Carbamoyl Phosphate Synthetase Research Articles

Evaluation of oxidative damage to biomolecules and inflammation in patients with urea cycle disorders

Urea cycle disorders (UCD) are inborn errors of metabolism that occur due to a loss of function in enzymes and transporters involved in the urea cycle, causing an intoxication by hyperammonemia and accumulation of metabolites. Patients can develop hepatic encephalopathy (HE), severe neurological and motor disabilities, and often death. The mechanisms involved in the pathophysiology of UCD are many and complex, but there are strong indications that oxidative stress and inflammation are present, being responsible for at least part of the cellular damage that occurs in these diseases. The aim of this study was to evaluate oxidative and nitrosative damage and inflammation in UCD, to better understand the pathophysiology mechanisms of these diseases. We evaluated the nitrite and nitrate content, thiobarbituric acid-reactive substances (TBARS), carbonyl protein content and a panel of cytokines in plasma sample of 14 patients. The UCD patients group consisted of individuals affected with ornithine transcarbamylase deficiency (n = 8), carbamoyl phosphate synthetase deficiency (n = 2), argininosuccinate synthetase deficiency (n = 2); arginase 1 deficiency (n = 1) and argininosuccinate lyase deficiency (n = 1). Patients mean age at diagnosis was 5.25 ± 9.86 years-old and mean concentrations were compared with healthy individuals of matched age and gender. We found a significant reduction in nitrogen reactive species in patients when compared to controls. TBARS was increased in patients, indicating lipid peroxidation. To evaluate protein oxidative damage in UCD, the carbonyl content was measured, and the results also demonstrated an increase in this biomarker. Finally, we found that UCD patients have enhanced concentrations of cytokines, with pro-inflammatory interleukins IL-6, IL-8, interferon-γ and TNF-α, and anti-inflammatory IL-10 being increased when compared to the control group. In conclusion, our results demonstrate that oxidative stress and inflammation occurs in UCD and probably contribute to the severe brain damage present in patients.

Complexed Crystal Structure of the Dihydroorotase Domain of Human CAD Protein with the Anticancer Drug 5-Fluorouracil.

Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.

A Tailored Strategy to Crosslink the Aspartate Transcarbamoylase Domain of the Multienzymatic Protein CAD.

CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization.

Dietary Isoleucine and Valine: Effects on Lipid Metabolism and Ureagenesis in Pigs Fed with Protein Restricted Diets.

A mixture of valine (Val) and isoleucine (Ile) not only decreases the negative impact of very low protein (VLP) diets on the growth of pigs, but also influences the nitrogen (N) balance and lipid metabolism; however, the underlying pathways are not well understood. This study aimed to investigate the effect of dietary Val and Ile on lipogenesis, lipolysis, and ureagenesis under protein restriction. After one week of acclimation, forty three-week-old pigs were randomly assigned to following dietary treatments (n = 8/group) for 5 weeks: positive control (PC): normal protein diet; negative control (NC): VLP diet; HV: NC supplemented with Val; HI: NC supplemented with Ile; and HVI: NC supplemented with both Val and Ile. HVI partially improved the body weight and completely recovered the feed intake (FI) of pigs fed with NC. HVI increased thermal radiation and improved the glucose clearance. HVI had a lower blood triglyceride than PC and blood urea N than NC. NC and HV promoted lipogenesis by increasing the transcript of fatty acid synthase (FAS) in the liver and lipoprotein lipase (LPL) in adipose tissue but reducing hormone-sensitive lipase (HSL) in the liver. HVI reduced the increased rate of lipogenesis induced by the NC group through normalizing the mRNA abundance of hepatic FAS, sterol regulatory element binding transcription factor 1, and HSL and LPL in adipose tissue. NC, HV, HI, and HVI reduced the ureagenesis by decreasing the protein abundance of carbamoyl phosphate synthetase I, ornithine transcarboxylase, and arginosuccinate lyase in the liver. Overall, HVI improved the growth, FI, and glucose clearance, and decreased the rate of lipogenesis induced by VLP diets.

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis.

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10-500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.

Mitochondrial carbonic anhydrase VA and VB: properties and roles in health and disease.

