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.

Carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC) deficiencies are rare urea cycle disorders, which can lead to life-threatening hyperammonemia. Liver transplantation (LT) provides a cure and offers an alternative to medical treatment and life-long dietary restrictions with permanent impending risk of hyperammonemia. Nevertheless, in most patients, metabolic aberrations persist after LT, especially low plasma citrulline levels, with questionable clinical impact. So far, little is known about these alterations and there is no consensus, whether l-citrulline substitution after LT improves patients\' symptoms and outcomes. In this multicentre, retrospective, observational study of 24 patients who underwent LT for CPS1 (n = 11) or OTC (n = 13) deficiency, 25% did not receive l-citrulline or arginine substitution. Correlation analysis revealed no correlation between substitution dosage and citrulline levels (CPS1, p = 0.8 and OTC, p = 1). Arginine levels after liver transplantation were normal after LT independent of citrulline substitution. Native liver survival had no impact on mental impairment (p = 0.67). Regression analysis showed no correlation between l-citrulline substitution and failure to thrive (p = 0.611) or neurological outcome (p = 0.701). Peak ammonia had a significant effect on mental impairment (p = 0.017). Peak plasma ammonia levels correlate with mental impairment after LT in CPS1 and OTC deficiency. Growth and intellectual impairment after LT are not significantly associated with l-citrulline substitution.

Carbamoyl phosphate synthetase 1 (CPS1) is the most abundant hepatocyte mitochondrial matrix protein. Hypoosmotic stress increases CPS1 release in isolated mouse hepatocytes without cell death. We hypothesized that increased CPS1 release during hypoosmosis is selective and associates with altered mitochondrial morphology. Both ex vivo and in vivo models were assessed. Mouse hepatocytes and livers were challenged with isotonic or hypoosmotic (35 mosM) buffer. Mice were injected intraperitoneally with water (10% body weight) with or without an antidiuretic. Mitochondrial and cytosolic fractions were isolated using differential centrifugation, then analyzed by immunoblotting to assess subcellular redistribution of four mitochondrial proteins: CPS1, ornithine transcarbamylase (OTC), pyrroline-5-carboxylate reductase 1 (PYCR1), and cytochrome c. Mitochondrial morphology alterations were examined using electron microscopy. Hypoosmotic treatment of whole livers or hepatocytes led to preferential or increased mitochondrial release, respectively, of CPS1 as compared with two mitochondrial matrix proteins (OTC/PYCR1) and with the intermembrane space protein, cytochrome c. Mitochondrial apoptosis-induced channel opening using staurosporine in hepatocytes led to preferential CPS1 and cytochrome c release. The CPS1-selective changes were accompanied by dramatic alterations in ultrastructural mitochondrial morphology. In mice, hypoosmosis/hyponatremia led to increased liver vascular congestion and increased CPS1 in bile but not blood, coupled with mitochondrial structural alterations. In contrast, isotonic increase of intravascular volume led to a decrease in mitochondrial size with limited change in bile CPS1 compared with hypoosmotic conditions and absence of the hypoosmosis-associated histological alterations. Taken together, hepatocyte CPS1 is selectively released in response to hypoosmosis/hyponatremia and provides a unique biomarker of mitochondrial injury.NEW & NOTEWORTHY Exposure of isolated mouse livers, primary cultured hepatocytes, or mice to hypoosmosis/hyponatremia conditions induces significant mitochondrial shape alterations accompanied by preferential release of the mitochondrial matrix protein CPS1, a urea cycle enzyme. In contrast, the intermembrane space protein, cytochrome c, and two other matrix proteins, including the urea cycle enzyme ornithine transcarbamylase, remain preferentially retained in mitochondria. Therefore, hepatocyte CPS1 manifests unique mitochondrial stress response compartmentalization and is a sensitive sensor of mitochondrial hypoosmotic/hyponatremic injury.

Tumor protein 53 (TP53) mutation in bladder carcinoma (BC), upregulates the transcription of carbamoyl phosphate synthetase 1 (CPS1), to reduce intracellular ammonia toxicity. To leverage ammonia combating BC, here, an intravesically perfusable nanoporter-encased hydrogel system is reported. A biomimetic fusogenic liposomalized nanoporter (FLNP) that is decorated with urea transporter-B (UT-B) is first synthesized with protonated chitosan oligosaccharide for bladder tumor-targeted co-delivery of urease and small interfering RNA targeting CPS1 (siCPS1). Mussel-inspired hydrogel featured with dual functions of bio-adhesion and injectability is then fabricated as the reservoir for intravesical immobilization of FLNP. It is found that FLNP-mediated UT-B immobilization dramatically induces urea transportation into tumor cells, and co-delivery of urease and siCPS1 significantly boosts ammonia accumulation in tumor inducing cell apoptosis. Treatment with hybrid system exhibits superior anti-tumor effect in orthotopic bladder tumor mouse model and patient-derived xenograft model, respectively. Combined with high-protein diet, the production of urinary urea increases, leading to an augmented intracellular deposition of ammonia in BC cells, and ultimately an enhanced tumor inhibition. Together, the work establishes that cascade modulation of ammonia in tumor cells could induce tumor apoptosis and may be a practical strategy for eradication of TP53-mutated bladder cancer.

