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The EMBO Journal Jun 2024The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC...
The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.
Topics: Decitabine; Humans; DNA (Cytosine-5-)-Methyltransferase 1; Ubiquitin-Protein Ligases; DNA Methylation; Antimetabolites, Antineoplastic; Animals; Sumoylation
PubMed: 38760575
DOI: 10.1038/s44318-024-00108-2 -
Journal of Biomolecular Structure &... Dec 2023Cancer is one of the leading causes of death in the U.S., and tumorous cancers such as cervical, lung, breast, and ovarian cancers are the most common types. APOBEC3B is...
Cancer is one of the leading causes of death in the U.S., and tumorous cancers such as cervical, lung, breast, and ovarian cancers are the most common types. APOBEC3B is a nonessential cytidine deaminase found in humans and theorized to defend against viral infection. However, overexpression of APOBEC3B is linked to cancer in humans, which makes APOBEC3B a potential cancer treatment target through competitive inhibition for several tumorous cancers. Computational studies can help reveal a small molecule inhibitor using high-throughput virtual screening of millions of candidates with relatively little cost. This study aims to narrow the field of potential APOBEC3B inhibition candidates for future assays and provide an effective scaffold for drug design studies. Another goal of this project is to provide critical amino acid targets in the active site for future drug design studies. This study simulated 7.8 million drug candidates using high-throughput virtual screening and further processed the top scoring 241 molecules from AutoDock Vina, DOCK 6, and design. Using virtual screening, design, and molecular dynamics simulations, a competitive inhibitor candidate was discovered with an average binding free energy score of -46.03 kcal/mol, more than 10 kcal/mol better than the substrate control (dCMP). These results indicate that this molecule (or a structural derivative) may be an effective inhibitor of APOBEC3B and prevent host genome mutagenesis resulting from protein overexpression. Another important finding is the confirmation of essential amino acid targets, such as Tyr250 and Gln213 within the active site of APOBEC3B. Therefore, study used novel computational methods to provide a theoretical scaffold for future drug design studies that may prove useful as a treatment for epithelial cancers.Communicated by Ramaswamy H. Sarma.
PubMed: 38109103
DOI: 10.1080/07391102.2023.2293269 -
Molecular Biology and Evolution Dec 2023The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine...
Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?
The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.
Topics: DCMP Deaminase; Gene Transfer, Horizontal; Escherichia coli; Amoeba; Thymidylate Synthase; Dictyostelium; Deoxycytidine Monophosphate
PubMed: 38064674
DOI: 10.1093/molbev/msad268 -
Scientific Reports Nov 2023Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine...
Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.
Topics: Cytidine; DCMP Deaminase; Deoxycytidine Kinase; Deoxycytidine; Metabolic Networks and Pathways; Cytidine Deaminase
PubMed: 37993628
DOI: 10.1038/s41598-023-47792-4 -
Archives of Biochemistry and Biophysics Sep 20222-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The...
2-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The large double-stranded DNA-containing chlorovirus PBCV-1 encodes a dCD family enzyme PBCV1dCD that was reported to be able to deaminize both dCMP and dCTP, which makes PBCV1dCD unique in the dCD family proteins. In this study, we report the crystal structure of PBCV1dCD in complex with dCTP/dCMP and dTTP/dTMP, respectively. We further proved the ability of PBCV1dCD in the deamination of dCDP, which makes PBCV1dCD a multi-functional deaminase. The structural basis for the versatility of PBCV1dCD is analyzed and discussed, with the finding of a unique Trp121 residue key to the deamination and substrate binding ability. Our findings may broaden the understanding of dCD family proteins and provide novel insights into the multi-functional enzyme.
Topics: Crystallography, X-Ray; DCMP Deaminase; Deoxycytidine Monophosphate; Substrate Specificity
PubMed: 35764100
DOI: 10.1016/j.abb.2022.109339 -
Molecular Microbiology Jul 2021We show that the ComEB protein is not required for transformation in Bacillus subtilis, despite its expression from within the comE operon under competence control, nor...
