Carbamoyl Phosphate Synthetase Research Articles

The role of Ashwagandha in modulating gut parameters in dogs—a randomized double-blind placebo-controlled trial

IntroductionThis study explored the role of Withania somnifera/Ashwagandha root extract (ARE) on important gut–microbiome parameters in healthy geriatric dogs. We hypothesized that ARE might promote a healthy gut by its adaptogenic and anti-inflammatory effects and improve vital parameters for healthy ageing.MethodsA randomized, double-blind, placebo-controlled trial was conducted in Telangana, India. Twelve healthy geriatric Beagle dogs aged 12–15 years were enrolled. The dogs were divided into two groups to receive ARE (15 mg/kg, once daily, orally, for 2 months) or a placebo control. Various parameters were assessed, including serum haematology, biochemical markers, stool parameters, and gut–microbiome parameters.ResultsThe erythrocyte counts and haemoglobin levels were significantly increased with ARE (p < 0.01 and p < 0.001). Moreover, a significant decrease in important serum liver biomarkers (alanine transaminase [ALT], aspartate transaminase [AST]; p < 0.01 and p < 0.001 at day 60) was observed in the ARE-treated dogs compared to that in the placebo control group. In addition, the levels of L-citrulline were significantly modulated by ARE intervention, whereas the intervention did not affect intestinal-type alkaline phosphatase (I-ALP), lactate, and carbamoyl-phosphate synthase (CPS). Interestingly, the faecal score reduced significantly with ARE (p < 0.001), while the faecal pH remained unaltered. Compared to the baseline, ARE significantly decreased two microbial metabolites, propionic acid, and total short chain fatty acids (SCFAs) levels after 60 days of intervention, whereas butyrate and acetic acid levels remained unchanged in the faecal samples.ConclusionIn summary, these findings suggest that ARE has gut health promoting benefits in healthy geriatric dogs by improving haematological and biochemical profiles; the levels of L-citrulline; propionic acid; and SCFA; thus, reducing age-related changes by modulating the microbiome and the associated metabolites.

N-acetylglutamate synthetase deficiency - literature review

Introduction and Objective: N-Acetylglutamate Synthetase Deficiency (NAGSD) is a rare urea cycle disorder, primarily caused by autosomal recessive genetic mutations. Most affected individuals are homozygous for the disease-causing allele. The aim of this publication is to discuss various aspects of N-acetylglutamate synthetase deficiency based on the latest literature.Review Methods: The PubMed database was searched to find scientific articles in which the terms "N-acetylglutamate synthetase," "NAGS deficiency," or "N-acetylglutamate synthetase deficiency" appear in the title, abstract, or keywords.Brief Description of the State of Knowledge: Symptoms of NAGSD typically appear in the neonatal period. The NAGS enzyme is essential for catalyzing the synthesis of N-acetylglutamate, an allosteric activator of carbamoylphosphate synthetase 1 (CPS1), crucial for the urea cycle. Key biochemical markers include elevated ammonia, glutamine, and alanine; decreased citrulline; and respiratory alkalosis. Hyperammonemia is the main clinical feature, manifesting as nausea, vomiting, cognitive impairment, seizures, and, in severe cases, coma or death. Genetic testing remains the cornerstone of diagnosis, enabling identification of specific mutations. Treatment involves carbamylglutamate, an NAG analog that activates CPS1.Summary: Expanding research aims to explore novel treatments in NAGSD, dietary strategies, and the safety of current therapies during pregnancy.

