ABSTRACT
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.
Subject(s)
Aspartate Carbamoyltransferase , Aspartic Acid , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Cryoelectron Microscopy , Proteins , Dihydroorotase/chemistry , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/metabolismABSTRACT
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.
Subject(s)
Aspartate Carbamoyltransferase , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , CD8-Positive T-Lymphocytes/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Cytokines , PyrimidinesABSTRACT
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.
Subject(s)
Aspartate Carbamoyltransferase , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) , Dihydroorotase , Epilepsy , Spasms, Infantile , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Epilepsy/genetics , Humans , Infant , Infant, Newborn , Iran , Mutation , Seizures , Spasms, Infantile/diagnosis , Spasms, Infantile/geneticsABSTRACT
Aspartate transcarbamoylase (ATC), an essential enzyme for de novo pyrimidine biosynthesis, is uniquely regulated in plants by feedback inhibition of uridine 5-monophosphate (UMP). Despite its importance in plant growth, the structure of this UMP-controlled ATC and the regulatory mechanism remain unknown. Here, we report the crystal structures of Arabidopsis ATC trimer free and bound to UMP, complexed to a transition-state analog or bearing a mutation that turns the enzyme insensitive to UMP. We found that UMP binds and blocks the ATC active site, directly competing with the binding of the substrates. We also prove that UMP recognition relies on a loop exclusively conserved in plants that is also responsible for the sequential firing of the active sites. In this work, we describe unique regulatory and catalytic properties of plant ATCs that could be exploited to modulate de novo pyrimidine synthesis and plant growth.
Subject(s)
Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/metabolism , Catalytic Domain/drug effects , Feedback/drug effects , Uridine Monophosphate/antagonists & inhibitors , Arabidopsis/genetics , Arabidopsis/metabolism , Aspartate Carbamoyltransferase/genetics , Aspartic Acid/metabolism , Binding Sites , Models, Molecular , Protein Conformation , PyrimidinesABSTRACT
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.
Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Drug Resistant Epilepsy/drug therapy , Drug Resistant Epilepsy/genetics , Uridine/pharmacology , Atrophy/pathology , Cerebellar Diseases/drug therapy , Cerebellar Diseases/genetics , Cerebellar Diseases/pathology , Child , Child, Preschool , Developmental Disabilities/drug therapy , Developmental Disabilities/genetics , Disease Progression , Female , Humans , Male , Pedigree , Siblings , Uridine/administration & dosageABSTRACT
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.
Subject(s)
Acetates/therapeutic use , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Epilepsy, Generalized/drug therapy , Epilepsy, Generalized/genetics , Uridine/analogs & derivatives , Child , Female , Humans , Mutation, Missense , Uridine/therapeutic useSubject(s)
Anemia, Dyserythropoietic, Congenital , Aspartate Carbamoyltransferase , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) , Dihydroorotase , Homozygote , Mutation , Uridine/administration & dosage , Anemia, Dyserythropoietic, Congenital/blood , Anemia, Dyserythropoietic, Congenital/drug therapy , Anemia, Dyserythropoietic, Congenital/genetics , Anemia, Dyserythropoietic, Congenital/pathology , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Child, Preschool , Dihydroorotase/genetics , Dihydroorotase/metabolism , Humans , MaleABSTRACT
Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.
