Adolescent-onset epilepsy and deterioration associated with CAD deficiency: A case report.

INTRODUCTION: CAD (MIM*114010) encodes a large multifunctional protein with the enzymatic activity of the first three enzymes initiating and controlling the de novo pyrimidine biosynthesis pathway. Biallelic pathogenic variants in CAD cause the autosomal recessive developmental and epileptic encephalopathy 50 (MIM #616457) or CAD deficiency presenting with epilepsy, status epilepticus (SE), neurological deterioration and anemia with anisopoikilocytosis. Mortality is around 9% of patients, mainly related to the no use of its specific treatment with uridine. Majority of reported cases have an early onset during infancy, with some few starting later in childhood. CASE REPORT: Here we report a deceased female patient with CAD deficiency whose epilepsy started at 14 years. She showed a rapid neurologic deterioration including cognitive decline, electroencephalographic background slowing which later evolved to a fatal refractory SE and supra and infratentorial atrophy on neuroimaging. Anemia developed after SE onset. METHODS AND RESULTS: her post-mortem whole exome sequencing identified biallelic missense variants in CAD (NM_004341.5): c.[2944G > A];[5366G > A] p.[(Asp982Asn)];[(Arg1789Gln)]. Our review of twenty-eight reported cases (2015-2023) revealed an epilepsy age onset from neonatal period to 7 years and the SE prevalence of 46 %. DISCUSSION: With our case, we highlight the relevance of suspecting this treatable condition in older patients and in SE with no evident etiology.

Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle.

Metabolism is a fundamental cellular process that is coordinated with cell cycle progression. Despite this association, a mechanistic understanding of cell cycle phase-dependent metabolic pathway regulation remains elusive. Here we report the mechanism by which human de novo pyrimidine biosynthesis is allosterically regulated during the cell cycle. Combining traditional synchronization methods and metabolomics, we characterize metabolites by their accumulation pattern during cell cycle phases and identify cell cycle phase-dependent regulation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD), the first, rate-limiting enzyme in de novo pyrimidine biosynthesis. Through systematic mutational scanning and structural modelling, we find allostery as a major regulatory mechanism that controls the activity change of CAD during the cell cycle. Specifically, we report evidence of two Animalia-specific loops in the CAD allosteric domain that involve sensing and binding of uridine 5'-triphosphate, a CAD allosteric inhibitor. Based on homology with a mitochondrial carbamoyl-phosphate synthetase homologue, we identify a critical role for a signal transmission loop in regulating the formation of a substrate channel, thereby controlling CAD activity.

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

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

SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their capacity to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting their rapid recall response are incompletely understood. Here, we identify a role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously activated CD8+ T cells. We found that CAD was rapidly phosphorylated upon naïve T cell activation in an mTORC1-dependent manner, yet remained phosphorylated long after initial activation. Previously activated CD8+ T cells displayed continued de novo pyrimidine synthesis in the absence of mitogenic signals, and interfering with this pathway diminished the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD did not affect cytokine transcript levels but diminished available pre-rRNA (ribosomal RNA), the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreased levels of detectable ribosomal proteins in previously activated CD8+ T cells. Conversely, overexpression of CAD improved both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

CAD gene and early infantile epileptic encephalopathy-50; three Iranian deceased patients and a novel mutation: case report.

BACKGROUND: Early infantile epileptic encephalopathy is a severe form of epilepsy that is genetically extremely heterogeneous and characterized by seizures or spasms at the beginning of infancy. Homozygous or compound heterozygous mutation in the CAD gene cause early infantile epileptic encephalopathy-50 (EIEE50). This case report describes the clinical and molecular features of three patients affected with early infantile epileptic encephalopathy. CASE PRESENTATION: In this report, we describe the clinical features of two deceased daughters and one recently deceased son affected with seizure, muscular hypotonia, and developmental delay. After genetic counseling, blood samples were obtained from the parents, and whole-exome sequencing was performed. Genomic DNA was extracted from whole blood, and mutation analysis was performed using PCR and sequencing methods for the CAD gene. Genetic analysis using the whole-exome sequencing method has detected a novel likely pathogenic mutation on CAD gene, c.2995G > A (p.Val999Met), in heterozygous states in asymptomatic parents and homozygous state in affected newborn son. This mutation has not been reported in the literature for its pathogenicity. CONCLUSIONS: The asymptomatic parents are carriers for the likely pathogenic variant in the CAD gene, and the recently deceased newborn son had the same mutation in a homozygous state. Given that, multiple lines of in silico computational analysis support the detrimental impact of the variant on the gene, and this variant is absent in population databases. Pathogenic mutations in the CAD gene are related to autosomal recessive EIEE50 with similar signs and symptoms to our patients. Ultimately, it is confirmed that this mutation is causative in our patients.

