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Abstract Argininosuccinate lyase (ASL) participates in arginine synthesis through catalysing a reversible reaction in which argininosuccinate (AS) converts into arginine and fumarate. ASL from Mycobacterium tuberculosis is essential for its growth. In this work, the crystal structure of the apo form of Mtb ASL was determined and reveals a tetrameric structure that is essential for its activity since the active sites are formed by residues from three different monomers. Subsequently, we determined the crystal structure of Mtb ASL-sulfate complex, and the ligand mimics the negatively charged intermediate. The complex structure and mutagenesis studies indicate that residues S282 and H161 might act as a catalytic dyad. A major conformational change in the Mtb ASL-SO 4 complex structure could be observed upon sulfate binding, and this movement facilitates the interaction between substrate and the residues involved in catalysis. A different conformational change in the C-terminal domain could be observed in the Mtb ASL-SO 4 complex compared with that in other homologues. This difference may be responsible for the lower activity of Mtb ASL, which is related to the slow growth rate of M. tuberculosis. The C-terminal domain is a potential allosteric site upon inhibitor binding. The various conformational changes and the diversity of the sequence of the potential allosteric site across the homologues might provide clues for designing selective inhibitors against M. tuberculosis. Highlights • Kinetic studies on Argininosuccinate lyase from Mycobacterium tuberculosis (Mtb ASL). • Crystal structure determination of apo form Mtb ASL and Mtb ASL-sulfate complex. • The structure of the complex and the mutagenesis studies identified the catalytic dyad. • The residues in the catalytic loop which are not conserved in Mtb ASL are proved to be involved. • The conformation changes of Mtb ASL induced by sulfate binding is different from its homologues. [ABSTRACT FROM AUTHOR]

Abstract Argininosuccinic aciduria (ASA) is the second most common genetic disorder affecting the urea cycle. The disease is caused by deleterious mutations in the gene encoding argininosuccinate lyase (ASL); total loss of ASL activity results in severe neonatal onset of the disease, which is characterized by hyperammonemia within a few days of birth that can rapidly progress to coma and death. The long-term complications of ASA, such as hypertension and neurocognitive deficits, appear to be resistant to the current treatment options of dietary restriction, arginine supplementation, and nitrogen scavenging drugs. Treatment-resistant disease is currently being managed by orthotopic liver transplant, which shows variable improvement and requires lifetime immunosuppression. Here, we developed a gene therapy strategy for ASA aimed at alleviating the symptoms associated with urea cycle disruption by providing stable expression of ASL protein in the liver. We designed a codon-optimized human ASL gene packaged within adeno-associated virus serotype 8 (AAV8) as a vector for targeted delivery to the liver. To evaluate the therapeutic efficacy of this approach, we utilized a murine hypomorphic model of ASA. Neonatal administration of AAV8 via the temporal facial vein extended survival in ASA hypomorphic mice, although not to wild-type levels. Intravenous injection into adolescent hypomorphic mice led to increased survival and body weight and correction of metabolites associated with the disease. Our results demonstrate that AAV8 gene therapy is a viable approach for the treatment of ASA. Highlights • AAV gene therapy extends survival of adolescent ASA hypomorphic mice. • AAV gene therapy extends survival of neonatal ASA hypomorphic mice. • AAV gene therapy normalizes plasma argininosuccinic acid and citrulline levels in ASA hypomorphic mice. • AAV gene therapy normalizes serum transaminase levels in ASA hypomorphic mice. [ABSTRACT FROM AUTHOR]