We show that the ComEB protein is not required for transformation in Bacillus subtilis, despite its expression from within the comE operon under competence control, nor is it required for the correct polar localization of ComGA. We show further that the synthesis of the putative channel protein ComEC is translationally coupled to the upstream comEB open reading frame, so that the translation of comEB and a suboptimal ribosomal-binding site embedded in its sequence are needed for proper comEC expression. Translational coupling appears to be a common mechanism in three major competence operons for the adjustment of protein amounts independent of transcriptional control, probably ensuring the correct stoichiometries for assembly of the transformation machinery. comEB and comFC, respectively, encode cytidine deaminase and a protein resembling type 1 phosphoribosyl transferases and we speculate that nucleotide scavenging proteins are produced under competence control for efficient reutilization of the products of degradation of the non-transforming strand during DNA uptake.
Topics: Bacillus subtilis; Bacterial Proteins; Cell Membrane; DCMP Deaminase; DNA Transformation Competence; DNA, Bacterial; DNA-Binding Proteins; Membrane Proteins; Multienzyme Complexes; Transformation, Bacterial
PubMed: 33527432
DOI: 10.1111/mmi.14690 -
ACS Infectious Diseases Feb 2021The maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis through synthesis and degradation is critical for accurate genomic and mitochondrial DNA...
The maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis through synthesis and degradation is critical for accurate genomic and mitochondrial DNA replication fidelity. makes use of both the salvage and pathways for the provision of pyrimidine dNTPs. In this respect, the sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) appears to be the most relevant dNTPase controlling dNTP/deoxynucleoside homeostasis in mammalian cells. Here, we have characterized the role of a unique trypanosomal SAMHD1 orthologue denominated TbHD52. Our results show that TbHD52 is a mitochondrial enzyme essential in bloodstream forms of . Knockout cells are pyrimidine auxotrophs that exhibit strong defects in genomic integrity, cell cycle progression, and nuclear DNA and kinetoplast segregation in the absence of extracellular thymidine. The lack of TbHD52 can be counteracted by the overexpression of human dCMP deaminase, an enzyme that is directly involved in dUMP formation yet absent in trypanosomes. Furthermore, the cellular dNTP quantification and metabolomic analysis of null mutants revealed perturbations in the nucleotide metabolism with a substantial accumulation of dCTP and cytosine-derived metabolites while dTTP formation was significantly reduced. We propose that this HD-domain-containing protein unique to kinetoplastids plays an essential role in pyrimidine dNTP homeostasis and contributes to the provision of deoxycytidine required for cellular dTTP biosynthesis.
Topics: Animals; Homeostasis; Humans; Mitochondria; Pyrimidines; SAM Domain and HD Domain-Containing Protein 1; Trypanosoma brucei brucei
PubMed: 33417760
DOI: 10.1021/acsinfecdis.0c00551 -
Molecular Microbiology May 2020Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase...
Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase ComGA during the state of competence. We show that ComEB plays an important role during competence, possibly because it is necessary for the recruitment of GomGA to the cell pole. ComEB localizes to the cell poles even upon expression during exponential phase, indicating that it can serve as polar marker. ComEB is also a deoxycytidylate monophosphate (dCMP) deaminase, for the function of which a conserved cysteine residue is important. However, cysteine-mutant ComEB is still capable of natural transformation, while a comEB deletion strain is highly impaired in competence, indicating that ComEB confers two independent functions. Single-molecule tracking (SMT) reveals that both proteins exchange at the cell poles between bound and unbound in a time scale of a few milliseconds, but turnover of ComGA increases during DNA uptake, whereas the mobility of ComEB is not affected. Our data reveal a highly dynamic role of ComGA during DNA uptake and an unusual role for ComEB as a mediator of polar localization, localizing by diffusion-capture on an extremely rapid time scale and functioning as a moonlighting enzyme.
