Carbamoyl Phosphate Synthetase Research Articles

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase, a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking phosphoribosylformylglycinamidine synthase or phosphoribosyl pyrophosphate amidotransferase, another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mammalian target of rapamycin complex 1 (mTORC1) suppression and was profoundly suppressed in cells deficient for tuberous sclerosis complex 2, which negatively regulates mTORC1 through inhibition of Ras homolog enriched in brain, suggesting that purines regulate autophagy through the tuberous sclerosis complex-Ras homolog enriched in brain-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase and dihydroorotate dehydrogenase activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

Mitochondrial dysfunction as a mechanistic biomarker in patients with non-alcoholic fatty liver disease (NAFLD)

BackgroundDysfunctional metabolism lies at the centre of the pathogenesis for Non-Alcoholic Fatty Liver Disease (NAFLD) and involves mitochondrial dysfunction, lipid dysmetabolism and oxidative stress. This study, for the first time, explores real-time energy changes in peripheral blood and corresponding metabolite changes, to investigate whether mitochondria-related immunometabolic biomarkers can predict progression in NAFLD. MethodsThirty subjects divided into 3 groups were assessed: NAFLD with biopsy-proven mild fibrosis (n = 10), severe fibrosis (n = 10) and healthy controls (HC, n = 10). Mitochondrial functional analysis was performed in a Seahorse XFp analyzer in live peripheral blood mononuclear cells (PBMCs). Global metabolomics quantified a broad range of human plasma metabolites. Mitochondrial carbamoyl phosphate synthase 1(CPS-1), Ornithine transcarbamoylase (OTC), Fibroblast growth factor-21 (FGF-21) and a range of cytokines in plasma were measured by ELISA. ResultsNAFLD patients with severe fibrosis demonstrated reduced maximal respiration (106 ± 25 versus 242 ± 62, p < 0.05) and reserve capacity (56 ± 16 versus 184 ± 42, p = 0.006) compared to mild/moderate fibrosis. Comparing mild/moderate vs severe liver fibrosis in patients with NAFLD, 14 out of 493 quantified metabolites were significantly changed (p < 0.05). Most of the amino acids modulated were the urea cycle (UC) components which included citrulline/ornithine ratio, arginine and glutamate. Plasma levels of CPS-1 and FGF-21 were significantly higher mild versus severe fibrosis in NAFLD patients. This novel panel generated an area under the ROC of 0.95, sensitivity of 100% and specificity 80% and p = 0.0007 (F1-F2 versus F3-F4). ConclusionProgression in NAFLD is associated with mitochondrial dysfunction and changes in metabolites associated with the urea cycle. We demonstrate a unique panel of mitochondrial-based, signatures which differentiate between stages of NAFLD. Lay summaryMitochondrial dysfunction in peripheral cells along with alterations in metabolites of urea cycle act as a sensor of hepatocyte mitochondrial damage. These changes can be measured in blood and together represent a unique panel of biomarkers for progression of fibrosis in NAFLD.

Multi-omics Analysis of CRISPRi-Knockdowns Identifies Mechanisms that Buffer Decreases of Enzymes in E.coli Metabolism.

Enzymes maintain metabolism, and their concentration affects cellular fitness: high enzyme levels are costly, and low enzyme levels can limit metabolic flux. Here, we used CRISPR interference (CRISPRi) to study the consequences of decreasing E.coli enzymes below wild-type levels. A pooled CRISPRi screen with 7,177 strains demonstrates that metabolism buffers fitness defects for hours after the induction of CRISPRi. We characterized the metabolome and proteome responses in 30 CRISPRi strains and elucidated three gene-specific buffering mechanisms: ornithine buffered the knockdown of carbamoyl phosphate synthetase (CarAB) by increasing CarAB activity, S-adenosylmethionine buffered the knockdown of homocysteine transmethylase (MetE) by de-repressing expression of the methionine pathway, and 6-phosphogluconate buffered the knockdown of 6-phosphogluconate dehydrogenase (Gnd) by activating a bypass. In total, this work demonstrates that CRISPRi screens can reveal global sources of metabolic robustness and identify local regulatory mechanisms that buffer decreases of specific enzymes. A record of this paper's transparent peer review process is included in the Supplemental Information.