Carbonic anhydrase V (CAV), a mitochondrial enzyme, was first isolated from guinea-pig liver and subsequently identified in mice and humans. Later, studies revealed that the mouse genome contains two mitochondrial CA sequences, named Car5A and Car5B. The CAVA enzyme is most highly expressed in the liver, whereas CAVB shows a broad tissue distribution. Car5A knockout mice demonstrated a predominant role for CAVA in ammonia detoxification, whereas the roles of CAVB in ureagenesis and gluconeogenesis were evident only in the absence of CAVA. Previous studies have suggested that CAVA is mainly involved in the provision of HCO3 - for biosynthetic processes. In children, mutations in the CA5A gene led to reduced CA activity, and the enzyme was sensitive to increased temperature. The metabolic profiles of these children showed a reduced supply of HCO3 - to the enzymes that take part in intermediary metabolism: carbamoylphosphate synthetase, pyruvate carboxylase, propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. Although the role of CAVB is still poorly understood, a recent study reported that it plays an essential role in human Sertoli cells, which sustain spermatogenesis. Metabolic disease associated with CAVA appears to be more common than other inborn errors of metabolism and responds well to treatment with N-carbamyl-l-glutamate. Therefore, early identification of hyperammonaemia will allow specific treatment with N-carbamyl-l-glutamate and prevent neurological sequelae. Carbonic anhydrase VA deficiency should therefore be considered a treatable condition in the differential diagnosis of hyperammonaemia in neonates and young children.

Effects of dietary cellulose supplementation on the intestinal health and ammonia tolerance in juvenile yellow catfish Pelteobagrus fulvidraco

Cellulose can reduce endogenous ammonia production of non-ruminant animals by altering intestinal microbiota structure. Although the utilization of cellulose in fish is limited, we speculate that cellulose has a potential role in regulating fish ammonia tolerance. Four experiment diets with different cellulose levels (Ctrl, 4.00%, 6.00% and 8.00%) were formulated and fed to yellow catfish for 84 days. This study found that dietary supplemented with 6.00% cellulose can improve growth performance (final body weight and weight gain rate), blood health (total protein, albumin and globulin), intestinal digestive enzyme activity (protease, lipase and cellulase), and ensure intestinal structure integrity (villi length), and maintain intestinal microbiota stability (Shannon and Chao1 indexes). After 96 h ammonia stress, serum ammonia content and cumulative mortality were negative correlated with Cetobacterium abundance. The activities of ammonia metabolism enzyme (glutamine synthetase, carbamyl phosphate synthetase, argininosuccinic acid synthase and arginase) and antioxidant enzyme (superoxide dismutase, catalase and glutathione peroxidase) in 4.00% and 6.00% cellulose groups were higher than those in Ctrl and 8.00% cellulose groups. The highest mRNA expression levels of antioxidant-related genes (Cu/Zn-SOD, CAT, GPX and GR) and inflammation-related genes (IL1, IL 8 and TNFɑ) were found in 8.00% cellulose group. This study indicates dietary supplemented with cellulose can increase the Cetobacterium abundance in intestinal tract, which is closely related to ammonia tolerance of yellow catfish. The levels of 4.00–6.00% cellulose in dietary is recommended in juvenile yellow catfish.

Ammonia detoxification promotes CD8+ T cell memory development by urea and citrulline cycles.

Amino acid metabolism is essential for cell survival, while the byproduct ammonia is toxic and can injure cellular longevity. Here we show that CD8+ memory T (TM) cells mobilize the carbamoyl phosphate (CP) metabolic pathway to clear ammonia, thus promoting memory development. CD8+ TM cells use β-hydroxybutyrylation to upregulate CP synthetase 1 and trigger the CP metabolic cascade to form arginine in the cytosol. This cytosolic arginine is then translocated into the mitochondria where it is split by arginase 2 to urea and ornithine. Cytosolic arginine is also converted to nitric oxide and citrulline by nitric oxide synthases. Thus, both the urea and citrulline cycles are employed by CD8+ T cells to clear ammonia and enable memory development. This ammonia clearance machinery might be targeted to improve T cell-based cancer immunotherapies.