Topics: Adenosine Triphosphatases; Bacillus subtilis; Bacterial Proteins; Cell Polarity; DCMP Deaminase; DNA, Bacterial; DNA-Binding Proteins; Green Fluorescent Proteins; Mutation; Protein Binding; Recombinant Fusion Proteins; Single Molecule Imaging; Transformation, Bacterial
PubMed: 31954084
DOI: 10.1111/mmi.14457 -
Science Translational Medicine Nov 2019Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the...
Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC.
Topics: Adenocarcinoma; Animals; Biphenyl Compounds; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; DCMP Deaminase; Dihydroorotate Dehydrogenase; Disease Progression; Enzyme Inhibitors; Humans; Lung Neoplasms; Mice; Molecular Targeted Therapy; Oxidoreductases Acting on CH-CH Group Donors; Pancreatic Neoplasms; Pyrimidines; Small Cell Lung Carcinoma; Survival Analysis; Xenograft Model Antitumor Assays
PubMed: 31694929
DOI: 10.1126/scitranslmed.aaw7852 -
MSphere Aug 2019Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we...
Cytidine deaminase (CDA) is a pyrimidine salvage enzyme that catalyzes cytidine and deoxycytidine hydrolytic deamination to yield uridine and deoxyuridine. Here we report the biochemical characterization of CDA as an enzyme within the tetrameric class of the CDA family that efficiently deaminates cytidine, deoxycytidine, and the nucleoside analogue 5-methyl-2'-deoxycytidine. In line with previous studies, we show that RNA interference (RNAi)-mediated CDA depletion impairs proliferation when grown in pyrimidine-deficient medium, while supplementation with thymidine or deoxyuridine restores growth, further underscoring the role of this enzyme in providing deoxyuridine for dUMP formation via thymidine kinase, the substrate required for thymidylate biosynthesis. This observation contrasts with the existence in of a dimeric deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), an essential enzyme that can produce dUMP via the hydrolysis of dUTP/dUDP. Thus, dUTPase-null mutants are thymidine auxotrophs, suggesting that dUTPase might have a role in providing dUMP for thymidylate biosynthesis. We show that overexpression of human dCMP deaminase (DCTD), an enzyme that provides directly dUMP through dCMP deamination, does not reverse the lethal phenotype of dUTPase knockout cells, which further supports the notion that in , CDA is uniquely involved in providing dUMP, while the main role of dUTPase would be the withdrawal of the excess of dUTP to avoid its incorporation into DNA. Furthermore, we report the mitochondrial localization of CDA, highlighting the importance of this organelle in pyrimidine metabolism. Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and deoxycytidine in the pyrimidine salvage pathway. In kinetoplastids, pyrimidine metabolism has been extensively studied as a source of potential drug targets, given the fact that many of the enzymes of the pathway are essential. Thymidylate (dTMP) synthesis in exhibits unique characteristics. Thus, it has been suggested that the production of dUMP, the substrate for dTMP formation, is solely dependent on cytidine deaminase and thymidine kinase. Here we characterize recombinant CDA (TbCDA) and present evidence that indeed the alternative route for dUMP formation via deoxyuridine 5'-triphosphate nucleotidohydrolase does not have a prominent role in dTMP formation. Furthermore, we provide a scheme for the compartmentalization of dTMP biosynthesis, taking into account the observation that CDA is located in the mitochondrion, together with available information on the intracellular localization of other enzymes involved in the dTTP biosynthetic pathway.
Topics: Cytidine Deaminase; DCMP Deaminase; Gene Knockdown Techniques; Humans; Kinetics; Protozoan Proteins; Pyrimidines; RNA Interference; Recombinant Proteins; Sequence Alignment; Thymidine Monophosphate; Thymine Nucleotides; Trypanosoma brucei brucei
PubMed: 31391279
DOI: 10.1128/mSphere.00374-19