Synergistic targeting of CHK1 and mTOR in MYC-driven tumors.

Deregulation of v-myc avian myelocytomatosis viral oncogene homolog (MYC) occurs in a broad range of human cancers and often predicts poor prognosis and resistance to therapy. However, directly targeting oncogenic MYC remains unsuccessful, and indirectly inhibiting MYC emerges as a promising approach. Checkpoint kinase 1 (CHK1) is a protein kinase that coordinates the G2/M cell cycle checkpoint and protects cancer cells from excessive replicative stress. Using c-MYC-mediated T-cell acute lymphoblastic leukemia (T-acute lymphoblastic leukemia) and N-MYC-driven neuroblastoma as model systems, we reveal that both c-MYC and N-MYC directly bind to the CHK1 locus and activate its transcription. CHIR-124, a selective CHK1 inhibitor, impairs cell viability and induces remarkable synergistic lethality with mTOR inhibitor rapamycin in MYC-overexpressing cells. Mechanistically, rapamycin inactivates carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD), the essential enzyme for the first three steps of de novo pyrimidine synthesis, and deteriorates CHIR-124-induced replicative stress. We further demonstrate that dual treatments impede T-acute lymphoblastic leukemia and neuroblastoma progression in vivo. These results suggest simultaneous targeting of CHK1 and mTOR as a novel and powerful co-treatment modality for MYC-mediated tumors.

Carbamoyl phosphate synthetase subunit Cpa1 interacting with Dut1, controls development, arginine biosynthesis, and pathogenicity of Colletotrichum gloeosporioides

Carbamoyl phosphate synthetase is involved in arginine biosynthesis in many organisms. In this study, we investigate the biological function of Cpa1, a small subunit of carbamoyl phosphate synthetase of Colletotrichum gloeosporioides. The deletion of the CPA1 gene affected vegetative growth, arginine biosynthesis, and fungal pathogenicity. Genetic complementation with native CPA1 fully recovered all these defective phenotypes. We observed that Cpa1-RFP fusion protein is localized at the mitochondria, which is consistent with Cpa2, a large subunit of carbamoyl phosphate synthetase. We identified the proteins that interact with Cpa1 by using the two-hybrid screen approach, and we showed that Dut1 interacts with Cpa1 but without Cpa2 in vivo. Dut1 is dispensable for hyphal growth, appressorial formation, and fungal pathogenicity. Interestingly, the Dut1-Cpa1 complex is localized at the mitochondria. Further studies showed that Dut1 regulates Cpa1-Cpa2 interaction in response to arginine. In summary, our studies provide new insights into how Cpa1 interacts with its partner proteins to mediate arginine synthesis.

Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Considering Proximal Urea Cycle Disorders in Expanded Newborn Screening.

Proximal urea cycle disorders (PUCDs) have adverse outcomes such as intellectual disability and death, which may benefit from newborn screening (NBS) through early detection and prevention with early treatment. Ornithine transcarbamylase deficiency (OTCD) and carbamoyl phosphate synthetase 1 deficiency (CPS1D) are screened in six and eight states in the United States. We analyzed current evidence to see if it supports inclusion of PUCDs in the NBS panels based upon prevention potential, medical, diagnostic, treatment, and public health rationales. A literature review was performed in PubMed using MESH terms for OTCD, CPS1D, and NAGSD. A systematic review was performed in the hallmark of NBS inclusion criteria. We reviewed 31 articles. Molecular and biochemical diagnosis is available to provide diagnostic evidence. Untreated PUCDs have a significant burden with considerable developmental delay and mortality that may improve with early treatment. Tandem mass spectrometry can be used for NBS for PUCDs; however, citrulline and glutamine alone are not specific. Medical treatments currently available for PUCDs meet existing medical, diagnostic, treatment, and public health rationales. Improvement in NBS algorithms to increase sensitivity and specificity will allow earlier diagnosis and treatment to potentially improve disability and mortality rates.