The Urea Cycle Disorders Consortium (UCDC) is a member of the NIH funded Rare Diseases Clinical Research Network and is performing a longitudinal study of 8 urea cycle disorders (UCDs) with initial enrollment beginning in 2006. The consortium consists of 14 sites in the U.S., Canada and Europe. This report summarizes data mining studies of 614 patients with UCDs enrolled in the UCDC's longitudinal study protocol. The most common disorder is ornithine transcarbamylase deficiency, accounting for more than half of the participants. We calculated the overall prevalence of urea cycle disorders to be 1/35,000, with 2/3rds presenting initial symptoms after the newborn period. We found the mortality rate to be 24% in neonatal onset cases and 11% in late onset cases. The most common precipitant of clinical hyperammonemic episodes in the post-neonatal period was intercurrent infections. Elevations in both blood ammonia and glutamine appeared to be biomarkers for neurocognitive outcome. In terms of chronic treatment, low protein diet appeared to result in normal weight but decreased linear growth while N-scavenger therapy with phenylbutyrate resulted in low levels of branched chain amino acids. Finally, we found an unexpectedly high risk for hepatic dysfunction in patients with ornithine transcarbamylase deficiency. This natural history study illustrates how a collaborative study of a rare genetic disorder can result in an improved understanding of morbidity and disease outcome. [ABSTRACT FROM AUTHOR]

Abstract: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). Study design: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6–17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. Results: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r=0.730, p<0.001) or as total 24-hour excretion (r=0.791 p<0.001), followed by plasma phenylacetylglutamine AUC24-hour, plasma phenylacetic acid AUC24-hour and phenylbutyric acid AUC24-hour. Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. Conclusion: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring. [Copyright &y& Elsevier]

Abstract: Objective: To compare the effects of combinatorial therapy with low-dose arginine and a nitrogen scavenging agent (sodium phenylbutyrate) vs. monotherapy with high-dose arginine on liver function tests in patients with argininosuccinic aciduria (ASA). Study design: Twelve patients with ASA were enrolled in a double-blind, placebo-controlled, cross-over study design. Subjects were randomized to receive either a low-dose of arginine therapy (100mg·kg−1·d−1) combined with sodium phenylbutyrate (500mg·kg−1·d−1) (LDA arm) or a high-dose of arginine alone (500mg·kg−1·d−1) (HDA arm) for one week. At the end of one week of therapy, liver function tests were assessed and metabolite fluxes were measured using a multi-tracer stable isotope protocol. Results: Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and measures of synthetic functions of the liver were the primary outcomes. Subjects had significantly increased levels of argininosuccinate (P <0.03) and AST levels (P <0.01) after treatment with high-dose arginine. In the subset of subjects with elevated AST or ALT, treatment with high-dose of arginine was associated with further increases in plasma levels of both aminotransferases. Whereas subjects had increased arginine and citrulline flux with high-dose arginine therapy, the glutamine flux was not different between the two treatment arms. The synthetic liver functions as assessed by prothrombin time, INR, and coagulation factor levels were not different between the HDA and LDA arms. Conclusions: Administering higher doses of arginine in subjects with ASA results in increases in AST and ALT levels, especially in the subset of patients with elevated baseline aminotransferases. Hence, low-dose arginine sufficient to normalize arginine levels in plasma combined with nitrogen scavenging therapy should be considered as a therapeutic option for treatment of ASA in patients with elevations of hepatic aminotransferases. [Copyright &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: Nutritional management is essential for patients with inborn errors of metabolism, such as urea cycle disorders (UCDs). Lack of appetite is common in these patients and can lead to underconsumption of calories, catabolism, and subsequently loss of metabolic control. The etiology of anorexia in these patients is largely unexplored. The neuroendocrine hormone peptide tyrosine tyrosine (PYY), secreted postprandially from endocrine cells of the ileum and colon, induces feelings of satiety and decreases food intake. While plasma PYY levels have been characterized in a number of populations, they have not been examined in UCD patients. In a retrospective study, plasma PYY concentrations were measured in UCD (n=42) patients and controls (n=28) via an ELISA to determine if levels of this anorexigenic hormone are altered in this patient population. Median PYY levels were significantly higher in UCD patients compared to controls (p=3.5×10−5). Body mass index was significantly associated with increased PYY levels in controls (p=0.02), while UCD diagnosis subtype was associated with PYY levels (p=1×10−3) in cases. Median PYY levels were significantly lower in ornithine carbamoyltransferase deficient patients compared with all other UCD subtypes (p=9×10−3), but significantly higher compared to controls (p=1.6×10−3). Overall, this study demonstrates that UCD cases have increased PYY levels compared to controls, suggesting that regulation of PYY may be altered in these patients. These observations may lead to a better understanding of the development of anorexia in UCD patients. [Copyright &y& Elsevier]

