Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures.

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.

Type I Interferon Signaling Disrupts the Hepatic Urea Cycle and Alters Systemic Metabolism to Suppress T Cell Function.

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.

The functional impact of 1,570 individual amino acid substitutions in human OTC.

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Individuals with neonatal onset appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma, and death, outcomes ameliorated by rapid diagnosis and treatment. Here, we develop a high-throughput functional assay for human OTC and individually measure the impact of 1,570 variants, 84% of all SNV-accessible missense mutations. Comparison to existing clinical significance calls, demonstrated that our assay distinguishes known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. This functional stratification allowed us to identify score ranges corresponding to clinically relevant levels of impairment of OTC activity. Examining the results of our assay in the context of protein structure further allowed us to identify a 13 amino acid domain, the SMG loop, whose function appears to be required in human cells but not in yeast. Finally, inclusion of our data as PS3 evidence under the current ACMG guidelines, in a pilot reclassification of 34 variants with complete loss of activity, would change the classification of 22 from variants of unknown significance to clinically actionable likely pathogenic variants. These results illustrate how large-scale functional assays are especially powerful when applied to rare genetic diseases.

p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis.

Cancer cells exhibit altered and usually increased metabolic processes to meet their high biogenetic demands1,2. Under these conditions, ammonia is concomitantly produced by the increased metabolic processing. However, it is unclear how tumour cells dispose of excess ammonia and what outcomes might be caused by the accumulation of ammonia. Here we report that the tumour suppressor p53, the most frequently mutated gene in human tumours, regulates ammonia metabolism by repressing the urea cycle. Through transcriptional downregulation of CPS1, OTC and ARG1, p53 suppresses ureagenesis and elimination of ammonia in vitro and in vivo, leading to the inhibition of tumour growth. Conversely, downregulation of these genes reciprocally activates p53 by MDM2-mediated mechanism(s). Furthermore, the accumulation of ammonia causes a significant decline in mRNA translation of the polyamine biosynthetic rate-limiting enzyme ODC, thereby inhibiting the biosynthesis of polyamine and cell proliferation. Together, these findings link p53 to ureagenesis and ammonia metabolism, and further reveal a role for ammonia in controlling polyamine biosynthesis and cell proliferation.

Revisiting the Roles of Catalytic Residues in Human Ornithine Transcarbamylase.

Human ornithine transcarbamylase (hOTC) is a mitochondrial transferase protein involved in the urea cycle and is crucial for the conversion of toxic ammonia to urea. Structural analysis coupled with kinetic studies of Escherichia coli, rat, bovine, and other transferase proteins has identified residues that play key roles in substrate recognition and conformational changes but has not provided direct evidence for all of the active residues involved in OTC function. Here, computational methods were used to predict the likely active residues of hOTC; the function of these residues was then probed with site-directed mutagenesis and biochemical characterization. This process identified previously reported active residues, as well as distal residues that contribute to activity. Mutation of active site residue D263 resulted in a substantial loss of activity without a decrease in protein stability, suggesting a key catalytic role for this residue. Mutation of predicted second-layer residues H302, K307, and E310 resulted in significant decreases in enzymatic activity relative to that of wild-type (WT) hOTC with respect to l-ornithine. The mutation of fourth-layer residue H107 to produce the hOTC H107N variant resulted in a 66-fold decrease in catalytic efficiency relative to that of WT hOTC with respect to carbamoyl phosphate and a substantial loss of thermal stability. Further investigation identified H107 and to a lesser extent E98Q as key residues involved in maintaining the hOTC quaternary structure. This work biochemically demonstrates the importance of D263 in hOTC catalytic activity and shows that residues remote from the active site also play key roles in activity.

Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system.

BACKGROUND: Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored. METHODS: We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). RESULTS: At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spfash mouse following elimination of residual endogenous murine OTC. CONCLUSIONS: The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.

Variant analysis and PGT-M of OTC gene in a Chinese family with ornithine carbamoyltransferase deficiency.