Pyrimidine Biosynthetic Enzyme CAD: Its Function, Regulation, and Diagnostic Potential.

CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamoylase, and Dihydroorotase) is a multifunctional protein that participates in the initial three speed-limiting steps of pyrimidine nucleotide synthesis. Over the past two decades, extensive investigations have been conducted to unmask CAD as a central player for the synthesis of nucleic acids, active intermediates, and cell membranes. Meanwhile, the important role of CAD in various physiopathological processes has also been emphasized. Deregulation of CAD-related pathways or CAD mutations cause cancer, neurological disorders, and inherited metabolic diseases. Here, we review the structure, function, and regulation of CAD in mammalian physiology as well as human diseases, and provide insights into the potential to target CAD in future clinical applications.

Deciphering CAD: Structure and function of a mega-enzymatic pyrimidine factory in health and disease.

CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein divided into different enzymatic domains, each catalyzing one of the initial reactions for de novo biosynthesis of pyrimidine nucleotides: glutaminase-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. The pathway for de novo pyrimidine synthesis is essential for cell proliferation and is conserved in all living organisms, but the covalent linkage of the first enzymatic activities into a multienzymatic CAD particle is unique to animals. In other organisms, these enzymatic activities are encoded as monofunctional proteins for which there is abundant structural and biochemical information. However, the knowledge about CAD is scarce and fragmented. Understanding CAD requires not only to determine the three-dimensional structures and define the catalytic and regulatory mechanisms of the different enzymatic domains, but also to comprehend how these domains entangle and work in a coordinated and regulated manner. This review summarizes significant progress over the past 10 years toward the characterization of CAD's architecture, function, regulatory mechanisms, and cellular compartmentalization, as well as the recent finding of a new and rare neurometabolic disorder caused by defects in CAD activities.

Uridine-responsive epileptic encephalopathy due to inherited variants in CAD: A Tale of Two Siblings.

We report two siblings with intractable epilepsy, developmental regression, and progressive cerebellar atrophy due to biallelic variants in the gene CAD. For the affected girl, uridine started at age 5 resulted in dramatic improvements in seizure control and development, cessation of cerebellar atrophy, and resolution of hematological abnormalities. Her older brother had a more severe course and only modest response to uridine started at 14 years old. Treatment of this progressive condition via uridine supplementation provides an example of precision diagnosis and treatment using clear outcome measures and biomarkers to monitor efficacy.

Structural insights on binding mechanism of CAD complexes (CPSase, ATCase and DHOase).

Pyrimidine biosynthetic pathway enzymes constitute an important target for the development of antitumor drugs. To understand the role of binding mechanisms underlying the inborn errors of pyrimidine biosynthetic pathway, structure and function of enzymes have been analyzed. Pyrimidine biosynthetic pathway is initiated by CAD enzymes that harbor the first three enzymatic activities facilitated by Carbamoyl Phosphate Synthetase (CPSase), Aspartate Transcarbamoylase (ATCase) and Dihydroorotase (DHOase). While being an attractive therapeutic target, the lack of data driven us to study the CPSase (CarA and CarB) and its mode of binding to ATCase and DHOase which are the major limitation for its structural optimization. Understanding the binding mode of CPSase, ATCase and DHOase could help to identify the potential interface hotspot residues that favor the mechanism behind it. The mechanistic insight into the CAD complexes were achieved through Molecular modeling, Protein-Protein docking, Alanine scanning and Molecular dynamics (MD) Studies. The hotspot residues present in the CarB region of carboxy phosphate and carbamoyl phosphate synthetic domains are responsible for the assembly of CAD (CPSase-ATCase-DHOase) complexes. Overall analysis suggests that the identified hotspot residues were confirmed by alanine scanning and important for the regulation of pyrimidine biosynthesis. MD simulations analysis provided the prolonged stability of the interacting complexes. The present study reveals the novel hotspot residues such as Glu134, Glu147, Glu154, Asp266, Lys269, Glu274, Asp333, Trp459, Asp526, Asp528, Glu533, Glu544, Glu546, Glu800, Val855, Asp877, Tyr884 and Gln919 which could be targeted for structure-based inhibitor design to potentiate the CAD mediated regulation of aggressive tumors.Communicated by Ramaswamy H. Sarma.