Abstract: The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a characteristic profile of plasma amino acid alterations that can be utilized for diagnosis. While enzyme assay is possible, an analysis of the underlying mutation is preferable for an accurate diagnosis. Mouse models for each of the urea cycle disorders exist (with the exception of NAGS deficiency), and for almost all of them, their clinical and biochemical phenotypes rather closely resemble the phenotypes seen in human patients. Consequently, all of the current mouse models are highly useful for future research into novel pharmacological and dietary treatments and gene therapy protocols for the management of urea cycle disorders. [Copyright &y& Elsevier]

Abstract: Nitric oxide (NO) is an important vasorelaxant produced along with l-citrulline from l-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of l-citrulline to l-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of l-citrulline to l-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of l-citrulline formed due to the efficiency of l-citrulline recycling via the citrulline–NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-l-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-l-citrulline formation were simultaneously determined. NO production exceeded apparent l-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, α-methyl-dl-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline–NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of l-citrulline recycling, via the citrulline–NO cycle, to endothelial NO production. [Copyright &y& Elsevier]

Abstract: Argininosuccinate-synthetase (ASS), argininosuccinate-lyase (ASL) and nitric oxide synthase (NOS) act in the l-arginine-NO-l-citrulline cycle. In the rat brain, ASS is expressed in neurons, ASL in neurons and astroglia in the striatum, both are co-expressed with nNOS in medium-sized neurons. Microglia cells express iNOS and ASS after activation but no information is available on ASL and on ASS/ASL/iNOS co-expression in this glial population. The present aim was to ascertain, by immunohistochemistry, whether the microglia cells of the rat striatum and fronto-parietal cortex express ASL and ASS in control conditions and after transient ischemia induced by middle cerebral artery occlusion, and whether ASL and ASS are co-expressed with iNOS. The study was conducted 24, 72 and 144 h after reperfusion in two groups of ischemic rats with different tissue damage and survival. ASS and ASL are not expressed by microglia cells in controls while are present in most of the activated microglia cells in the ischemic rats. In those animals with longer survival, ASS and ASL were no more detectable at 144 h, while, in the animals with shorter survival, they were co-expressed with iNOS, but only at 72 h. In the cortex, at variance with the striatum, almost all of nNOS-positive neurons co-expressed ASS and ASL. In conclusion, only activated microglia cells express ASS and ASL, this expression precedes that of iNOS and does not necessarily imply its appearance. Therefore, local factors such as the NO produced by nNOS/ASS/ASL-positive neurons, could influence ASS/ASL-positive microglia cells avoiding or allowing the induction, in these cells, of iNOS. [Copyright &y& Elsevier]

Abstract: In neurodegenerative diseases, an increased number of neuronal nitric oxide synthase (nNOS)-positive neurons was reported, but nothing is known on which are the neurons induced to express nNOS. Argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and nNOS act in the l-arginine–NO–l-citrulline cycle permitting a correct NO production. In the brain, nNOS-positive neurons co-expressing ASS were known, while those co-expressing ASL were not demonstrated. We investigated by immunohistochemistry the presence of these types of neurons in the rat striatum to verify whether there was a correlation between their changes due to neurotoxic insults and animal survival. Transient ischemia, a neurodegenerative insult model, was induced in rat brain by 2 h of middle cerebral artery occlusion. The striatum, the core of ischemia, was examined at 24, 72 and 144 h after reperfusion and compared with that of rats in normal condition. ASS, ASL and nNOS-positive neurons, some of the latter also expressing ASS and ASL, were present both in normal and ischemic conditions. At 24 h after reperfusion, the number of the nNOS-positive neurons and the percentage of those co-expressing ASS and ASL were significantly increased in the animals with a longer survival and at 144 h after ischemia there was an almost complete restore of the number and/or percentage of these neurons. We hypothesize that the neurons induced to express nNOS were the ASS- and ASL-positive ones and that the neurons co-expressing nNOS, ASS and ASL, since having the enzymes necessary to maintain a correct NO production, might protect from neurotoxic insults. [Copyright &y& Elsevier]

