Affinage

CAD

DNA fragmentation factor subunit beta · UniProt O76075

Length
338 aa
Mass
39.1 kDa
Annotated
2026-06-09
100 papers in source corpus 27 papers cited in narrative 26 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

CAD is a multienzymatic protein that catalyzes the first three rate-limiting steps of de novo pyrimidine biosynthesis through an N-to-C domain arrangement of GLNase-CPSase-DHOase-ATCase (PMID:3345746), with the N-terminal dihydroorotase domain carrying a single catalytic zinc atom (PMID:2871022) and the C-terminal ATCase domain forming homotrimers that provide the trimer-of-trimers interface underlying CAD oligomerization (PMID:24316846). Pathway flux is governed by sequential phosphorylation: ERK1/2 (MAP kinase) activates CAD at Thr-456 (PMID:15890648, PMID:17206380, PMID:17485345), an event antagonized by PKA phosphorylation at Ser-1406 through steric interference between stably bound kinases, while PKC delta/epsilon phosphorylation at Ser-1873 promotes ERK access (PMID:17206380, PMID:17485345); in tumors, oncogenic beta-catenin drives an AKT2-dependent CAD phosphorylation at Ser-1406/Ser-1859 to potentiate proliferation (PMID:36122209). Activation requires CAD to be imported into the nucleus, where phospho-Thr-456 CAD associates with the nuclear matrix and nuclear localization is needed for optimal pyrimidine synthesis, nucleotide pools, and growth (PMID:15890648), and across the cell cycle activity is set allosterically by UTP binding to two Animalia-specific loops that gate the substrate channel (PMID:36747088). CAD enzymatic activity is essential for cell survival without exogenous uridine, and its complementation assay defines pathogenicity of disease-associated CAD missense variants (PMID:32461667). Entirely separately, CAD also denotes the apoptotic endonuclease CAD/DFF40, held inactive by its inhibitor ICAD/DFF45 until caspase-3 cleaves ICAD to release CAD homodimers (PMID:9422513, PMID:15149602) that, as a Mg2+-dependent enzyme acting specifically on double-stranded DNA, cleave internucleosomal linker DNA to drive oligonucleosomal laddering and final chromatin condensation (PMID:11577114, PMID:11330826). Beyond these roles, CAD physically engages partners that co-opt its pyrimidine output or non-metabolic activity, including the androgen receptor to promote its nuclear translocation (PMID:21982950), NOD2 to suppress antibacterial NF-kappaB/p38 signaling (PMID:22387394), and Ebola virus NP, which recruits CAD via its glutaminase domain to supply pyrimidines for viral replication (PMID:32370067).

Mechanistic history

Synthesis pass · year-by-year structured walk · 26 steps
  1. 1983 High

    Establishing that CAD's catalytic activities reside in separable, autonomously folding domains was the first step in defining how one polypeptide performs multiple enzymatic steps.

    Evidence Controlled proteolysis and kinetic/thermal characterization of the released ~40 kDa ATCase domain

    PMID:6300078

    Open questions at the time
    • Did not establish full domain order
    • Stabilizing inter-domain contacts inferred from stability differences, not structurally resolved
  2. 1986 High

    Localizing the dihydroorotase domain to the extreme N-terminus and identifying its catalytic zinc clarified the chemistry of one CAD active site.

    Evidence Elastase proteolysis, 65Zn radiolabeling, atomic absorption, and chelator inhibition of the isolated 44 kDa domain

    PMID:2871022

    Open questions at the time
    • Catalytic mechanism of zinc not resolved at atomic level here
    • Did not address regulation of the domain in intact CAD
  3. 1988 High

    Resolving the definitive GLNase-CPSase-DHOase-ATCase domain order and demonstrating PKA phosphorylation that abolishes UTP protection linked CAD architecture to its regulation.

    Evidence Limited proteolysis with domain-specific antibodies plus in vitro PKA phosphorylation and phosphopeptide mapping

    PMID:3345746

    Open questions at the time
    • Second PKA phosphosite unmapped
    • Physiological consequence of PKA phosphorylation on flux not measured
  4. 1988 Medium

    Subcellular localization addressed whether CAD channels its intermediate to mitochondria, showing it is cytosolic and punctate rather than mitochondria-associated.

    Evidence Differential centrifugation and dual immunocytochemical localization in BHK cells

    PMID:2903106

    Open questions at the time
    • Did not resolve the nature of the punctate structures
    • Predates discovery of activation-dependent nuclear import
  5. 1998 High

    Identifying caspase-3 cleavage of ICAD as the trigger that releases CAD/DFF40 explained how apoptotic DNA fragmentation is switched on.