Tandem mass spectrometry screening and genetic analysis of neonates with Urea cycle disorders

To explore the results of four types of Urea cycle disorders (UCDs) in newborns from the Xuzhou region, assess the efficacy of newborn screening by tandem mass spectrometry (MS/MS), and analyze their genetic characteristics. A retrospective analysis was performed using tandem mass spectrometry to screen for inherited metabolic disorders in 691 712 newborns at the Maternal and Child Health Care Hospital of Xuzhou from November 2015 to December 2023. Ten children (cases 1-10) were diagnosed with Ornithine transcarbamylase deficiency (OTCD), Carbamoylphosphate synthase 1 deficiency (CPS1D), Arginase deficiency (ARGD), and Argininosuccinate synthase deficiency (ASSD) based on MS/MS and genetic testing. This study was approved by the Medical Ethics Committee of Xuzhou Maternity and Child Health Care Hospital (Ethics No.XZFY2024-051K-01J). A total of 691 712 neonates were screened for UCDs using MS/MS, which identified 1 237, 1 237, 510, and 1 009 initial positive cases for OTCD, CPS1D, ASSD, and ARGD, respectively. After genetic testing, 1 case of OTCD, 1 case of CPS1D, 1 case of ASSD, and 7 cases of ARGD were confirmed. The overall positive predictive value for these four UCDs was 0.362%. Among the 10 diagnosed UCD cases, four novel variants were identified, which included OTC: c.1024C>A (p.L342M) and ASS1: c.826A>G (p.M276V), c.695C>T (p.P232L) and c.694C>T (p.P232S). Bioinformatic analysis has rated these as variants of uncertain clinical significance or likely pathogenic based on guidelines from the American College of Medical Genetics and Genomics (ACMG). The incidence of four UCDs in neonates from the Xuzhou area is relatively low, and there is a correlation between genetic variants and clinical phenotypes. For novel variants with uncertain clinical significance or suspected pathogenicity, their pathogenicity should be clarified in conjunction with clinical and biochemical indicators. The four novel pathogenic variants of UCDs identified in this study have enriched the mutational spectrum of UCDs-associated genes in the Xuzhou region.

Virtual Screening for Molecular Targets of Emodin Against Red Complex Pathogens

Objective: Periodontitis is a chronic inflammatory disease affecting teeth' supporting tissues. It is caused by specific bacterial species, including Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), and Treponema denticola, known as the "red complex" group. These bacteria manipulate the immune response and promote tissue destruction, making them key players in periodontal pathogenesis. The present study aims to identify the potential molecular targets of Emodin against the red complex pathogens. Method: The interaction between the phytocompound Emodin and red complex pathogens was identified using the STITCH tool. The proteins identified were then classified into functional categories using the VICMPred. The virulent proteins identified were then subjected to Bepired prediction, which provided information about the epitopes in the virulent proteins. Finally, the subcellular location of the proteins was demonstrated with the pSORTb tool. Results: Carbamoyl-phosphate synthase is a large subunit identified as a virulence protein in Pg and Tf. DNA topoisomerase IV subunit A was found to be the common virulence protein for Pg and Td. The DNA gyrase subunit A and ATPase/histidine kinase/DNA gyrase B/HSP90 domain-containing protein were found to be identified in Td and Tf. It was the only protein predicted to be in the cytoplasmic membrane, while others were found in the cytoplasm. The four virulent proteins targeted by Emodin were found to harbor multiple epitopes. Conclusion: Emodin was found to interact with all three pathogens of the red complex group. However, further experimental validation is warranted to prove the antimicrobial effect of Emodin against periodontal pathogens.

Rhizophagus irregularis regulates RiCPSI and RiCARI expression to influence plant drought tolerance.