Adaptation of the carbamoyl-phosphate synthetase enzyme in an extremophile fish.

Tetrapods and fish have adapted distinct carbamoyl-phosphate synthase (CPS) enzymes to initiate the ornithine urea cycle during the detoxification of nitrogenous wastes. We report evidence that in the ureotelic subgenus of extremophile fish Oreochromis Alcolapia, CPS III has undergone convergent evolution and adapted its substrate affinity to ammonia, which is typical of terrestrial vertebrate CPS I. Unusually, unlike in other vertebrates, the expression of CPS III in Alcolapia is localized to the skeletal muscle and is activated in the myogenic lineage during early embryonic development with expression remaining in mature fish. We propose that adaptation in Alcolapia included both convergent evolution of CPS function to that of terrestrial vertebrates, as well as changes in development mechanisms redirecting CPS III gene expression to the skeletal muscle.

The conversion of formate into purines stimulates mTORC1 leading to CAD-dependent activation of pyrimidine synthesis

BackgroundMitochondrial serine catabolism to formate induces a metabolic switch to a hypermetabolic state with high rates of glycolysis, purine synthesis and pyrimidine synthesis. While formate is a purine precursor, it is not clear how formate induces pyrimidine synthesis.MethodsHere we combine phospho-proteome and metabolic profiling to determine how formate induces pyrimidine synthesis.ResultsWe discover that formate induces phosphorylation of carbamoyl phosphate synthetase (CAD), which is known to increase CAD enzymatic activity. Mechanistically, formate induces mechanistic target of rapamycin complex 1 (mTORC1) activity as quantified by phosphorylation of its targets S6, 4E-BP1, S6K1 and CAD. Treatment with the allosteric mTORC1 inhibitor rapamycin abrogates CAD phosphorylation and pyrimidine synthesis induced by formate. Furthermore, we show that the formate-dependent induction of mTOR signalling and CAD phosphorylation is dependent on an increase in purine synthesis.ConclusionsWe conclude that formate activates mTORC1 and induces pyrimidine synthesis via the mTORC1-dependent phosphorylation of CAD.

Multi-omics analysis reveals that co-exposure to phthalates and metals disturbs urea cycle and choline metabolism

This study aimed to evaluate the response of HepaRG cells after co-exposure to phthalates and heavy metals, using a high-dimensional biology paradigm (HDB). Liver is the main metabolism site for the majority of xenobiotics. For this reason, the HepaRG cell line was used as an in vitro model, and cells were exposed to two characteristic mixtures of phthalates and heavy metals containing phthalates (DEHP, DiNP, BBzP) and metals (lead, methylmercury, total mercury) in a concentration-dependent manner. The applied chemical mixtures were selected as the most abundant pollutants in the REPRO_PL and PHIME cohorts, which were studied using the exposome-wide approach in the frame of the EU project HEALS. These studies investigated the environmental causation of neurodevelopmental disorders in neonates and across Europe. The INTEGRA computational platform was used for the calculation of the effective concentrations of the chemicals in the liver through extrapolation from human biomonitoring data and this dose (and a ten-times higher one) was applied to the hepatocyte model. Multi-omics analysis was performed to reveal the genes, proteins, and metabolites affected by the exposure to these chemical mixtures. By extension, we could detect the perturbed metabolic pathways. The generated data were analyzed using advanced bioinformatic tools following the HEALS connectivity paradigm for multi-omics pathway analysis. Co-mapped transcriptomics and proteomics data showed that co-exposure to phthalates and heavy metals leads to perturbations of the urea cycle due to differential expression levels of arginase-1 and -2, argininosuccinate synthase, carbamoyl-phosphate synthase, ornithine carbamoyltransferase, and argininosuccinate lyase. Joint pathway analysis of proteomics and metabolomics data revealed that the detected proteins and metabolites, choline phosphate cytidylyltransferase A, phospholipase D3, group XIIA secretory phospholipase A2, α-phosphatidylcholine, and the a 1,2-diacyl-sn-glycero-3-phosphocholine, are responsible for the homeostasis of the metabolic pathways phosphatidylcholine biosynthesis I, and phospholipases metabolism. The urea, phosphatidylcholine biosynthesis I and phospholipase metabolic pathways are of particular interest since they have been identified also in human samples from the REPRO_PL and PHIME cohorts using untargeted metabolomics analysis and have been associated with impaired psychomotor development in children at the age of two. In conclusion, this study provides the mechanistic evidence that co-exposure to phthalates and metals disturb biochemical processes related to mitochondrial respiration during critical developmental stages, which are clinically linked to neurodevelopmental perturbations.