O-linked N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of intracellular proteins is a dynamic process broadly implicated in age-related disease, yet it remains uncharacterized whether and how O-GlcNAcylation contributes to the natural aging process. O-GlcNAc transferase (OGT) and the opposing enzyme O-GlcNAcase (OGA) control this nutrient-sensing protein modification in cells. Here, we show that global O-GlcNAc levels are increased in multiple tissues of aged mice. In aged liver, carbamoyl phosphate synthetase 1 (CPS1) is among the most heavily O-GlcNAcylated proteins. CPS1 O-GlcNAcylation is reversed by calorie restriction and is sensitive to genetic and pharmacological manipulations of the O-GlcNAc pathway. High glucose stimulates CPS1 O-GlcNAcylation and inhibits CPS1 activity. Liver-specific deletion of OGT potentiates CPS1 activity and renders CPS1 irresponsive to further stimulation by a prolonged fasting. Our results identify CPS1 O-GlcNAcylation as a key nutrient-sensing regulatory step in the urea cycle during aging and dietary restriction, implying a role for mitochondrial O-GlcNAcylation in nutritional regulation of longevity.

Carbamoyl phosphate synthetase 1 (CPS1), which is the antigen for the hepatocyte paraffin 1 antibody, exhibits focal immunoreactivity in adenocarcinoma from the gastrointestinal tract, but its expression profiles and roles in gastric cancer (GC) remain largely unknown. The present study aimed to determine the expression pattern and prognostic value of CPS1 in Correa\'s cascade using tissues from 32 patients with chronic atrophic gastritis with intestinal metaplasia (IM), 62 patients with low- or high-grade intraepithelial neoplasia (IN) and 401 patients with GC. The expression of CPS1 was diffuse and strongly positive in 32 cases (100%) of IM of the glandular epithelium, and gradually downregulated in Correa\'s cascade, with a strongly positive ratio of 21 (70%) in low-grade IN and 4 (12.5%) in high-grade IN. The levels of CPS1 expression were significantly higher in diffuse-type GC, with 37 (26%) cases strongly positive for CPS1, compared with 14 (8%) in intestinal-type and 11 (13%) cases in mixed-type GC. In intestinal-type GC, CPS1 expression was completely lost in 107 (62%) of cases, which was associated with an advanced Tumor-Node-Metastasis stage (P=0.031) and depth of invasion (P=0.037). Kaplan-Meier analysis suggested that low CPS1 expression levels were independently associated with a short overall survival (OS) time in the three types of GC (P<0.001 in intestinal-type, P=0.003 in diffuse-type and P=0.018 in mixed-type GC). Furthermore, low levels of CPS1 mRNA and high methylation levels in the CPS1 promoter were associated with a short OS time in patients with GC. These results suggested that the expression of CPS1 was progressively downregulated in Correa\'s cascade, and that CPS1 may serve as a prognostic marker for patients with GC, regardless of tumor type.

Urea cycle disorders (UCDs) are inherited metabolic diseases that lead to hyperammonemia with variable clinical manifestations. Using data from a nationwide study, we investigated the onset time, gene variants, clinical manifestations, and treatment of patients with UCDs in Japan. Of the 229 patients with UCDs diagnosed and/or treated between January 2000 and March 2018, identified gene variants and clinical information were available for 102 patients, including 62 patients with ornithine transcarbamylase (OTC) deficiency, 18 patients with carbamoyl phosphate synthetase 1 (CPS1) deficiency, 16 patients with argininosuccinate synthetase (ASS) deficiency, and 6 patients with argininosuccinate lyase (ASL) deficiency. A total of 13, 10, 4, and 5 variants in the OTC, CPS1, ASS, and ASL genes were respectively identified as novel variants, which were neither registered in ClinVar databases nor previously reported. The onset time and severity in patients with UCD could be predicted based on the identified gene variants in each patient from this nationwide study and previous studies. This genetic information may help in predicting the long-term outcome and determining specific treatment strategies such as liver transplantation in patients with UCDs.

Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as \"heterozygous-only\" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.

The aim of the present report was to describe the clinical presentation, diagnosis, and treatment of a case of carbamoyl phosphate synthetase 1 (CPS1) deficiency in a neonate, specifically, a 3 day-old female who visited Hunan Provincial People\'s Hospital due to anorexia and lethargy for 1 day. Physical and laboratory examination, and MRI were undertaken. Whole exome sequencing (WES) was applied for molecular etiology identification. Sanger sequencing was utilized to validate the variants detected by WES. Structural modeling was conducted for pathogenic analysis. Clinical examination revealed increased intracranial pressure, hyperammonemia, reduced citrulline, and increased glutamic acid levels. WES identified compound heterozygosity of c.713G>C, p.Arg238Pro and c.2339G>A, p.Arg780His in CPS1 (NCBI reference sequence, NM_001875.4) as candidate pathogenic variants. Sanger sequencing validated these variants. Structural modeling further confirmed the pathogenesis of these mutations. In conclusion, CPS1 deficiency in neonates is a serious condition that may be misdiagnosed due to severe infection. WES can be a helpful tool in facilitating the diagnosis of this disease.

Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.