Arbuscular mycorrhizal fungi (AMF) can transfer inorganic nitrogen (N) from the soil to host plants to cope with drought stress, with arginine synthesis and NH4+ transport being pivotal processes. However, the regulatory mechanism underlying these processes remains unclear. Here, we found that drought stress upregulated expression of genes involved in the N transfer pathway and putrescine and glutathione synthesis in the mycorrhizal structures of Rhizophagus irregularis within alfalfa (Medicago sativa) roots, i.e., carbamoyl-phosphate synthase (RiCPSI), arginase (RiCARI), urease (RiURE), ornithine decarboxylase (RiODC), and glutamate-cysteine ligase (RiGCL). Furthermore, we confirmed that RiCPSI is a carbamoyl phosphate synthase. Silencing RiCARI via host-induced gene silencing inhibited arbuscule formation, suppressed putrescine and glutathione synthesis, and altered arginine metabolism within R. irregularis-plant symbiosis, leading to a substantial reduction in the drought tolerance of M. sativa. Conversely, silencing RiCPSI decreased arginine, putrescine, and glutathione synthesis in R. irregularis but did not adversely affect NH4+ transfer from fungi to the host plant and drought tolerance of M. sativa. Interestingly, overexpressing RiCPSI via our host-induced gene overexpressing system enhanced arginine, putrescine, and glutathione synthesis in R. irregularis, reduced arbuscule abundance, and improved drought tolerance of M. sativa. Our findings demonstrate that, under drought stress, R. irregularis-plant symbiosis facilitates improved NH4+ transfer from AMF to the host plant. This is accompanied by increased arginine, putrescine, and glutathione synthesis within R. irregularis, driven by the upregulation of RiCPSI and RiCARI expression in mycorrhizal structures within the roots. These molecular adjustments collectively contribute to enhanced drought tolerance in R. irregularis-plant symbiosis.

Single-cell and machine learning approaches uncover intrinsic immune-evasion genes in the prognosis of hepatocellular carcinoma

Background and aimsHepatocellular carcinoma (HCC) is a tumor of high heterogeneity and complexity, which poses significant challenges to effective treatment and patient prognosis because of its immune evasion characteristics. To address these issues, single-cell technology and machine learning methods have emerged as a promising approach to identify genes associated with immune escape in HCC. This study aimed to develop a prognostic risk score model for HCC by identifying intrinsic immune-evasion genes (IIEGs) through single-cell technology and machine learning, providing insights into immune infiltration, enhancing predictive accuracy, and facilitating the development of more effective treatment strategies. Materials and methodsThe study utilized data from The Cancer Genome Atlas database to analyze gene expression profiles and clinical data related to intrinsic immune evasion in patients with HCC. Various tools, including the Human Protein Atlas, cBioPortal, single-cell analysis, machine learning, and Kaplan-Meier plot, were used to analyze IIEGs. Functional enrichment analysis was conducted to explore potential mechanisms. In addition, the abundance of infiltrating cells in the tumor microenvironment was investigated using single-sample gene set enrichment analysis, CIBERSORT, xCELL, and tumor immunophenotype algorithms. The expression of glycosylphosphatidylinositol anchor attachment 1 (GPAA1) was examined in the clinical sample of HCC by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. ResultsUnivariate Cox analysis identified 63 IIEGs associated with the prognosis of HCC. Using random forest, least absolute shrinkage and selection operator regression analysis, and support vector machine, a risk score model consisting of six IIEGs (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), phosphatidylinositol glycan anchor biosynthesis class U (PIGU), endoplasmic reticulum membrane protein complex subunit 3 (EMC3), centrosomal protein 55 (CEP55), autophagy-related 10 (ATG10), and GPAA1) developed, which was validated using 10 pairs of HCC and adjacent non-cancerous samples. Based on the calculated median risk score, HCC samples were categorized into high- and low-risk groups. The Kaplan-Meier curve analysis showed that the high-risk group had a worse prognosis compared with the low-risk group. Time-dependent receiver operating characteristic analysis demonstrated the accurate predictive capability of the risk score model for HCC prognosis. Furthermore, immune infiltration analysis showed a positive correlation between the risk score model and 40 immune checkpoint genes as well as Th2 cells. ConclusionsA prognostic risk score model was formulated by six IIEG signatures and showed promise in predicting the prognosis of patients diagnosed with HCC. The utilization of the IIEG risk score as a novel prognostic index, together with its significance as a valuable biomarker for immunotherapy in HCC, provides benefit for patients with HCC in determining therapeutic strategies for clinical application.