Triacetyluridine treats epileptic encephalopathy from CAD mutations: a case report and review.

Refractory epilepsy and encephalopathy are frequently encountered in patients with inborn errors of metabolism. We report a case of an 8-year-old girl with history of developmental delay, autism and intractable epilepsy that was found to have a pathogenic variant in CAD. We briefly review the biochemical pathway of CAD and the preclinical and clinical studies that suggest uridine supplementation can rescue the CAD deficiency phenotypes. Our case demonstrates a relatively late-onset case of refractory epilepsy with a rapid response to treatment using the uridine pro-drug triacetyluridine (TAU), the FDA-approved treatment for hereditary orotic aciduria.

CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

OBJECTIVES: Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected. MATERIALS AND METHODS: We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine ß-synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells. RESULTS: We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine-mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway-associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S-adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus. CONCLUSIONS: Taken together, these studies demonstrate that CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy.

PURPOSE: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. RESULTS: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. CONCLUSIONS: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.

The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription.

Ebola virus (EBOV) is a zoonotic pathogen causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and extent of outbreaks has increased, highlighting the importance of better understanding the molecular aspects of EBOV infection and host cell interactions to control this virus more efficiently. Many viruses, including EBOV, have been shown to recruit host proteins for different viral processes. Based on a genome-wide siRNA screen, we recently identified the cellular host factor carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being involved in EBOV RNA synthesis. However, mechanistic details of how this host factor plays a role in the EBOV life cycle remain elusive. In this study, we analyzed the functional and molecular interactions between EBOV and CAD. To this end, we used siRNA knockdowns in combination with various reverse genetics-based life cycle modelling systems and additionally performed co-immunoprecipitation and co-immunofluorescence assays to investigate the influence of CAD on individual aspects of the EBOV life cycle and to characterize the interactions of CAD with viral proteins. Following this approach, we could demonstrate that CAD directly interacts with the EBOV nucleoprotein NP, and that NP is sufficient to recruit CAD into inclusion bodies dependent on the glutaminase (GLN) domain of CAD. Further, siRNA knockdown experiments indicated that CAD is important for both viral genome replication and transcription, while substrate rescue experiments showed that the function of CAD in pyrimidine synthesis is indeed required for those processes. Together, this suggests that NP recruits CAD into inclusion bodies via its GLN domain in order to provide pyrimidines for EBOV genome replication and transcription. These results define a novel mechanism by which EBOV hijacks host cell pathways in order to facilitate genome replication and transcription and provide a further basis for the development of host-directed broad-spectrum antivirals.

The multienzymatic protein CAD leading the de novo biosynthesis of pyrimidines localizes exclusively in the cytoplasm and does not translocate to the nucleus.

CAD, the multienzymatic protein that initiates and controls the de novo biosynthesis of pyrimidines, plays a major role in nucleotide homeostasis, cell growth and proliferation. Despite its interest as a potential antitumoral target, there is a lack of understanding on CAD's structure and functioning mechanisms. Although mainly identified as a cytosolic complex, different studies support the translocation of CAD into the nucleus, where it could have a yet undefined function. Here, we track the subcellular localization of CAD by using fluorescent chimeras, cell fractionation and immunoblotting with specific antibodies. Contradicting previous studies, we demonstrate that CAD is exclusively localized at the cytosol and discard a possible translocation to the nucleus.

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

CAD, A Multienzymatic Protein at the Head of de Novo Pyrimidine Biosynthesis.

CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein that carries the enzymatic activities for the first three steps in the de novo biosynthesis of pyrimidine nucleotides: glutamine-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase and Dihydroorotase. This metabolic pathway is essential for cell growth and proliferation and is conserved in all living organisms. However, the fusion of the first three enzymatic activities of the pathway into a single multienzymatic protein only occurs in animals. In prokaryotes, by contrast, these activities are encoded as distinct monofunctional enzymes that function independently or by forming more or less transient complexes. Whereas the structural information about these enzymes in bacteria is abundant, the large size and instability of CAD has only allowed a fragmented characterization of its structure. Here we retrace some of the most significant efforts to decipher the architecture of CAD and to understand its catalytic and regulatory mechanisms.