The Application of Next-Generation Sequencing (NGS) in Neonatal-Onset Urea Cycle Disorders (UCDs): Clinical Course, Metabolomic Profiling, and Genetic Findings in Nine Chinese Hyperammonemia Patients.

During Jan. 2016–Dec. 2019, nine Chinese patients from eight unrelated families were diagnosed with neonatal-onset UCDs by targeted panel sequencing or whole-exome sequencing (WES). Their clinical manifestations, biochemical features, 180-day-age outcomes, and molecular genetic characteristics were reviewed retrospectively. NGS-based tests revealed 7 patients diagnosed with ornithine transcarbamylase deficiency (OTCD) and 2 with carbamoylphosphate synthetase I deficiency (CPS1D). The spectrum of the clinical presentation of nine affected individuals progressed from unspecific symptoms like poor feeding to somnolence, coma, and death. All patients presented with an acute hyperammonemia. The most robust metabolic pattern in OTCD was hyperglutaminemic hyperammonemia with high concentration of urine orotic acid, and it was reported in six patients. Of ten variants found on the OTC gene and CPS1 gene, 3 were novel: (c.176T>C (p.L59P)) in the OTC gene, c.2938G>A (p.G980S) and c.3734T>A (p.L1245H) in the CPS1 gene. There was a high mortality rate of 77.78% (7/9) for all the defects combined. An OTC-deficient male and a CPS1-deficient female survived from episodes of hyperammonemia. Although prompt recognition of UCD and the use of alternative pathway therapy in addition to provision of appropriate nutrition and dialysis improved survival, the overall outcomes for the neonatal-onset type are poor in China.

Effect of dietary l-arginine of broiler breeder hens on embryonic development, apparent metabolism, and immunity of offspring

This study investigated the effects of supplemented l-arginine (l-Arg) in broiler breeder hens' diets on the embryonic development and physiological changes of offspring during the hatching period. A total of 480 35-wk-old healthy female Arbor Acres broiler breeders were randomly divided into 6 groups and fed a corn and soybean meal diet with 6 digestible Arg levels (0.96%, 1.16%, 1.35%, 1.55%, 1.74%, and 1.93%). After a 10-wk experiment, eggs were collected for incubation. At embryonic day (E) 11 to E21, eggs, embryos, and organs (liver, breast muscle, and thigh muscle) were weighed. Total protein, urea nitrogen, creatinine, cholesterol, and triglyceride in plasma, were measured. Plasma level of immunoglobulin G (IgG), immunoglobulin M (IgM), and nitric oxide synthase (NOS) were measured at E13, E17, and E21. Messenger RNA expression of carbamoyl phosphate synthase I (CPS1), ornithine transcarbamylase (OTC), and argininosuccinate synthase (ASS) in liver and breast muscle tissues was assessed at E13, E17, and E21. The results showed that 1.16% Arg in maternal diet increased egg weight (P < 0.05). The level of Arg in maternal diet has a significant effect on organ index and embryo weight of multiple embryonic days (P < 0.05). Embryonic plasma total protein concentration was significantly affected by maternal dietary Arg level (P < 0.05) and exhibited quadratic responses at E11, E15, E17, and E21 (P < 0.01). Plasma urea nitrogen, creatinine, triglyceride, and cholesterol level were also significantly affected by the level of maternal Arg at different embryonic ages (P < 0.05). Dietary digestible Arg levels quadratically influenced plasma urea nitrogen level at E21 (P < 0.05) and cholesterol concentration at E17 and E19 (P < 0.01). L-Arg supplementation in maternal diet significantly improved the IgG level at E17 and E21 (1.16%, 1.35%, 1.55%, and 1.74%; P < 0.05), the IgM level at E13 (1.35%, 1.55%, 1.74%, and 1.93%) and E17 (P < 0.05) and the NOS level at E13, E17, and E21 (P < 0.05). Maternal dietary L-Arg supplementation significantly improved the expression of CPS1 gene, OTC gene (1.16%, 1.35%, and 1.55%), and ASS gene (1.35% and 1.55%) in the liver (P < 0.05), and also enhanced the CPS1 gene (except 1.35%) and OTC gene (1.55% and 1.74%) expression in the breast muscle (P < 0.05). In conclusion, maternal Arg level affected the embryonic development of offspring and regulated the apparent metabolic programming and immunity state of the embryo. Arginine level of 1.55% in hens' diet was beneficial to the protein synthesis and immunity of the offspring in the embryonic period, and it was recommended to obtain healthy offspring.