Loss of Carbamoyl Phosphate Synthetase 1 Potentiates Hepatocellular Carcinoma Metastasis by Reducing Aspartate Level.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. Numerous studies have shown that metabolic reprogramming is crucial for the development of HCC. Carbamoyl phosphate synthase 1 (CPS1), a rate-limiting enzyme in urea cycle, is an abundant protein in normal hepatocytes, however, lacking systemic research in HCC. It is found that CPS1 is low-expressed in HCC tissues and circulating tumor cells, negatively correlated with HCC stage and prognosis. Further study reveals that CPS1 is a double-edged sword. On the one hand, it inhibits the activity of phosphatidylcholine-specific phospholipase C to block the biosynthesis of diacylglycerol (DAG), leading to the downregulation of the DAG/protein kinase C pathway to inhibit invasion and metastasis of cancer cells. On the other hand, CPS1 promotes cell proliferation by increasing intracellular S-adenosylmethionin to enhance the m6A modification of solute carrier family 1 member 3 mRNA, a key transporter for aspartate intake. Finally, CPS1 overexpressing adeno-associated virus can dampen HCC progression. Collectively, this results uncovered that CPS1 is a switch between HCC proliferation and metastasis by increasing intracellular aspartate level.

Functional and Proteomic Analyses of a Putative Carbamoyl Phosphate Synthase Large Subunit in Relation to Virulence, Arginine and Pyrimidine Biosynthesis, and Siderophore Production in Erwinia amylovora

The apple is a significant global fruit cultivated extensively worldwide. Fire blight, caused by Erwinia amylovora (Ea), poses a significant threat to global apple production. To control this disease, characterizing the virulence mechanisms/factors is imperative. Carbamoyl phosphate synthase is an essential enzyme in the biosynthesis of arginine and pyrimidine. However, the functions of this protein in Ea remains poorly understood. This study aimed to investigate the functions of the carbamoyl phosphate synthase large subunit in Ea (CarBEa). In a virulence assay using fruitlets, an Ea strain lacking CarBEa exhibited significantly reduced virulence on fruitlets. In the auxotrophy assay, this mutant failed to grow in minimal media lacking both arginine and pyrimidine, but growth was restored when both compounds were supplemented. The comparative proteomic analysis suggests that CarBEa is involved in diverse biological processes, including amino acid and nucleotide metabolism, and inorganic ion transport. Finally, we demonstrated that CarBEa is related to siderophore secretion/production by the chrome azurol S agar plate assay. This report provides valuable insights into the functions of carbamoyl phosphate synthase large subunit, which serves as a potential target for developing efficient anti-virulence substances to control fire blight.

Phylogeography of the ambrosia beetle Euwallacea interjectus (Coleoptera: Curculionidae: Scolytinae): an emerging poplar pest and its Fusarium mutualists from poplar plantations in China.

Native to Asia, Euwallacea interjectus (Blandford) (Coleoptera: Curculionidae: Scolytinae) is a destructive and invasive pest of live trees, and now it has been found in the United States and Argentina. In recent years, this pest appeared in high densities in poplar monocultures from Eastern China (Jiangsu and Shanghai) and Argentina and caused significant poplar mortality. However, the origin of the pests related to tree damage and the Fusarium mutualists from some poplar zones in China remained unclear. Here, we provided a broader phylogeographic analysis of E. interjectus based on the mitochondrial gene (cytochrome c oxidase I) to determine the global genetic structure of this species. Five mitochondrial lineages were found in the native area. Populations introduced to the United States were originated from 4 localities. The Argentine population was derived from Japan. The species was observed with strikingly high level of cytochrome c oxidase I intraspecific divergence that exceeded interspecific divergence, but the high intraspecific variation was correlated with geographical locations among the native populations. Two nuclear genes (arginine kinase and carbamoyl-phosphate synthetase 2-aspartate transcarbamylase-dihydroorotase) were more conservative, and intraspecific differences were lower than interspecific differences. The mitochondrial genetic variation was probably caused by evolution of lineages among geographically isolated populations. But it is immature to infer the existence of cryptic species based on cytochrome c oxidase I differences. All samples collected from poplar populations were indigenous and formed close relationship with a specimen from eastern and southern China. Surprisingly, pests from poplar populations in Jiangsu and Shanghai showed different haplotypes and mutualists. This suggested that the control strategies should consider the genetic and mutualistic diversity of beetles at different poplar localities.