BACKGROUND: Ornithine carbamoyltransferase deficiency (OTCD) is a kind of X-linked metabolic disease caused by a deficiency in ornithine transcarbamylase leading to urea cycle disorders. The main reason is that the OTC gene variants lead to the loss or decrease of OTC enzyme function, which hinders the ammonia conversion to urea, resulting in hyperammonemia and severe neurological dysfunction. Here, we studied one Chinese family of three generations who consecutively gave birth to two babies with OTCD. This study aims to explore the pathogenicity of two missense variants in the OTC gene and investigate the application of preimplantation genetic testing for monogenic (PGT-M) for a family troubled by Ornithine carbamoyltransferase deficiency (OTCD). METHODS: The retrospective method was used to classify the pathogenicity of two missense variants in the OTC gene in a family tortured by OTCD. Sanger sequencing was used to validate the variants in the OTC gene, and then the pathogenicity of variants was confirmed through family analysis and bioinformatics software. We used PGT-M to target the OTC gene and select a suitable embryo for transplantation. Prenatal diagnosis was recommended to confirm previous results using Sanger sequencing and karyotyping at an appropriate gestational stage. Tandem mass spectrometry (MS-MS) and gas chromatography-mass spectrometry (GC-MS) were used to detect fetal metabolism after birth. The number of the study cohort is ChiCTR2100053616. RESULTS: Two missense variants, c.959G > C (p.Arg320Pro) and c.634G > A (p.Gly212Arg), were validated in the OTC gene in this family. According to the ACMG genetic variation classification criteria, the missense variant c.959G > C can be considered as "pathogenic", and the missense variant c.634G > A can be regarded as "likely benign." PGT-M identified a female embryo carrying the heterozygous variant c.959G > C (p.Arg320Pro), which was selected for transplantation. Prenatal diagnosis revealed the same variant in the fetus, and continued pregnancy was recommended. A female baby was born, and her blood amino acid testing and urine organic acid testing were regular. Follow-up was conducted after six months and indicated the girl was healthy. CONCLUSION: Our research first validated the segregation of both c.959G > C and c.634G > A variants in the OTC gene in a Chinese OTCD family. Then, we classified variant c.959G > C as "pathogenic" and variant c.634G > A as "likely benign", providing corresponding theoretical support for genetic counseling and fertility guidance in this family. PGT-M and prenatal diagnosis were recommended to help the couple receive a female baby successfully with a six-month follow-up.

Implications of Genetic Factors Underlying Mouse Hydronephrosis: Cautionary Considerations on Phenotypic Interpretation in Genetically Engineered Mice.

Hydronephrosis, the dilation of kidneys due to abnormal urine retention, occurs spontaneously in certain inbred mouse strains. In humans, its occurrence is often attributed to acquired urinary tract obstructions in adults, whereas in children, it can be congenital. However, the genetic factors underlying hydronephrosis pathogenesis remain unclear. We investigated the cause of hydronephrosis by analyzing tetraspanin 7 (Tspan7) gene-modified mice, which had shown a high incidence of hydronephrosis-like symptoms. We found that these mice were characterized by low liver weights relative to kidney weights and elevated blood ammonia levels, suggesting liver involvement in hydronephrosis. Gene expression analysis of the liver suggested that dysfunction of ornithine transcarbamylase (OTC), encoded by the X chromosome gene Otc and involved in the urea cycle, may contribute as a congenital factor in hydronephrosis. This OTC dysfunction may be caused by genomic mutations in X chromosome genes contiguous to Otc, such as Tspan7, or via the genomic manipulations used to generate transgenic mice, including the introduction of Cre recombinase DNA cassettes and cleavage of loxP by Cre recombinase. Therefore, caution should be exercised in interpreting the hydronephrosis phenotype observed in transgenic mice as solely a physiological function of the target gene.

Excretion of excess nitrogen and increased survival by loss of SLC6A19 in a mouse model of ornithine transcarbamylase deficiency.

Protein catabolism ultimately yields toxic ammonia, which must be converted to urea by the liver for renal excretion. In extrahepatic tissues, ammonia is temporarily converted primarily to glutamine for subsequent hepatic extraction. Urea cycle disorders (UCDs) are inborn errors of metabolism causing impaired ureagenesis, leading to neurotoxic accumulation of ammonia and brain glutamine. Treatment includes dietary protein restriction and oral "ammonia scavengers." These scavengers chemically combine with glutamine and glycine to yield excretable products, creating an alternate pathway of waste nitrogen disposal. The amino acid transporter SLC6A19 is responsible for >95% of absorption and reabsorption of free neutral amino acids in the small intestine and kidney, respectively. Genetic SLC6A19 deficiency causes massive neutral aminoaciduria but is typically benign. We hypothesized that inhibiting SLC6A19 would open a novel and effective alternate pathway of waste nitrogen disposal. To test this, we crossed SLC6A19 knockout (KO) mice with spfash mice, a model of ornithine transcarbamylase (OTC) deficiency. Loss of SLC6A19 in spfash mice normalized plasma ammonia and brain glutamine and increased median survival in response to a high protein diet from 7 to 97 days. While induced excretion of amino acid nitrogen is likely the primary therapeutic mechanism, reduced intestinal absorption of dietary free amino acids, and decreased muscle protein turnover due to loss of SLC6A19 may also play a role. In summary, the results suggest that SLC6A19 inhibition represents a promising approach to treating UCDs and related aminoacidopathies.