Subject(s)
Antimalarials , Aspartate Carbamoyltransferase/genetics , Naphthyridines/pharmacology , Plasmodium falciparum , Protozoan Proteins/genetics , Antimalarials/pharmacology , Aspartic Acid , Plasmodium falciparum/enzymology , Plasmodium falciparum/geneticsABSTRACT
The control of pyrimidine nucleotide formation in the bacterium Pseudomonas aurantiaca ATCC 33663 by pyrimidines was studied. The activities of the pyrimidine biosynthetic pathway enzymes were investigated in P. aurantiaca ATCC 33663 cells and from cells of an auxotroph lacking orotate phosphoribosyltransferase activity under selected culture conditions. All activities of the pyrimidine biosynthetic pathway enzymes in ATCC 33663 cells were depressed by uracil addition to the minimal medium when succinate served as the carbon source. In contrast, all pyrimidine biosynthetic pathway enzyme activities in ATCC 33663 cells were depressed by orotic acid supplementation to the minimal medium when glucose served as the carbon source. The orotidine 5'-monophosphate decarboxylase activity in the phosphoribosyltransferase mutant strain increased by more than sixfold in succinate-grown cells and by more than 16-fold in glucose-grown cells after pyrimidine limitation showing possible repression of the decarboxylase by a pyrimidine-related compound. Inhibition by ATP, GTP, UTP and pyrophosphate of the in vitro activity of aspartate transcarbamoylase in ATCC 33663 was observed. The findings demonstrated control at the level of pyrimidine biosynthetic enzyme synthesis and activity for the P. aurantiaca transcarbamoylase. The control of pyrimidine synthesis in P. aurantiaca seemed to differ from what has been observed previously for the regulation of pyrimidine biosynthesis in related Pseudomonas species. This investigation could prove helpful to future work studying pseudomonad taxonomic analysis as well as to those exploring antifungal and antimicrobial agents produced by P. aurantiaca.
Subject(s)
Aspartate Carbamoyltransferase/metabolism , Pseudomonas/metabolism , Pyrimidine Nucleotides/biosynthesis , Pyrimidines/metabolism , Aspartate Carbamoyltransferase/genetics , Biosynthetic Pathways , Diphosphates , Gene Expression Regulation, Bacterial , Orotate Phosphoribosyltransferase/genetics , Orotidine-5'-Phosphate Decarboxylase/metabolism , Pseudomonas/enzymology , Pyrimidine Nucleotides/metabolism , Succinic Acid/metabolism , Uracil/metabolismABSTRACT
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.
Subject(s)
Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/metabolism , Dihydroorotase/chemistry , Dihydroorotase/metabolism , Pseudomonas aeruginosa/enzymology , Amino Acid Motifs , Aspartate Carbamoyltransferase/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biocatalysis , Catalytic Domain , Circular Dichroism , Dihydroorotase/genetics , Models, Molecular , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/genetics , ThermodynamicsABSTRACT
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.
Subject(s)
Aspartate Carbamoyltransferase/genetics , Aspartic Acid/analogs & derivatives , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Estrogen Receptor alpha/metabolism , Fulvestrant/pharmacology , Hepatitis D, Chronic/drug therapy , Phosphonoacetic Acid/analogs & derivatives , Pyrimidines/biosynthesis , Antiviral Agents/pharmacology , Aspartate Carbamoyltransferase/antagonists & inhibitors , Aspartate Carbamoyltransferase/metabolism , Aspartic Acid/pharmacology , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/antagonists & inhibitors , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Cell Line , Dihydroorotase/antagonists & inhibitors , Dihydroorotase/metabolism , Estrogen Receptor Antagonists/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Gene Silencing , Hepatitis D, Chronic/genetics , Hepatitis D, Chronic/metabolism , Hepatitis Delta Virus/physiology , Hepatocytes , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Insulin Resistance , Life Cycle Stages , Loss of Function Mutation , Phosphonoacetic Acid/pharmacology , RNA Interference , RNA, Small Interfering/genetics , RNA, Viral/metabolism , Signal Transduction , Virus ReplicationABSTRACT
The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis.
Subject(s)
Aspartate Carbamoyltransferase/chemistry , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry , Dihydroorotase/chemistry , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Catalysis , Catalytic Domain , Dihydroorotase/genetics , Humans , Molecular Dynamics Simulation , Mutagenesis , Mutation , Phenylalanine/chemistry , Protein Conformation , Protein DomainsABSTRACT
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.