Prokaryotic-virus-encoded auxiliary metabolic genes throughout the global oceans

BackgroundProkaryotic microbes have impacted marine biogeochemical cycles for billions of years. Viruses also impact these cycles, through lysis, horizontal gene transfer, and encoding and expressing genes that contribute to metabolic reprogramming of prokaryotic cells. While this impact is difficult to quantify in nature, we hypothesized that it can be examined by surveying virus-encoded auxiliary metabolic genes (AMGs) and assessing their ecological context.ResultsWe systematically developed a global ocean AMG catalog by integrating previously described and newly identified AMGs and then placed this catalog into ecological and metabolic contexts relevant to ocean biogeochemistry. From 7.6 terabases of Tara Oceans paired prokaryote- and virus-enriched metagenomic sequence data, we increased known ocean virus populations to 579,904 (up 16%). From these virus populations, we then conservatively identified 86,913 AMGs that grouped into 22,779 sequence-based gene clusters, 7248 (~ 32%) of which were not previously reported. Using our catalog and modeled data from mock communities, we estimate that ~ 19% of ocean virus populations carry at least one AMG. To understand AMGs in their metabolic context, we identified 340 metabolic pathways encoded by ocean microbes and showed that AMGs map to 128 of them. Furthermore, we identified metabolic “hot spots” targeted by virus AMGs, including nine pathways where most steps (≥ 0.75) were AMG-targeted (involved in carbohydrate, amino acid, fatty acid, and nucleotide metabolism), as well as other pathways where virus-encoded AMGs outnumbered cellular homologs (involved in lipid A phosphates, phosphatidylethanolamine, creatine biosynthesis, phosphoribosylamine-glycine ligase, and carbamoyl-phosphate synthase pathways).ConclusionsTogether, this systematically curated, global ocean AMG catalog and analyses provide a valuable resource and foundational observations to understand the role of viruses in modulating global ocean metabolisms and their biogeochemical implications.7DrB1a6JQbvrw3a2P4Hm3dVideo

Clinical features and CPS1 variants in Chinese patients with carbamoyl phosphate synthetase 1 deficiency

BackgroundCarbamoyl phosphate synthetase 1 (CPS1) deficiency (OMIM 237300), an autosomal recessive rare and severe urea cycle disorder, is associated with hyperammonemia and high mortality.MethodsHerein we present 12 genetic variants identified in seven clinically well-characterized Chinese patients with CPS1 deficiency who were admitted to the Children’s Medical Center of Peking University First Hospital from September 2014 to August 2023.ResultsSeven patients (two male and five female patients including two sisters) experienced symptoms onset between 2 days and 13 years of age, and they were diagnosed with CPS1 deficiency between 2 months and 20 years. Peak blood ammonia levels ranged from 160 to 1,000 µmol/L. Three patients showed early-onset CPS1 deficiency, with only one surviving after treatment with sodium phenylbutyrate, N-carbamoyl-L-glutamate, and liver transplantation at 4 months, showing a favorable outcome. The remaining four patients had late-onset CPS1 deficiency, presenting with mental retardation, psychiatric symptoms, and self-selected low-protein diets. Among the 12 CPS1 variants identified in these patients, 10 were novel, with all patients exhibiting compound heterozygosity for CPS1 mutant alleles. Seven variants (c.149T > C, c.616 A > T, c.1145 C > T, c.1294G > A, c.3029 C > T, c.3503 A > T, and c.3793 C > T) resulted in single amino acid substitutions. Three frameshift variations (c.2493del, c.3067dup, and c.3241del) were identified, leading to enzyme truncation. One mutation (c.3506_3508del) caused an in-frame single amino acid deletion, while another (c.2895 + 2T > C) resulted in aberrant splicing.ConclusionsExcept for two known variants, all other variants were identified as novel. No hotspot variants were observed among the patients. Our data contribute to expanding the mutation spectrum of CPS1.