Deficiency of Carbamoyl Phosphate Synthetase 1 Engenders Radioresistance in Hepatocellular Carcinoma via Deubiquitinating c-Myc

Tumor radiation resistance is the main obstacle to effective radiation therapy for patients with hepatocellular carcinoma (HCC). We identified the role of urea cycle key enzyme carbamoyl phosphate synthetase 1 (CPS1) in radioresistance of HCC and explored its mechanism, aiming to provide a novel radiosensitization strategy for the CPS1-deficiency HCC subtype. The expression of CPS1 was measured by western blot and immunohistochemistry. Cell growth assay, EdU assay, cell apoptosis assay, cell cycle assay, clone formation assay, and subcutaneous tumor assay were performed to explore the relationship between CPS1 and radioresistance of HCC cells. Lipid metabonomic analysis was used for investigating the effects of CPS1 on lipid synthesis of HCC cells. RNA sequencing and coimmunoprecipitation assay were carried out to reveal the mechanism of CPS1 participating in the regulation of HCC radiation therapy resistance. Furthermore, 10074-G5, the specific inhibitor of c-Myc, was administered to HCC cells to investigate the role of c-Myc in CPS1-deficiency HCC cells. We found that urea cycle key enzyme CPS1 was frequently lower in human HCC samples and positively associated with the patient's prognosis. Functionally, the present study proved that CPS1 depletion could accelerate the development of HCC and induce radiation resistance of HCC in vitro and in vivo, and deficiency of CPS1 promoted the synthesis of some lipid molecules. Regarding the mechanism, we uncovered that inhibition of CPS1 upregulated CyclinA2 and CyclinD1 by stabilizing oncoprotein c-Myc at the posttranscriptional level and generated radioresistance of HCC cells. Moreover, inactivation of c-Myc using 10074-G5, a specific c-Myc inhibitor, could partially attenuate the proliferation and radioresistance induced by depletion of CPS1. Our results recapitulated that silencing CPS1 could promote HCC progression and radioresistance via c-Myc stability mediated by the ubiquitin-proteasome system, suggesting that targeting c-Myc in CPS1-deficiency HCC subtype may be a valuable radiosensitization strategy in the treatment of HCC.

Identification and expression pattern analysis of PtCarA and PtCarB genes in Populus trichocarpa under different nitrogen treatments.

Carbamoyl phosphate synthetase (CPS) catalyses the synthesis of ammonia carbamoyl phosphate (CP), which plays a key role in the biosynthesis of arginine and pyrimidine nucleotides. There are two subunits of the CPS enzyme in Populus trichocarpa, CarA (small subunit) and CarB (large subunit). Only when they coexist can CPS catalyse synthesis of CP. However, it is not clear how CPS responds to nitrogen (N) to affect arginine and pyrimidine nucleotide biosynthesis. In this study, bioinformatics methods were used to analyse the expression patterns of genes encoding CarA and CarB, and qRT-PCR and RNA-seq were used to investigate their molecular responses under different N concentrations. Phylogenetic analysis revealed that the phylogenetic trees of CarA and CarB had similar topologies. qRT-PCR showed that the PtCarA and PtCarB genes were regulated by N, while their N-regulated patterns differed in different tissues. The expression patterns of PtCarA and PtCarB show a significant positive correlation according to qRT-PCR and RNA-seq. The analysis of promoter cis-acting elements showed that the promoter regions of PtCarA1, PtCarA2 and PtCarB contained some identical cis-acting elements. According to analysis of the phylogenetic tree, expression patterns and promoter elements, we speculate that there might be coevolution among PtCarA1, PtCarA2 and PtCarB. This study provides valuable information for further understanding the function of CPS in poplar, especially for N response, and provides new ideas for studying the evolution of gene families related to heteromultimers.

Exonuclease 1 Drives Growth and Genomic Instability in Multiple Myeloma through Interaction with CAD, a Key Protein Involved in De Novo Pyrimidine Biosynthesis