A simple screening method for heterozygous female patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD), caused by X-linked OTC mutations, is characterized by life-threatening hyperammonemia. Heterozygous female patients are often asymptomatic and usually have milder disease than affected male patients, but can have higher morbidity and mortality rates if the disease progresses prior to diagnosis. Our purpose was to establish a screening method for female heterozygotes with OTCD. We retrospectively identified female patients who underwent plasma amino acid analysis at the National Center for Child Health and Development, using data from electronic medical records from March 2002 to September 2021. We extracted patient age, medical history, and biochemical data, including plasma amino acid levels. Patients were categorized into several groups according to their underlying diseases; those with underlying diseases that could potentially affect plasma amino acid levels, such as mitochondrial disease or short bowel syndrome, were excluded, except for untreated OTCD. Biochemical values were compared between OTCD patients and others using the Mann-Whitney U test. The receiver operator characteristic analysis was performed to assess the diagnostic capability for detecting OTCD in each subject. For patients with multiple test data, the most recent of the measurement dates was used in the analysis. The data sets of 976 patients were included. There were significant differences in values of glutamine, citrulline, arginine, and ammonia, but the diagnostic capacity of each alone was inadequate. By contrast, the (glutamine + glycine)/(citrulline + arginine) ratio was appropriate for discriminating heterozygous female patients with OTCD, with a sensitivity of 100% and specificity of 98.6% when the cutoff level was 15.8; the AUC for this discrimination was 0.996 (95% confidence interval, 0.992 to 1.000). These findings could help identify heterozygous female patients with OTCD before the onset of clinical disease.

Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.

A promoter variant in the OTC gene associated with late and variable age of onset hyperammonemia.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked inborn error caused by loss of function variants in the OTC gene typically associated with severe neonatal hyperammonemia. Rare examples of late-onset OTCD have also been described. Here, we describe an OTC promoter variant, c.-106C>A, in a conserved HNF4a binding site, identified in two male siblings in Family 1 whose first and only recognized episodes of severe hyperammonemia occurred at ages 14 and 39 years, respectively. We identified the same OTC variant segregating in a large family with late-onset OTCD with variable expressivity (Family 2). We show that this OTC promoter variant reduces expression >5-fold in a dual-luciferase assay that tests promoter function. Addition of an upstream OTC enhancer increases expression of both the wild type and the c.-106C>A variant promoter constructs >5-fold with the mutant promoter still about fourfold lower than the wild type. Thus, in both contexts, the promoter variant results in substantially lower OTC expression. Under normal demand on urea cycle function, OTC expression in hemizygous males, although reduced, is sufficient to meet the demand for waste nitrogen excretion. However, in response to severe metabolic stress with attendant increased requirements on urea cycle function, the impaired promoter function results in inadequate OTC expression with resultant hyperammonemia. In the absence of precipitating events, hemizygotes with this allele are asymptomatic, explaining the late age of onset of hyperammonemia in affected individuals and the incomplete penetrance observed in some individuals in Family 2.

Glutaminase 2 knockdown reduces hyperammonemia and associated lethality of urea cycle disorder mouse model.

Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otcspf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otcspf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otcspf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Common polymorphic OTC variants can act as genetic modifiers of enzymatic activity.

Understanding the role of common polymorphisms in modulating the clinical phenotype when they co-occur with a disease-causing lesion is of critical importance in medical genetics. We explored the impact of apparently neutral common polymorphisms, using the gene encoding the urea cycle enzyme, ornithine transcarbamylase (OTC), as a model system. Distinct combinations of genetic backgrounds embracing two missense polymorphisms were created in cis with the pathogenic p.Arg40His replacement. In vitro enzymatic assays revealed that the polymorphic variants were able to modulate OTC activity both in the presence or absence of the pathogenic lesion. First, we found that the combination of the minor alleles of polymorphisms p.Lys46Arg and p.Gln270Arg significantly enhanced enzymatic activity in the wild-type protein. Second, enzymatic assays revealed that the minor allele of the p.Gln270Arg polymorphism was capable of ameliorating OTC activity when combined in cis with the pathogenic p.Arg40His replacement. Structural analysis predicted that the minor allele of the p.Gln270Arg polymorphism would serve to stabilize the OTC wild-type protein, thereby corroborating the results of the experimental assays. Our findings demonstrate the potential importance of cis-interactions between common polymorphic variants and pathogenic missense mutations and illustrate how standing genetic variation can modulate protein function.