End Point Multiplex PCR for Diagnosis of Haemoprotozoan Diseases in Cattle.

Theileriosis, trypanosomosis and babesiosis are the three major haemoprotozoan diseases causing huge economic losses worldwide. Difficulty in diagnosis of these diseases lies with the detection of carrier state with low parasitemia and concurrent infection. The present study was conducted to standardize and evaluate multiplex PCR assay for specific, fast and simultaneous detection of Theileria annulata, Trypanosoma evansi and Babesia bovis in bovines. Positive parasitic DNA was obtained from microscopically positive samples. Simplex PCR assay was developed targeting repetitive nucleotide sequences for Trypanosoma evansi and gene coding enzyme carbamoyl phosphate synthetase II for Babesia bovis. For theileriosis conditions already standardized targeting cytochrome b gene was used. Gradient PCR assay was used to determine common amplification conditions and develop multiplex PCR assay. Limit of detection was determined using tenfold serial dilution of parasitic DNA. Blood samples collected from 117 bovines suspected for haemoparasite infection was tested by simplex and multiplex PCR assay. Simplex PCR assay was able to detect Theileria annulata, Trypanosoma evansi and Babesia bovis at dilution 10-9, 10-8 and 10-8 which corresponds to copy number 1, 10 and 10, respectively, whereas of multiplex PCR assay was found to be 10-7 dilution corresponding to 100 copy number. PCR products bands obtained in multiplex PCR assay at 257, 312 and 446bp were easily distinguishable. Results of simplex PCR assay for detection of individual parasites revealed 48 (41.02%), 27 (23.07%) and 5 (4.27%) samples positive for T. annulata, T. evansi and B. bovis, respectively. Sixty-three (53.8%) samples were found positive by multiplex PCR assay with 15 samples (23.8%) showing mixed infection. Multiplex PCR assay was found to be highly specific and can be used for easy, early, sensitive, specific and simultaneous diagnosis of haemoprotozoan diseases in epidemiological survey as a robust tool.

Sustained Virologic Remission in an 8‐Month‐old Pediatric Patient With Carbamoyl Phosphate Synthetase I Deficiency and Hepatitis C Infection Using Direct‐acting Antivirals Prior to Liver Transplant

Sustained Virologic Remission in an 8‐Month‐old Pediatric Patient With Carbamoyl Phosphate Synthetase I Deficiency and Hepatitis C Infection Using Direct‐acting Antivirals Prior to Liver Transplant

Molecular docking studies of natural compounds of naringin on enzymes involved in the urea cycle pathway in hyperammonemia

Purpose: To investigate the anti-hyperammonemic activity of naringin by molecular docking via in silico studies.Methods: Urea cycle proteins were docked to the natural compound naringin as well as a standard drug, sodium benzoate. Hydrogen bonds and binding energy were obtained using Catalytic Site Atlas and Cast P Finder Software Tool.Results: There were six urea cycle enzymes, including N-acetyl glutamate synthase, carbamoyl phosphate synthase I, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase I. On evaluating protein interactions with naringin, which is dynamically connected to the urea cycle pathway with hyperammonemia, naringin showed more hydrogen bonds and also produced higher binding energy when compared to the standard drug, sodium benzoate.Conclusion: The results of the molecular docking study show that naringin interacts with urea cycle enzymes with more hydrogen bonds and higher bonding energy than the standard drug, sodium benzoate. This supports the hypothesis that naringin can prevent experimental hyperammonemia.&#x0D; Keywords: Naringin, Sodium benzoate, Hyperammonemia, Urea cycle enzymes, In silico studies&#x0D;

