Beyond genetics: Deciphering the impact of missense variants in CAD deficiency.

CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.

Complexed Crystal Structure of the Dihydroorotase Domain of Human CAD Protein with the Anticancer Drug 5-Fluorouracil.

Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.

Crystal structure of Methanococcus jannaschii dihydroorotase.

In this paper, we report the structural analysis of dihydroorotase (DHOase) from the hyperthermophilic and barophilic archaeon Methanococcus jannaschii. DHOase catalyzes the reversible cyclization of N-carbamoyl-l-aspartate to l-dihydroorotate in the third step of de novo pyrimidine biosynthesis. DHOases form a very diverse family of enzymes and have been classified into types and subtypes with structural similarities and differences among them. This is the first archaeal DHOase studied by x-ray diffraction. Its structure and comparison with known representatives of the other subtypes help define the structural features of the archaeal subtype. The M. jannaschii DHOase is found here to have traits from all subtypes. Contrary to expectations, it has a carboxylated lysine bridging the two Zn ions in the active site, and a long catalytic loop. It is a monomeric protein with a large ß sandwich domain adjacent to the TIM barrel. Loop 5 is similar to bacterial type III and the C-terminal extension is long.

Oncogenic ß-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer.

CTNNB1, encoding ß-catenin protein, is the most frequently altered proto-oncogene in hepatic neoplasms. In this study, we studied the significance and pathological mechanism of CTNNB1 gain-of-function mutations in hepatocarcinogenesis. Activated ß-catenin not only triggered hepatic tumorigenesis but also exacerbated Tp53 deletion or hepatitis B virus infection-mediated liver cancer development in mouse models. Using untargeted metabolomic profiling, we identified boosted de novo pyrimidine synthesis as the major metabolic aberration in ß-catenin mutant cell lines and livers. Oncogenic ß-catenin transcriptionally stimulated AKT2, which then phosphorylated the rate-limiting de novo pyrimidine synthesis enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase) on S1406 and S1859 to potentiate nucleotide synthesis. Moreover, inhibition of ß-catenin/AKT2-stimulated pyrimidine synthesis axis preferentially repressed ß-catenin mutant cell proliferation and tumor formation. Therefore, ß-catenin active mutations are oncogenic in various preclinical liver cancer models. Stimulation of ß-catenin/AKT2/CAD signaling cascade on pyrimidine synthesis is an essential and druggable vulnerability for ß-catenin mutant liver cancer.

Structural Analysis of Saccharomyces cerevisiae Dihydroorotase Reveals Molecular Insights into the Tetramerization Mechanism.

Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.

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.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

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.

Structural basis for the interaction modes of dihydroorotase with the anticancer drugs 5-fluorouracil and 5-aminouracil.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies.

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.

Characterization and assembly of the Pseudomonas aeruginosa aspartate transcarbamoylase-pseudo dihydroorotase complex.

Pseudomonas aeruginosa is a virulent pathogen that has become more threatening with the emergence of multidrug resistance. The aspartate transcarbamoylase (ATCase) of this organism is a dodecamer comprised of six 37 kDa catalytic chains and six 45 kDa chains homologous to dihydroorotase (pDHO). The pDHO chain is inactive but is necessary for ATCase activity. A stoichiometric mixture of the subunits associates into a dodecamer with full ATCase activity. Unlike other known ATCases, the P. aeruginosa catalytic chain does not spontaneously assemble into a trimer. Chemical-crosslinking and size-exclusion chromatography showed that P. aeruginosa ATCase is monomeric which accounts for its lack of catalytic activity since the active site is a composite comprised of residues from adjacent monomers in the trimer. Circular dichroism spectroscopy indicated that the ATCase chain adopts a structure that contains secondary structure elements although neither the ATCase nor the pDHO subunits are very stable as determined by a thermal shift assay. Formation of the complex increases the melting temperature by about 30°C. The ATCase is strongly inhibited by all nucleotide di- and triphosphates and exhibits extreme cooperativity. Previous studies suggested that the regulatory site is located in an 11-residue extension of the amino end of the catalytic chain. However, deletion of the extensions did not affect catalytic activity, nucleotide inhibition or the assembly of the dodecamer. Nucleotides destabilized the dodecamer which probably accounts for the inhibition and apparent cooperativity of the substrate saturation curves. Contrary to previous interpretations, these results suggest that P. aeruginosa ATCase is not allosterically regulated by nucleotides.

Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

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.

Pyrimidine biosynthesis in pathogens - Structures and analysis of dihydroorotases from Yersinia pestis and Vibrio cholerae.

The de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-l-aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis (YpDHO), the plague-causing pathogen, and Vibrio cholerae (VcDHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum.

Pressure-induced activation of latent dihydroorotase from Aquifex aeolicus as revealed by high pressure protein crystallography.

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site-directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure-induced reactivation was due to the decrease in the volume of the system, -ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme.

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.

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.

The Sda and Cad glycan antigens and their glycosyltransferase, ß1,4GalNAcT-II, in xenotransplantation.

Antibody-mediated rejection is a barrier to the clinical application of xenotransplantation, and xenoantigens play an important role in this process. Early research suggested that N-acetyl-D-galactosamine (GalNAc) could serve as a potential xenoantigen. GalNAc is the immunodominant glycan of the Sda antigen. Recently, knockout of ß1,4-N-acetylgalactosaminyltransferase 2 (ß1,4GalNAcT-II) from the pig results in a decrease in binding of human serum antibodies to pig cells. It is believed that this is the result of the elimination of the GalNAc on the Sda antigen, which is catalyzed by the enzyme, ß1,4GalNAcT-II. However, research into human blood group antigens suggests that only a small percentage (1%-2%) of people express anti-Sda antibodies directed to Sda antigen, and yet a majority appear to have antibodies directed to the products of pig B4GALNT2. Questions can therefore be asked as to (i) whether the comprehensive structure of the Sda antigen in humans, that is, the underlying sugar structure, is identical to the Sda antigen in pigs, (ii) whether the human anti-Sda antibody binds ubiquitously to pig cells, but not to human cells, and (iii) what role the Sda++ (also called Cad) antigen is playing in this discrepancy. We review what is known about these antigens and discuss the discrepancies that have been noted above.