Identification of rare variants causing urea cycle disorders: A clinical, genetic, and biophysical study.

Urea cycle disorders (UCDs) are a group of rare metabolic conditions characterized by hyperammonemia and a broad spectrum of phenotypic severity. They are caused by the congenital deficiency in the eight biomolecules involved in urea cycle. In the present study, five cases of UCD were recruited and submitted to a series of clinical, biochemical, and genetic analysis with a combination of high throughput techniques. Moreover, in silico analysis was conducted on the identified missense genetic variants. Various clinical and biochemical indications (including profiles of amino acids and urinary orotic acids) of UCD were manifested by the five probands. Sequence analysis revealed nine diagnostic variants, including three novel ones, which caused Argininosuccinic aciduria (ASA) in one case, Carbamoyl phosphate synthetase 1deficiency (CPS1D) in two cases, Ornithine transcarbamylase deficiency (OTCD) in one case, and Citrin deficiency in 1case. Results of in silico biophysical analysis strongly suggested the pathogenicity of each the five missense variants and provided insight into their intramolecular impacts. In conclusion, this study expanded the genetic variation spectrum of UCD, gave solid evidence for counselling to the affected families, and should facilitate the functional study on the proteins in urea cycle.

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies.

BACKGROUND: Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. METHODS: Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. RESULTS: Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5'ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5'ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. CONCLUSIONS: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Circadian oscillation expression of ornithine carbamoyltransferase and its significance in sleep disturbance.

Ornithine transcarbamylases (OTC), a key enzyme in urea cycle, is an important marker for some liver injury or diseases. However, whether OTC could be a sensitive indicator for liver dysfunction under sleep disturbance condition remains unknown. The present study aimed to explore the circadian oscillation expression of OTC and its significance in disturbed sleep condition. Sleep disturbance was conducted by a sleep deprivation (SD) instrument. Our results found that SD for 72h induced abnormal increasing of OTC levels in serum and liver of rats. And, serum OTC concentration and liver OTC expression could return to normal levels after recovery sleep following SD. Moreover, hepatic OTC expression showed circadian oscillation in day and night, characterized with occurrence of a peak between ZT 22 and ZT 2, and a nadir between ZT 14 and ZT 18. Further analysis suggested the existence of ROR response element (RORE) for potential RORɑ binding sites in OTC promoter region, and elevated RORɑ expression in rat livers under sleep disturbance condition. Additionally, oscillation expression of OTC induced by serum shock in HepG2 cells was characterized with a peak occurred between ZT 12 and ZT 16, and RORɑ knockdown at ZT 16 significantly lowered OTC expression. The results together indicate that OTC is closely correlated with circadian clock, and could be a sensitive indicator for sleep disturbance stress.

Variants associated with urea cycle disorders in Japanese patients: Nationwide study and literature review.

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.

Ornithine carbamoyltransferase from psychrophiles to thermophiles: structural evolution of catalytic fold to accommodate physiological diversity.

Here, we have analyzed the enzyme ornithine carbamoyltransferase (OCTase) in different classes of microorganisms belonging to psychrophiles, mesophiles and thermophiles. This OCTase catalyzes the formation of citrulline from carbamoyl phosphate (CP) and ornithine (ORN) in arginine biosynthesis pathway and has certain unique adaptations to regulate metabolic pathways in extreme conditions. The tertiary structure of OCTase showed two binding domains, the CP domain and ORN-binding domain at N and C terminals, respectively. We propose general acid-base catalysis in Pseudomonas gessardii between His259 and Asp220 in which later may act as a recipient of proton in the process. The comparative docking analysis showed that substrate-binding loops have been evolved to accommodate their lifestyles across the physiological temperature range where two substrates bind on two distinct loops in psychrophiles and mesophiles, whereas both the substrates bind on a single-substrate-binding loop in thermophiles and bring down the flexibility of the active site pocket to improve its evolutionary fitness.