Subject(s)
Genomics , Metabolomics , Neoplasms/pathology , Urea/metabolism , Amino Acid Transport Systems, Basic/metabolism , Animals , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Cell Line, Tumor , Dihydroorotase/genetics , Dihydroorotase/metabolism , Female , Humans , Mice , Mice, Inbred C57BL , Mice, SCID , Mitochondrial Membrane Transport Proteins , Neoplasms/metabolism , Ornithine Carbamoyltransferase/antagonists & inhibitors , Ornithine Carbamoyltransferase/genetics , Ornithine Carbamoyltransferase/metabolism , Phosphorylation/drug effects , Pyrimidines/biosynthesis , Pyrimidines/chemistry , RNA Interference , RNA, Small Interfering/metabolism , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolismABSTRACT
Chemotherapy drugs interfere with cellular processes to generate genotoxic lesions that activate cell death pathways. Sustained DNA damage induced by these drugs can provoke mutations in surviving non-cancerous cells, potentially increasing the risk of therapy-related cancers. Ligation of death receptors by ligands such as TRAIL, and subsequent activation of extrinsic apoptotic pathways, also provokes mutations. In this study, we show that executioner caspase activation of the apoptotic nuclease CAD/DFF40 is essential for TRAIL-induced mutations in surviving cells. As exposure to chemotherapy drugs also activates apoptotic caspases and presumably CAD, we hypothesized that these pathways may also contribute to the mutagenesis induced by conventional chemotherapy drugs, perhaps augmenting the mutations that arise from direct DNA damage provoked by these agents. Interestingly, vincristine-mediated mutations were caspase and CAD dependent. Executioner caspases accounted for some of the mutations caused by the topoisomerase poisons doxorubicin and SN38, but were dispensable for mutagenesis following treatment with cisplatin or temozolomide. These data highlight a non-apoptotic role of caspases in mutagenesis mediated by death receptor agonists, microtubule poisons and topoisomerase inhibitors, and provide further evidence for a potential carcinogenic consequence of sublethal apoptotic signaling stimulated by anticancer therapies.
Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Caspases/genetics , Dihydroorotase/genetics , Neoplasms/genetics , TNF-Related Apoptosis-Inducing Ligand/genetics , Apoptosis/drug effects , Camptothecin/administration & dosage , Camptothecin/adverse effects , Camptothecin/analogs & derivatives , Cell Line, Tumor , Cell Survival/drug effects , Cisplatin/administration & dosage , Cisplatin/adverse effects , DNA Damage/drug effects , Dacarbazine/administration & dosage , Dacarbazine/adverse effects , Dacarbazine/analogs & derivatives , Doxorubicin/administration & dosage , Doxorubicin/adverse effects , Enzyme Activation/drug effects , Humans , Irinotecan , Mutagenesis/drug effects , Mutation , Neoplasms/drug therapy , Neoplasms/pathology , Signal Transduction/drug effects , Temozolomide , Vincristine/administration & dosage , Vincristine/adverse effectsABSTRACT
A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations.
Subject(s)
Amino Acid Substitution , Aspartate Carbamoyltransferase/chemistry , Aspartic Acid/chemistry , Allosteric Regulation , Amino Acid Motifs , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Aspartic Acid/metabolism , Biocatalysis , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Substrate Specificity , ThermodynamicsABSTRACT
CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into â¼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD.
Subject(s)
Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Dihydroorotase/chemistry , Dihydroorotase/metabolism , Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Carbamyl Phosphate/chemistry , Carbamyl Phosphate/metabolism , Chaetomium/enzymology , Crystallography, X-Ray , Dihydroorotase/genetics , Humans , Microscopy, Electron , Models, Molecular , Mutagenesis, Site-Directed , Protein Domains , Protein Multimerization , Pyrimidines/biosynthesisABSTRACT
BACKGROUND: Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. PATIENTS AND METHODS: We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying a CAD-ALK translocation during treatment with an ALK inhibitor. RESULTS: Using a custom next generation sequencing panel we identified the genomic CAD-ALK rearrangement and a TP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of the CAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient's clinical course. Detection of the CAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identified ALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. CONCLUSION: We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.