Energy and nitrogenous waste from glutamate/glutamine catabolism facilitates acute osmotic adjustment in non-neuroectodermal branchial cells

Maintenance of homeostasis is one of the most important physiological responses for animals upon osmotic perturbations. Ionocytes of branchial epithelia are the major cell types responsible for active ion transport, which is mediated by energy-consuming ion pumps (e.g., Na+-K+-ATPase, NKA) and secondary active transporters. Consequently, in addition to osmolyte adjustments, sufficient and immediate energy replenishment is essenttableial for acclimation to osmotic changes. In this study, we propose that glutamate/glutamine catabolism and trans-epithelial transport of nitrogenous waste may aid euryhaline teleosts Japanese medaka (Oryzias latipes) during acclimation to osmotic changes. Glutamate family amino acid contents in gills were increased by hyperosmotic challenge along an acclimation period of 72 hours. This change in amino acids was accompanied by a stimulation of putative glutamate/glutamine transporters (Eaats, Sat) and synthesis enzymes (Gls, Glul) that participate in regulating glutamate/glutamine cycling in branchial epithelia during acclimation to hyperosmotic conditions. In situ hybridization of glutaminase and glutamine synthetase in combination with immunocytochemistry demonstrate a partial colocalization of olgls1a and olgls2 but not olglul with Na+/K+-ATPase-rich ionocytes. Also for the glutamate and glutamine transporters colocalization with ionocytes was found for oleaat1, oleaat3, and olslc38a4, but not oleaat2. Morpholino knock-down of Sat decreased Na+ flux from the larval epithelium, demonstrating the importance of glutamate/glutamine transport in osmotic regulation. In addition to its role as an energy substrate, glutamate deamination produces NH4+, which may contribute to osmolyte production; genes encoding components of the urea production cycle, including carbamoyl phosphate synthetase (CPS) and ornithine transcarbamylase (OTC), were upregulated under hyperosmotic challenges. Based on these findings the present work demonstrates that the glutamate/glutamine cycle and subsequent transepithelial transport of nitrogenous waste in branchial epithelia represents an essential component for the maintenance of ionic homeostasis under a hyperosmotic challenge.

Carbamoyl-Phosphate Synthase 1 as a Novel Target of Phomoxanthone A, a Bioactive Fungal Metabolite.

Phomoxanthone A, a bioactive xanthone dimer isolated from the endophytic fungus Phomopsis sp., is a mitochondrial toxin weakening cellular respiration and electron transport chain activity by a fast breakup of the mitochondrial assembly. Here, a multi-disciplinary strategy has been developed and applied for identifying phomoxanthone A target(s) to fully address its mechanism of action, based on drug affinity response target stability and targeted limited proteolysis. Both approaches point to the identification of carbamoyl-phosphate synthase 1 as a major phomoxanthone A target in mitochondria cell lysates, giving also detailed insights into the ligand/target interaction sites by molecular docking and assessing an interesting phomoxanthone A stimulating activity on carbamoyl-phosphate synthase 1. Thus, phomoxanthone A can be regarded as an inspiring molecule for the development of new leads in counteracting hyperammonemia states.