Carbamoyl phosphate synthase 1 (CPS1), a mitochondrial enzyme of the urea cycle, is involved in LDL receptor modulation: In vitro studies

Carbamoyl phosphate synthase 1 (CPS1), a mitochondrial enzyme of the urea cycle, is involved in LDL receptor modulation: In vitro studies

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Advances in human glutamine-hydrolyzing synthetases and their therapeutic potential

Bifunctional enzymes, characterized by their dual active sites, enable efficient chemical conversion and substrate channeling using elegant coupling mechanisms to coordinate the two active sites. In humans, several bifunctional enzymes synthesize de novo carbon-nitrogen bonds by hydrolyzing glutamine and ATP in distinct active sites. Notable examples include guanosine monophosphate synthetase, cytidine triphosphate synthetase, phosphoribosylformyl-glycinamidine synthase, asparagine synthetase, and nicotinamide adenine dinucleotide synthetase. A more complex example of multifunctional glutamine-hydrolyzing synthetases in humans is carbamoyl phosphate synthetase. These enzymes are crucial for the biosynthesis of amino acids, nucleic acids, and co-factors, thereby playing pivotal roles in human health. This review delineates recent progress in understanding the structural characteristics, regulatory mechanisms, and disease relevance of glutamine-hydrolyzing synthetases in humans. Insights into their catalysis and activity regulation offer potential pathways for developing novel therapeutics.

Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased Km Glutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

Urea cycle promotion via ammonia-upregulated CPS1 is involved in arsenite-induced pulmonary fibrosis through enhancing collagen synthesis

Arsenic exposure is connected with lung toxicity and is related to lung fibrotic changes. Idiopathic pulmonary fibrosis (IPF) is characterized by extracellular matrix (ECM) deposition. Various genetic mechanisms and environmental factors induce or exacerbate pulmonary fibrosis. Collagen synthesis induced by sodium arsenite (NaAsO2) is closely associated with IPF. Fibroblasts tend to fine-tune their metabolic networks to support their synthetic requirements in response to environmental stimuli. Alterations in metabolism have an influential role in the pathogenesis of IPF. However, it is unclear how arsenic affects the metabolism in IPF. The urea cycle (UC) is needed for collagen formation, which provides adequate levels of proline (Pro) for biosynthesis of collagen. Carbamoyl phosphate synthetase 1 (CPS1) converts the ammonia to carbamoyl phosphate, which controls the first reaction of the UC. We show that, in arsenite-exposed mice, high amounts of ammonia in the lung microenvironment promotes the expression levels of CPS1 and the Pro metabolism. Reduction of ammonia and CPS1 ablation inhibit collagen synthesis and ameliorate IPF phenotypes induced by arsenite. This work takes advantage of multi-omics data to enhance understanding of the underlying pathogenic mechanisms, the key molecules and the complicated cellular responses to this pollutant, which provide a target for the prevention of pulmonary fibrosis caused by arsenic.

Prognostic and Immunotherapeutic Predictive Value of CAD Gene in Hepatocellular Carcinoma: Integrated Bioinformatics and Experimental Analysis.

Hepatocellular carcinoma (HCC) is a common type of cancer that ranks first in cancer-associated death worldwide. Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) are the key components of the pyrimidine pathway, which promotes cancer development. However, the function of CAD in HCC needs to be clarified. In this study, the clinical and transcriptome data of 424 TCGA-derived HCC cases were analyzed. The results demonstrated that high CAD expression was associated with poor prognosis in HCC patients. The effect of CAD on HCC was then investigated comprehensively using GO annotation analysis, KEGG enrichment analysis, Gene Set Enrichment Analysis (GSEA), and CIBERSORT algorithm. The results showed that CAD expression was correlated with immune checkpoint inhibitors and immune cell infiltration. In addition, low CAD levels in HCC patients predicted increased sensitivity to anti-CTLA4 and PD1, while HCC patients with high CAD expression exhibited high sensitivity to chemotherapeutic and molecular-targeted agents, including gemcitabine, paclitaxel, and sorafenib. Finally, the results from clinical sample suggested that CAD expression increased remarkably in HCC compared with non-cancerous tissues. Loss of function experiments demonstrated that CAD knockdown could significantly inhibit HCC cell growth and migration both in vitro and in vivo. Collectively, the results indicated that CAD is a potential oncogene during HCC metastasis and progression. Therefore, CAD is recommended as a candidate marker and target for HCC prediction and treatment.