Subject(s)
Aspartate Carbamoyltransferase/genetics , Benzamides/therapeutic use , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Dihydroorotase/genetics , Gene Rearrangement , Indazoles/therapeutic use , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Anaplastic Lymphoma Kinase , Biomarkers, Tumor , Colorectal Neoplasms/blood , Colorectal Neoplasms/urine , Drug Resistance, Neoplasm , Female , Gene Fusion , Humans , Middle Aged , Polymerase Chain Reaction/methods , Receptor Protein-Tyrosine Kinases/geneticsABSTRACT
Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues.
Subject(s)
Aspartate Carbamoyltransferase/metabolism , Bacteria/enzymology , Bacterial Proteins/metabolism , Dihydroorotase/metabolism , Protein Multimerization/physiology , Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/genetics , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalytic Domain/physiology , Dihydroorotase/chemistry , Dihydroorotase/genetics , Enzyme Activation/physiology , Hydrostatic PressureABSTRACT
Unexplained global developmental delay and epilepsy in childhood pose a major socioeconomic burden. Progress in defining the molecular bases does not often translate into effective treatment. Notable exceptions include certain inborn errors of metabolism amenable to dietary intervention. CAD encodes a multifunctional enzyme involved in de novo pyrimidine biosynthesis. Alternatively, pyrimidines can be recycled from uridine. Exome sequencing in three families identified biallelic CAD mutations in four children with global developmental delay, epileptic encephalopathy, and anaemia with anisopoikilocytosis. Two died aged 4 and 5 years after a neurodegenerative disease course. Supplementation of the two surviving children with oral uridine led to immediate cessation of seizures in both. A 4-year-old female, previously in a minimally conscious state, began to communicate and walk with assistance after 9 weeks of treatment. A 3-year-old female likewise showed developmental progress. Blood smears normalized and anaemia resolved. We establish CAD as a gene confidently implicated in this neurometabolic disorder, characterized by co-occurrence of global developmental delay, dyserythropoietic anaemia and seizures. While the natural disease course can be lethal in early childhood, our findings support the efficacy of uridine supplementation, rendering CAD deficiency a treatable neurometabolic disorder and therefore a potential condition for future (genetic) newborn screening.
Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Mutation/genetics , Spasms, Infantile/drug therapy , Spasms, Infantile/genetics , Uridine/therapeutic use , Anemia/complications , Anemia/drug therapy , Anemia/genetics , Brain/diagnostic imaging , Child , Child, Preschool , DNA Mutational Analysis , Developmental Disabilities/complications , Developmental Disabilities/genetics , Female , Humans , Infant , Magnetic Resonance Imaging , Male , Spasms, Infantile/complications , Spasms, Infantile/diagnostic imagingABSTRACT
Army ant colonies host a diverse community of arthropod symbionts. Among the best-studied symbiont communities are those of Neotropical army ants of the genus Eciton. It is clear, however, that even in these comparatively well studied systems, a large proportion of symbiont biodiversity remains unknown. Even more striking is our lack of knowledge regarding the nature and specificity of these host-symbiont interactions. Here we surveyed the diversity and host specificity of rove beetles of the genus Tetradonia Wasmann, 1894 (Staphylinidae: Aleocharinae). Systematic community sampling of 58 colonies of the six local Eciton species at La Selva Biological Station, Costa Rica, combined with an integrative taxonomic approach, allowed us to uncover species diversity, host specificity, and co-occurrence patterns of symbionts in unprecedented detail. We used an integrative taxonomic approach combining morphological and genetic analyses, to delineate species boundaries. Mitochondrial DNA barcodes were analyzed for 362 Tetradonia specimens, and additional nuclear markers for a subset of 88 specimens. All analyses supported the presence of five Tetradonia species, including two species new to science. Host specificity is highly variable across species, ranging from generalists such as T. laticeps, which parasitizes all six local Eciton species, to specialists such as T. lizonae, which primarily parasitizes a single species, E. hamatum. Here we provide a dichotomous key along with diagnostic molecular characters for identification of Tetradonia species at La Selva Biological Station. By reliably assessing biodiversity and providing tools for species identification, we hope to set the baseline for future studies of the ecological and evolutionary dynamics in these species-rich host-symbiont networks.