Sex-Specific Differences in the Control of Serum Concentrations of Glycine in Subjects with Metabolic Syndrome and Mendelian Randomization Analysis for Obesity

Abstract Objectives Glycine is a novel circulating marker for metabolic diseases associated with lower type-2 diabetes and protection against obesity in some studies. Circulating glycine levels are genetically determined under the control of several loci. The locus most strongly associated is the carbamoyl-phosphate synthase I (CPS1). Interestingly, some sex-specific genome-wide association studies (GWAS) showed differences in the effect of the main single nucleotide polymorphisms (SNPs) in this locus on glycine levels (significant sex * CPS1 interactions). However, the potential mechanisms explaining this sex-heterogeneity are unknown. Therefore, our aims were: 1) to analyze whether the sex * CPS1 interaction for serum glycine is present in subjects from a Mediterranean population, and 2) to test, in another population, the glycine sex-specific associations with obesity, using a Mendelian randomization approach. Methods We studied 426 subjects (aged 55–75 y) with metabolic syndrome from the PREDIMED Plus-Valencia Study. Serum glycine was determined by NMR spectroscopy. We screened the CPS1 gene for SNP associations. In another Mediterranean population (PREDIMED-Valencia participants, n = 1009) we analyzed the sex-specific associations between the genetically determined glycine (CPS1-SNP) and obesity, using Mendelian randomization. Results In the PREDIMED Plus-Valencia subjects, serum glycine levels were higher in women than in men (280 vs 240 micromol/L, respectively; P = 1.4 × 1E15). The CPS1-rs7684 (3’-UTR) SNPs was strongly associated with glycine levels in women but not in men (P for sex * CPS1 interaction = 0.004), replicating previous findings. The minor allele was associated with lower serum glycine. In the Mendelian randomization study in the population with unmeasured glycine levels, the CPS1-rs7684 SNPs presented a sex-specific interaction for obesity (P &amp;lt; 0.05). Women with the minor allele (lower glycine levels) were more likely to be obese than homozygous women for the major allele (P &amp;lt; 0.05). No associations were detected in men. Conclusions We replicated the sex * CPS1 interaction in determining glycine levels and also detected sex * CPS1 interactions for obesity risk. Funding Sources Generalitat Valenciana (PROMETEO2017/017, APOSTD/2019/136), Fundació La Marató de TV3 (538/U/2016), and CIBEROBN.

Discovery of 2,6-Dimethylpiperazines as Allosteric Inhibitors of CPS1.

Carbamoyl phosphate synthetase 1 (CPS1) is a potential synthetic lethal target in LKB1-deficient nonsmall cell lung cancer, where its overexpression supports the production of pyrimidine synthesis. In other cancer types, CPS1 overexpression and activity may prevent the accumulation of toxic levels of intratumoral ammonia to support tumor growth. Herein we report the discovery of a novel series of potent and selective small-molecule inhibitors of CPS1. Piperazine 2 was initially identified as a promising CPS1 inhibitor through a high-throughput screening effort. Subsequent structure-activity relationship optimization and structure-based drug design led to the discovery of piperazine H3B-616 (25), a potent allosteric inhibitor of CPS1 (IC50 = 66 nM).

Retrospective evaluation of 85 patients with urea cycle disorders: one center experience, three new mutations.

Objectives Urea cycle disorders (UCDs) are rare hereditary diseases. This study was conducted to help identify the characteristics of UCDs in Turkey. Methods The primary outcome was to determine patient characteristics. Investigating the relationships between the patient outcomes and ammonia levels were the secondary outcomes. Eighty five patients from 79 families, diagnosed with UCD at a single metabolic referral center between 1979 and 2017, were included. Clinical and laboratory data were retrieved retrospectively from hospital records. Results Classical citrullinemia was the most common type of UCD; citrin deficiency and carbamoyl phosphate synthase 1 deficiency (CPS1D) were the rarest. One thirty one hyperammonemic episodes were recorded. The peak ammonia levels were found to be significantly associated with polycythemia and hypocalcemia at presentation. The median peak ammonia values of the patients who died were higher than those of the survivors. The highest mortality rate was in the classical citrullinemia group. The mortality rate of the first hyperammonemic crisis was 28.6%, while it was 6.7% in subsequent episodes with an odds ratio of 4.28 (95% CI: 1.67-11.0) (p=0.001). Forty-four patients underwent genetic analysis and genetic variants were detected in 42 patients (95%). Three of the detected variants have not been previously reported. Conclusions This is the largest UCD series in Turkey and may serve as a guide to clinical, biochemical and genetic features of UCDs in our country. Prevention of hyperammonemia may be the most influential measure to improve long term survival.