    Evidence Site-directed mutagenesis of ICAD cleavage sites and rescue in Fas/staurosporine-treated Jurkat cells

    PMID:9422513

    Open questions at the time
    • Did not resolve the structural basis of CAD activation
    • Did not distinguish CAD's contribution from other apoptotic nucleases
  6. 1998 Medium

    Demonstrating that CAD binds adenovirus pTP at nuclear-matrix replication sites was the first link between CAD and viral DNA replication machinery.

    Evidence Far-Western, reciprocal Co-IP, and confocal co-localization with BrdU incorporation sites

    PMID:9525610

    Open questions at the time
    • Functional requirement of CAD for viral replication not tested
    • Which CAD function (metabolic vs structural) is involved unresolved
  7. 2000 High

    Defining the CAD-domain ubiquitin superfold and its tight binding to ICAD established the molecular basis of nuclease inhibition and folding.

    Evidence Multidimensional NMR structure and SPR binding measurement (Kd ~55 nM)

    PMID:10764577

    Open questions at the time
    • Did not capture the full active dimer
    • Mechanism coupling inhibition to folding inferred, not directly observed
  8. 2001 High

    Genetic knockout assigned CAD/DFF40 specifically to oligonucleosomal fragmentation and late nuclear condensation, revealing distinct earlier-acting nucleases.

    Evidence CAD-/- DT40 cells with full ORF deletion, DNA electrophoresis, and nuclear morphology across apoptotic stimuli

    PMID:11577114

    Open questions at the time
    • Identity of the high-molecular-weight cleavage nucleases not established
    • Did not address non-apoptotic functions
  9. 2001 High

    Biochemical reconstitution defined CAD/DFF40 as a Mg2+-dependent dsDNA endonuclease tuned to apoptotic ionic conditions.

    Evidence In vitro endonuclease assays varying K+, divalent metals, and pH with purified enzyme

    PMID:11330826

    Open questions at the time
    • Single lab
    • In vitro conditions may not fully recapitulate chromatin context
  10. 2003 Medium

    Identifying CIIA as a direct CAD-binding inhibitor that acts downstream of ICAD cleavage revealed an additional layer of nuclease control.

    Evidence Reciprocal Co-IP, in vitro nuclease assay, and antisense knockdown in L929 cells

    PMID:14557248

    Open questions at the time
    • Single lab without structural mapping of the CIIA-CAD interface
    • Physiological dominance over ICAD regulation unclear
  11. 2004 High

    The activated CAD/DFF40 crystal structure showed it is a homodimer with a linker-DNA-cleaving crevice and clarified how ICAD assembles or disassembles it.

    Evidence X-ray crystallography of activated CAD/DFF40 with biochemical dimer assembly/disassembly assays

    PMID:15149602

    Open questions at the time
    • Did not capture DNA-bound state
    • Catalytic mechanism at the metal site not resolved
  12. 2005 High

    Showing that ERK-mediated Thr-456 phosphorylation activates CAD and that nuclear localization is required for optimal pathway flux connected signaling, localization, and metabolic output.

    Evidence Phospho-specific antibodies, Thr-456 mutagenesis, NLS/NES-CAD constructs, HPLC nucleotide pools, and growth assays

    PMID:15890648

    Open questions at the time
    • Mechanism driving CAD nuclear import not identified
    • Nature of nuclear-matrix association unresolved
  13. 2006 Medium

    Mapping polyanion inhibition to the alpha-4 helices of the DFF40 dimer explained how RNA and other polyanions block dsDNA binding.

    Evidence In vitro nuclease inhibition assays with structural surface mapping

    PMID:16699957

    Open questions at the time
    • Single lab, single method
    • Physiological relevance of polyanion inhibition in vivo not tested
  14. 2008 High

    Demonstrating exclusive dsDNA cleavage with competitive inhibition by other nucleic acids refined CAD/DFF40 substrate specificity.

    Evidence In vitro nuclease assays with synthetic ribo/deoxyribo oligonucleotides plus apoptosis RNA correlation

    PMID:18283539

    Open questions at the time
    • Did not structurally define the DNA-binding site
    • In vivo competition by cellular RNA not quantified
  15. 2007 High

    Establishing reciprocal antagonism among ERK, PKA, and PKC at distinct CAD phosphosites defined the integrated kinase code controlling pyrimidine synthesis.