Abstract: δ-Crystallin is the major structural protein in avian and reptilian eye lenses but its sequence is highly homologous with the urea cycle enzyme, argininosuccinate lyase (ASL). In previous studies the multi-step unfolding process of this protein in the presence of GdmCl was sensitively probed using tryptophan fluorescence. In this study the contribution of single tryptophan residues to the stability of the local environment was monitored by mutation of two highly conservative tryptophan residues in goose δ-crystallin, Trp 74 and Trp 169. These residues behaved differently in terms of fluorescence intensity and maxima emission wavelength, consistent with their structural location in buried or solvent accessible regions. No gross changes in the secondary structure after mutation were observed, as judged by far-UV CD. The side chains of tryptophan residues in the structure of wild-type goose δ-crystallin possess both hydrophobic and hydrogen bonding interactions. Replacement of the side chain with phenylalanine or alanine led to expose of a hydrophobic area and a reduction in thermal stability; W169A particularly has a Tm value that was 10°C lower than the wild type enzyme. In the presence of GdmCl, a sharp red shift in fluorescence wavelength due to subunit dissociation can be sensitively detected using a single tryptophan, with the region surrounding W74 undergoing the first transition with a [GdmCl]1/2 of 0.45M. Further measurement of unfolding curves by CD revealed that the W169A mutant was most unstable with a [GdmCl]1/2 of 0.22M. From sedimentation velocity analysis, the unstable conformation of the W169A mutant affected the assembly of the quaternary structure. Our studies demonstrate the critical role for the tryptophan residues in stabilizing protein conformations and subunit assembly in δ-crystallin. [Copyright &y& Elsevier]

Abstract: Arginine deprivation causes many types of tumour cells to die, often because they cannot recover or convert urea cycle intermediates into arginine. The powerful homeostatic mechanisms that kicks in to restore arginine levels in vivo are lacking in vitro, where there is no supply of citrulline. Comparison between cells deprived of arginine by direct elimination methods or indirectly via arginine degrading enzymes should show differences depending on their ability to handle alternative intermediates (ornithine, citrulline and argininosuccinate) of the urea cycle. The internal state of cells that can, versus those that cannot, use intermediates will metabolically be quite different. These differences should provide clear indicators regarding the sensitivity (susceptibility) of cells to arginine deprivation, from which we will be in a much better position to judge which tumours to treat, and possibly how to design the best treatment to eliminate them. [Copyright &y& Elsevier]

Argininosuccinate lyase (AL) has several roles in intermediary metabolism. It is an essential component of the urea cycle, providing a pathway for the disposal of excess nitrogen in mammals. AL links the urea cycle to the tricarboxylic acid (TCA) cycle by generating fumarate. Finally, AL is required for the endogenous production of arginine. In this latter role it may function outside ureagenic organs to provide arginine as a substrate for nitric oxide synthases (NOS). Increasing evidence suggests that argininosuccinate synthetase (AS) and AL are more globally expressed, and the coordinate regulation of AS and AL gene expression with that of the inducible form of NOS (iNOS) provides evidence that this may facilitate the regulation of NOS activity. Deficiency of AL leads to the human urea cycle disorder argininosuccinic aciduria. We produced an AL deficient mouse by gene targeting in order to investigate the role of AL in endogenous arginine production. This mouse also provides a model of the human disorder to explore the pathogenesis of the disorder and possible new treatments. Metabolic studies of these mice demonstrated that they have the same biochemical phenotype as humans, with hyperammonemia, elevated plasma argininosuccinic acid and low plasma arginine. Plasma nitrites, derived from NO, were not reduced in AL deficient mice and there was no significant difference is the level of cyclic GMP, the second messenger induced by NO. [Copyright &y& Elsevier]