Human exonuclease 1 (Exo1) plays important roles in DNA replication and repair including the resection of DNA ends during homologous recombination (HR). Based on our previous data demonstrating the role of HR in genomic instability and growth of cancer cells, we investigated role of EXO1 in multiple myeloma (MM). Expression of EXO1 and pRPA32 (a marker of DNA end resection) was elevated in all myeloma cell lines tested (N=10) relative to normal peripheral blood mononuclear (PBMC) cell samples. We developed a fluorescence-based EXO1 activity assay and observed elevated (~1.5- to > 5-fold; p < 0.05) Exo1 activity in all twelve MM cell lines tested relative to normal PBMCs. Moreover., elevated expression of EXO1 correlated with poor overall and event-free survival in MM patients (MMRF dataset; p < 1x10-5). Transgenic suppression of EXO1 in MM cell lines caused a time-dependent loss in cell viability. Evaluation at day 5 after selection indicated that, relative to control cells, EXO1 suppression in RPMI, MM1S, MM1R and U266 cells was associated with ∼95%, 85%, 60% and 40% cell death, respectively. Apoptosis, as evaluated by annexin-labeling, was detected in ~50% of EXO1-knockdown cells compared to 11.9% of control MM1S cells. Conditional knockdown in H929 cells evaluation at 48 h after adding doxycycline demonstrated apoptosis in 18% of EXO1-suppressed cells vs. 4.9% of control cells. To investigate the mechanisms by which EXO1 impacts MM cell viability, the proteins interacting with EXO1 in MM cell lines (MM1S and H929) were identified using Exo1 pull-down and mass spectrometry. Interaction of EXO1 with CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a trifunctional protein involved in the first three steps of de novo pyrimidine biosynthesis, was identified and its binding confirmed with co-immunoprecipitation. We found that EXO1 overexpression increased the growth rate of non-cancerous cell types (HS5 - bone marrow stromal cells and normal fibroblasts), whereas suppression of CAD inhibited the spontaneous and EXO1-mediated increase in growth rate as well as DNA replication in both normal cell types. Consistently, the CAD-knockdown increased apoptosis in HS5 cells by ∼5-fold, whereas EXO1 overexpression reduced it by ~2-fold. Conversely, suppression of CAD also increased apoptosis in EXO1-overexpressing cells by 3-fold. In normal diploid fibroblast cells with a limited lifespan, EXO1 overexpression increased DNA replication by 32%, whereas CAD-knockdown reduced it by ∼65%. Importantly, CAD-knockdown in EXO1-overexpressing cells also reduced DNA replication by 26%. These data indicate that CAD is involved in EXO1-dependent cell proliferation and survival. Investigating the molecular events associated with EXO1, we observed that its suppression in MM cells inhibits DNA end resection, DNA breaks, HR activity and genomic instability (as assessed by a micronucleus assay); whereas overexpression in normal cells increased those activities. Moreover, inhibition of CAD reduced spontaneous as well as EXO1-induced HR activity and genomic instability in both hematopoietic and solid tumor cell types. Since CAD is involved in the first three steps of pyrimidine biosynthesis, our data suggest that interaction with CAD enables EXO1 to drive DNA replication, HR and associated genomic instability. Functional relevance of this interaction and possible regulation of de novo pyrimidine biosynthesis by EXO1 is being further investigated. Inhibitors of CAD may provide a novel therapeutic target in myeloma as well as other Exo1 overexpressing malignancies.

