASL expression and clinicopathologic features and prognosis in HCC patients. Immunohistochemistry was used to determine the expression of ASL in HCC tissues from 61 patients who had undergone hepatic tumor resection. The correlation of ASL expression in HCC with background liver status, viral status, tumor size, portal vein invasion, histopathologic differentiation, early tumor recurrence, sex, and age were assessed with the χ(2) test. Patient survival and survival differences were determined by the Kaplan-Meier method and log-rank test. Cox regression (proportional hazard model) was used for multivariate analysis of prognostic factors. Strong positive staining was found in 39/61 HCCs and normal liver tissues, and reduced ASL staining was found in 22/61 HCCs (36.1%). Patients with low ASL expression had a significantly poorer overall survival and disease-free survival (both P<0.001). Reduced ASL expression in carcinoma tissues was also significantly associated with the tumor-node-metastasis stage and early tumor recurrence, and histopathologic differentiation and portal vein invasion (P<0.05). Cox regression analysis showed that ASL is an independent prognostic marker for HCC. Therefore, reduced ASL expression may be a novel maker for poor prognosis in HCC patients.

Mitochondrial diseases arise as a result of dysfunction of the respiratory chain, leading to inadequate ATP production required to meet the energy needs of various organs. On the other hand, nitric oxide (NO) deficiency can occur in mitochondrial diseases and potentially play major roles in the pathogenesis of several complications including stroke-like episodes, myopathy, diabetes, and lactic acidosis. NO deficiency in mitochondrial disorders can result from multiple factors including decreased NO production due to endothelial dysfunction, NO sequestration by cytochrome c oxidase, NO shunting into reactive nitrogen species formation, and decreased availability of the NO precursors arginine and citrulline. Arginine and citrulline supplementation can result in increased NO production and hence potentially have therapeutic effects on NO deficiency-related manifestations of mitochondrial diseases. Citrulline is a more efficient NO donor than arginine as it results in a greater increase in de novo arginine synthesis, which plays a major role in driving NO production. This concept is supported by the observation that the three enzymes responsible for recycling citrulline to NO (argininosuccinate synthase and lyase, and nitric oxide synthase) function as a complex that can result in compartmentalizing NO synthesis and channeling citrulline efficiently to NO synthesis. Clinical research evaluating the effect of arginine and citrulline in mitochondrial diseases is limited to uncontrolled open label studies demonstrating that arginine administration to subjects with MELAS syndrome results in improvement in the clinical symptoms associated with stroke-like episodes and a decrease in the frequency and severity of these episodes. Therefore, controlled clinical studies of the effects of arginine or citrulline supplementation on different aspects of mitochondrial diseases are needed to explore the potential therapeutic effects of these NO donors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Argininosuccinic aciduria (ASA) is a urea cycle disorder with a complex phenotype. In spite of a lower risk for recurrent hyperammonemic episodes as compared to the proximal disorders of ureagenesis, subjects with ASA are at risk for long-term complications including, poor neurocognitive outcome, hepatic disease and systemic hypertension. These complications can occur in spite of current standard therapy that includes dietary modifications and arginine supplementation suggesting that the presently available therapy is suboptimal. In this article, we discuss the natural history of ASA and the recent mechanistic insights from animal studies that have shown the requirement of argininosuccinate lyase, the enzyme deficient in ASA, for systemic nitric oxide production. These findings may have therapeutic implications and may help optimize therapy in ASA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Members of the aspartase/fumarase superfamily share a common tertiary and quaternary fold, as well as a similar active site architecture; the superfamily includes aspartase, fumarase, argininosuccinate lyase, adenylosuccinate lyase, δ-crystallin, and 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE). These enzymes all process succinyl-containing substrates, leading to the formation of fumarate as the common product (except for the CMLE-catalyzed reaction, which results in the formation of a lactone). In the past few years, X-ray crystallographic analysis of several superfamily members in complex with substrate, product, or substrate analogues has provided detailed insights into their substrate binding modes and catalytic mechanisms. This structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate (or enediolate) intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.