Cytotoxicity of Quantum Dots in Receptor-Mediated Endocytic and Pinocytic Pathways in Yeast.

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.

The histone demethylase JMJD1C regulates CPS1 expression and promotes the proliferation of paroxysmal nocturnal haemoglobinuria clones through cell metabolic reprogramming.

Paroxysmal nocturnal haemoglobinuria (PNH) is a disorder resulting from erythrocyte membrane deficiencies caused by PIG-A gene mutations. While current treatments alleviate symptoms, they fail to address the underlying cause of the disease-the pathogenic PNH clones. In this study, we found that the expression of carbamoyl phosphate synthetase 1 (CPS1) was downregulated in PNH clones, and the level of CPS1 was negatively correlated with the proportion of PNH clones. Using PIG-A knockout K562 (K562 KO) cells, we demonstrated that CPS1 knockdown increased cell proliferation and altered cell metabolism, suggesting that CPS1 participates in PNH clonal proliferation through metabolic reprogramming. Furthermore, we observed an increase in the expression levels of the histone demethylase JMJD1C in PNH clones, and JMJD1C expression was negatively correlated with CPS1 expression. Knocking down JMJD1C in K562 KO cells upregulated CPS1 and H3K36me3 expression, decreased cell proliferation and increased cell apoptosis. Chromatin immunoprecipitation analysis further demonstrated that H3K36me3 regulated CPS1 expression. Finally, we demonstrated that histone demethylase inhibitor JIB-04 can suppressed K562 KO cell proliferation and reduced the proportion of PNH clones in PNH mice. In conclusion, aberrant regulation of the JMJD1C-H3K36me3-CPS1 axis contributes to PNH clonal proliferation. Targeting JMJD1C with a specific inhibitor unveils a potential strategy for treating PNH patients.

L-Ornithine-l-Aspartate (LOLA) Normalizes Metabolic Parameters in Models of Steatosis, Insulin Resistance and Metabolic Syndrome.

l-Ornithine- l-aspartate (LOLA) reduces toxic ammonium (NH3) plasma levels in hepatic encephalopathy. NH3 detoxification/excretion is achieved by its incorporation into urea and glutamine via activation of carbamoyl phosphate synthetase 1 (CSP1) by l-ornithine and stimulation of arginase by l-aspartate. We aimed at identifying additional molecular targets of LOLA as a potential treatment option for non-alcoholic fatty liver disease (NAFLD). In primary hepatocytes from NAFLD patients, urea cycle enzymes CSP1 and ornithine transcarbamylase (OTC) increase, while the catabolism of branched-chain amino acids (BCAAs) decreases with disease severity. In contrast, LOLA increased the expression rates of the BCAA enzyme transcripts bcat2, bckdha, and bckdk. In untreated HepG2 hepatoblastoma cells and HepG2-based models of steatosis, insulin resistance, and metabolic syndrome (the latter for the first time established herein), LOLA reduced the release of NH3; beneficially modulated the expression of genes related to fatty acid import/transport (cd36, cpt1), synthesis (fasn, scd1, ACC1), and regulation (srbf1); reduced cellular ATP and acetyl-CoA; and favorably modulated the expression of master regulators/genes of energy balance/mitochondrial biogenesis (AMPK-α, pgc1α). Moreover, LOLA reconstituted the depolarized mitochondrial membrane potential, while retaining mitochondrial integrity and avoiding induction of superoxide production. Most effects were concentration-dependent at ≤40 mM LOLA. We demonstrate for l-ornithine-l-aspartate a broad range of reconstituting effects on metabolic carriers and targets of catabolism/energy metabolism impaired in NAFLD. These findings strongly advocate further investigations to establish LOLA as a safe, efficacious, and cost-effective basic medication for preventing and/or alleviating NAFLD.