Identifying Mechanisms of Resistance to DHODH Inhibition in Cancer

All transcriptionally active and rapidly dividing cells require high amounts of pyrimidines for DNA replication and repair and RNA synthesis. The only two known mechanisms by which cells can acquire pyrimidines are the de novo pyrimidine biosynthesis pathway and the pyrimidine salvage pathway. The rate-limiting enzyme in de novo synthesis is dihydroorotate dehydrogenase (DHODH), a critical metabolic enzyme that converts dihydroorotate (DHO) to orotate, which is then converted to the pyrimidine precursor uridine monophosphate (UMP). The rate-limiting enzyme in the salvage pathway is uridine-cytidine kinase 2 (UCK2), a ribonucleoside kinase that converts uridine to UMP and cytidine to cytidine monophosphate (CMP). Under physiologic conditions, salvage pathway substrates are limiting, and many cancer types, most notably acute myeloid leukemia (AML), are highly dependent on DHODH activity for proliferation and survival. Based on these observations, several DHODH inhibitors have been developed as anti-cancer therapeutics and have been tested clinically. Unfortunately, to date, these drugs have failed to show significant anti-cancer activity in early-phase clinical trials. In order to understand how leukemia cells that are highly sensitive to DHODH inhibitors can bypass their dependence on DHODH activity, we conducted positive-selection whole-genome CRISPR-activation drug resistance screens in TF-1 human AML cells using BAY 2402234, a highly potent and highly specific DHODH inhibitor. In preliminary studies, we found that 1 nM BAY 2402234 fully inhibits TF-1 cells whereas TF-1 cells that overexpress high levels of DHODH require 50 nM BAY 2402234 to achieve the same effect. Importantly, the effects of 1 nM and 50 nM BAY 2402234 are fully rescued by addition of uridine to the culture media, demonstrating that the anti-leukemic activity of BAY 2402234 is on-target at both drug doses. To perform the drug resistance screens, we stably expressed nuclease-deactivated Cas9 fused to a transcriptional activator (dCas9-VP64) in TF-1 cells and then infected the cells with the human whole-genome Calabrese sgRNA lentiviral library. We then passaged the cells in media containing vehicle control (DMSO), 1 nM or 50 nM BAY 2402234 for 18 days, collecting cells on day 0 and day 18 of drug treatment. Genomic DNA was then extracted and was subjected to next generation sequencing to assess the relative abundance of each sgRNA in each sample. We reasoned that activation of genes that enhance de novo pathway activity at the level of, or downstream of, DHODH would promote resistance to 1 nM BAY 2402234 but not 50 nM BAY 2402234, whereas activation of genes that act through de novo pathway-independent mechanisms would promote resistance to both 1 nM and 50 nM BAY 2402234. As predicted, sgRNAs targeting DHODH conferred resistance to 1 nM but not 50 nM BAY 2402234 whereas sgRNAs targeting UCK2 conferred resistance to both 1 and 50 nM BAY 2402234 (Figure 1). A number of other known salvage pathway genes scored in both screens, including uracil phosphoribosyltransferase (UPRT) and the nucleoside transporters SLC4A1 and SLC29A1. Unexpectedly, the screens also identified carbamoyl-phosphate synthetase 2/aspartate transcarbamoylase/dihydroorotase (CAD) as a potential mechanism of resistance to DHODH inhibition. CAD is a trifunctional multi-domain enzyme that acts upstream of DHODH, catalyzing the first three steps in de novopyrimidine biosynthesis. How upregulation of CAD activity can bypass the requirement for DHODH activity is not clear, although CAD is a very large enzyme complex about which very little is known besides its de novo pyrimidine synthesis activity. The screens also identified several genes that are not known to play a role in pyrimidine metabolism as potential resistance mechanisms. These include the serine-threonine kinase MARK3, the serine-threonine phosphatase CTDSPL2, and the RNA-binding protein NANOS2. Finally, the screens identified several genes whose upregulation sensitized cells to BAY 2402234, including the urokinase receptor PLAUR, the adaptor protein BCAR3, the scaffold protein SHOC2, and the deoxynucleoside phosphohydrolase SAMHD1. Studies are currently underway to validate the results of the CRISPRa screens and elucidate the mechanisms by which the candidate resistance genes help cells to bypass their requirement for DHODH activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phylogenetic Analyses Support the Monophyly of the Genus Lispe Latreille (Diptera: Muscidae) with Insights into Intrageneric Relationships.

Lispe Latreille (Diptera: Muscidae) are a widespread group of predatory flies that inhabit semi-aquatic environments. Previous studies on this genus have mainly focused on morphological classification, so molecular data are entirely lacking, and there has been no attempt at a phylogenetic placement of the genus or the resolution of intragenic relationships. To address the phylogenetic placement of Lispe and to fill its gap in the Tree of Life Web Project, 58 Lispe spp. (covering 11 out of 13 acknowledged Lispe species groups) were selected to reconstruct a phylogeny using Maximum likelihood (ML) estimates, Maximum Parsimony (MP) analyses, and Bayesian inference (BI) based on two mitochondrial protein-coding genes (cytochrome c oxidase subunit I (COXI) and cytochrome b gene (CYTB)) and one nuclear gene (a fragment of the carbamoyl phosphate synthetase region of the CAD gene). The phylogenetic analyses indicated that the monophyletic Lispe is the sister group of the monophyletic Limnophora, together forming the tribe Limnophorini under the subfamily Coenosiinae. Three generic categories are proven obsolete: Chaetolispa Malloch, Lispacoenosia Snyder, and Xenolispa Malloch. Within the genus, the validity of 11 species groups is clarified by both molecular and morphological data. This study provides a sound basis for continuing intergeneric and intrageneric research into this fascinating and widespread genus.

Novel pathogenic variant (c.2947C > T) of the carbamoyl phosphate synthetase 1 gene in neonatal-onset deficiency.

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder characterized by hyperammonaemia. The biochemical measurement of the intermediate metabolites is helpful for CPS1D diagnosis; it however cannot distinguish CPS1D from N-acetylglutamate synthetase deficiency. Therefore, next-generation sequencing (NGS) is often essential for the accurate diagnosis of CPS1D. NGS was performed to identify candidate gene variants of CPS1D in a Asian neonatal patient presented with poor feeding, reduced activity, tachypnea, lethargy, and convulsions. The potential pathogenicity of the identified variants was predicted by various types of bioinformatical analyses, including evolution conservation, domain and 3D structure simulations. Compound heterozygosity of CPS1D were identified. One was in exon 24 with a novel heterozygous missense variant c.2947C > T (p.P983S), and another was previously reported in exon 20 with c.2548C > T (p.R850C). Both variants were predicted to be deleterious. Conservation analysis and structural modeling showed that the two substituted amino acids were highly evolutionarily conserved, resulting in potential decreases of the binding pocket stability and the partial loss of enzyme activity. In this study, two pathogenic missense variants were identified with NGS, expanding the variants pectrum of the CPS1 gene. The variants and related structural knowledge of CPS enzyme demonstrate the applicability for the accurate diagnosis of CPS1D.

Opposing effects of chronic glucagon receptor agonism and antagonism on amino acids, hepatic gene expression, and alpha cells

The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.

Identification and validation in a novel quantification system of the glutamine metabolism patterns for the prediction of prognosis and therapy response in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has one of the highest mortality rates worldwide. Abnormal glutamine metabolism (GM) has been reported to be involved in HCC progression. The current study sought to examine the predictive value of GM in HCC patient's prognosis and therapy response. The RNA-sequencing data and clinical information of HCC samples were obtained from The Cancer Genome Atlas (TCGA) database (N=377) and Gene Expression Omnibus (GEO) database (N=242). By analyzing a data set from TCGA, we showed that the GM landscape of HCC patients was developed based on the non-negative matrix factorization (NMF) algorithm. Univariate Cox regression and least absolute shrinkage and selection operator (LASSO)-penalized Cox regression analyses were used to construct a risk model. The accuracy of the model, which was based on the GM-related genes (GMRGs), was verified by Kaplan-Meier (K-M) and receiver operating characteristic (ROC) curves. We also verified the reliability of the model based on GEO data. Finally, the immune infiltration analysis, pathway enrichment analysis, and treatment response prediction results were compared to each other in the 2risk groups. In our study, the HCC samples were divided into 2 GM-related patterns; that is, C1 and C2. The multi-analysis revealed that the GM-related patterns were associated with the pathologic stage, T stages, N stages, histologic grade, and the tumor immune microenvironment (TIME). Next, the prognostic model containing 5 GMRGs (i.e., aldehyde dehydrogenase 5 family member A1, ASNSD1, carbamoyl-phosphate synthetase 1, GMPS, and PPAT) was constructed to calculate the risk score. The high-risk group of HCC patients had significantly worse overall survival (OS) than the low-risk group in both datasets (P<0.001). Multivariate Cox regression uncover the riskScores may serve as an independent prognostic marker for HCC patients [TCGA: hazard ratio (HR) =2.909 (1.940-4.362), P<0.001; GEO: HR =2.911 (1.753-5.848), P=0.043]. Finally, we found that the prognostic model was significantly correlated with the pathologic stage and TIME of the HCC patients in both databases. Moreover, the prognostic model may guide the immunotherapy, chemotherapy, and targeted drugs choice. In summary, we developed a GM-related 5-gene risk-score model, which may be a useful tool for predicting prognosis and guiding the treatment of HCC patients.

Clinical findings of patients with hyperammonemia affected by urea cycle disorders with hepatic encephalopathy.

Urea cycle disorders (UCD) are a group of genetic diseases caused by deficiencies in the enzymes and transporters involved in the urea cycle. The impairment of the cycle results in ammonia accumulation, leading to neurological dysfunctions and poor outcomes to affected patients. The aim of this study is to investigate and describe UCD patients' principal clinical and biochemical presentations to support professionals on urgent diagnosis and quick management, aiming better outcomes for patients. We explored medical records of 30 patients diagnosed in a referral center from Brazil to delineate UCD clinical and biochemical profile. Patients demonstrated a range of signs and symptoms, such as altered levels of consciousness, acute encephalopathy, seizures, progressive loss of appetite, vomiting, coma, and respiratory distress, in most cases combined with high levels of ammonia, which is an immediate biomarker, leading to a UCD suspicion. The most prevalent UCD detected were ornithine transcarbamylase deficiency, followed by citrullinemia type 1, hyperargininemia, carbamoyl phosphate synthase 1 deficiency, and argininosuccinic aciduria. Clinical symptoms were highly severe, being the majority developmental and neurological disabilities, with 20% of death rate. Laboratory analysis revealed high levels of ammonia (mean ± SD: 860 ± 470 μmol/L; reference value: ≤80 μmol/L), hypoglycemia, metabolic acidosis, and high excretion of orotic acid in the urine (except in carbamoyl phosphate synthetase 1 [CPS1] deficiency). We emphasize the need of urgent identification of UCD clinical and biochemical conditions, and immediate measurement of ammonia, to enable the correct diagnosis and increase the chances of patients' survival, minimizing neurological and psychomotor damage caused by hepatic encephalopathy.

Oncogenic β-catenin stimulation of AKT2–CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer

CTNNB1, encoding β-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated β-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in β-catenin mutant cell lines and livers. Oncogenic β-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of β-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed β-catenin mutant cell proliferation and tumor formation. Therefore, β-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of β-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for β-catenin mutant liver cancer.

Ornithine aminotransferase and carbamoyl phosphate synthetase 1 involved in ammonia metabolism serve as novel targets for early stages of gastric cancer.

ObjectiveThe sensitivity and specificity of current biomarkers for gastric cancer were insufficient. The aim of the present study was to screen novel biomarkers and determine the diagnostic values of ornithine aminotransferase (OAT) and carbamoyl phosphate synthetase 1 (CPS1) for detecting gastric cancer.MethodsWith stable isotope tags, we labelled an initial discovery group of four paired gastric cancer tissue samples and identified with LC‐ESI‐MS/MS. A validation group of 159 gastric cancer samples and 30 healthy controls were used to validate the candidate targets. GSEA was used to explore the pathways activated in gastric cancer.ResultsFour hundred and thirty one proteins were found differentially expressed in gastric cancer tissues. Of these proteins, OAT and CPS1 were found over‐expressed in gastric cancer patients, with sensitivity of 70.4% (95% CI: 63.3%–77.6%) and specificity of 80.5% (95% CI: 74.3%–86.7%) for ornithine aminotransferase, and with sensitivity of 68.6% (95% CI: 61.3%–75.8%) and specificity of 73% (95% CI: 66%–79.9%) for carbamoyl phosphate synthetase 1. The co‐expression of OAT and CPS1 in gastric cancer tissues has a sensitivity of 81% (95% CI: 73.2%–88.8%) and specificity of 89% (95% CI: 83%–95%). Furthermore, both OAT and CPS1 were overexpressed in patients with local invasion T3 and T4 stages than those in patients with T1 and T2 stages. The co‐expression of OAT and CPS1 was strongly correlated with histological grade I 68% (95% CI: 58.7%–77.3%) and TNM stage I/II 52% (95% CI: 42%–62%). The areas under ROC curves were up to 0.758 for the co‐expression of OAT and CPS1 in gastric cancer. GSEA results showed that two gene sets and 30 gene sets were activated in OAT high‐ and CPS1 high‐expression patients with gastric cancer, respectively.ConclusionsThe present findings indicated a tight correlation between the co‐expression of OAT and CPS1 and the histological grade, local invasion, and TNM stages of gastric cancer. Therefore, OAT and CPS1 might be predictors for gastric cancer invasion and potential targets for anticancer drug design for gastric cancer.

Anesthesia management in living-donor liver transplantation in a patient with carbamoyl phosphate synthetase deficiency: a case report

BackgroundCarbamoyl phosphate synthetase deficiency (CPS1D) is a urea-cycle disorder (UCD). We report successful perioperative management of pediatric living donor liver transplantation (LDLT) in a CPS1D patient.Case presentationA 10-year-old female patient with CPS1D underwent LDLT. Proper administration of dextrose 50% and 60 kcal/kg/day with l-arginine and l-carnitine resulted in the avoidance of intraoperative hyperammonemia induced by hypercatabolism. Serum ammonia level transiently increased to 61 mmol/L in the anhepatic phase and decreased to 44 mmol/L after reperfusion.ConclusionsWe suggest anesthesia management with administration of dextrose to avoid hyperammonemia during LDLT in patients with CPS1D.