    Evidence In vitro kinase assays, LC/MS phosphosite mapping, PMA/inhibitor treatments, kinase-CAD Co-IP, and deletion mutagenesis

    PMID:17206380 PMID:17485345

    Open questions at the time
    • Quantitative contribution of each phosphosite to in vivo flux not fully resolved
    • Structural basis of steric kinase interference inferred, not visualized
  16. 2011 Medium

    Linking CAD to androgen receptor nuclear translocation revealed a non-metabolic function in nuclear receptor signaling.

    Evidence AR immunopurification from xenografts, Co-IP in prostate cells, AR reporter assays, and localization imaging

    PMID:21982950

    Open questions at the time
    • Single lab without structural interaction mapping
    • Whether CAD enzymatic activity is required for AR effect untested
  17. 2012 Medium

    Identifying CAD as a direct NOD2 binder that suppresses antibacterial signaling assigned it a negative-regulatory role in innate immunity.

    Evidence IP-MS interactome, Co-IP, NF-kappaB/p38 reporters, gentamicin protection, siRNA, and CAD inhibitor treatment

    PMID:22387394

    Open questions at the time
    • Single lab
    • Whether the inhibitory effect depends on CAD catalytic activity not resolved
  18. 2013 Medium

    Solving the human ATCase domain homotrimer structure established the structural basis for CAD hexamer assembly.

    Evidence Recombinant domain expression, gel filtration, and X-ray crystallography at 2.1 Å

    PMID:24316846

    Open questions at the time
    • Isolated domain only; full-length hexamer not solved
    • Preliminary diffraction data, single lab
  19. 2017 Medium

    Showing that caspase-activated CAD/DFF40 drives mutagenesis in surviving cells revealed a non-apoptotic, pro-mutagenic role.

    Evidence Genetic loss-of-function of executioner caspases and CAD with mutation-frequency assays after TRAIL or vincristine

    PMID:28981092

    Open questions at the time
    • Single lab
    • Mechanism by which sublethal CAD activity produces mutations not detailed
  20. 2020 Medium

    Ebola NP was shown to recruit CAD via its glutaminase domain to supply pyrimidines required for viral replication and transcription.

    Evidence Co-IP, co-immunofluorescence, siRNA knockdown, reverse genetics, GLN-domain deletion, and uridine rescue

    PMID:32370067

    Open questions at the time
    • Single lab
    • Structural basis of NP-GLN domain binding unresolved
  21. 2020 High

    CRISPR knockout established CAD enzymatic activity as essential without exogenous uridine and validated a complementation assay for variant pathogenicity, linking CAD to human disease genetics.

    Evidence CRISPR/Cas9 knockout, uridine-dependent growth complementation, and transient transfection of variant constructs across 34 variants

    PMID:32461667

    Open questions at the time
    • Did not enumerate the clinical disease phenotype mechanistically
    • Variant effects on specific catalytic steps not dissected
  22. 2021 Medium

    Identifying CAD as a direct afatinib target explained an EGFR-kinase-independent immunosuppressive effect on CD8+ T cells.

    Evidence Biochemical target identification, T cell proliferation assays, and in vivo LLC/MC38 tumor models with TIL quantification

    PMID:33771897

    Open questions at the time
    • Single lab
    • Afatinib binding site on CAD not structurally defined
  23. 2022 High

    Defining a beta-catenin/AKT2/CAD phosphorylation axis at Ser-1406/Ser-1859 linked oncogenic signaling to pyrimidine-driven hepatocellular carcinoma growth.

    Evidence Untargeted metabolomics, reporter assays, MS phosphosite mapping, siRNA, mouse liver tumor models, and pharmacological inhibition

    PMID:36122209

    Open questions at the time
    • Interplay with ERK/PKA phosphorylation not resolved
    • Whether AKT2 acts on the same residues across tissues untested
  24. 2022 Medium

    Demonstrating sustained mTORC1-dependent CAD activity in memory CD8+ T cells coupled pyrimidine synthesis to ribosome biogenesis and recall responses.

    Evidence CAD phosphorylation/activity assays, metabolomics, inhibition, overexpression, pre-rRNA and ribosomal protein quantification, and cytokine assays

    PMID:35622902

    Open questions at the time
    • Single lab
    • Direct connection between pyrimidine pools and rRNA synthesis mechanistically partial
  25. 2023 High

    Identifying UTP-sensing Animalia-specific allosteric loops controlling the substrate channel established allostery as the primary driver of cell-cycle-dependent CAD activity.

    Evidence Cell synchronization with metabolomics, systematic mutational scanning, structural modeling, and in cellulo activity assays

    PMID:36747088

    Open questions at the time
    • Allosteric mechanism modeled rather than crystallographically resolved in full-length CAD
    • Interplay between allostery and phosphorylation not integrated
  26. 2023 High

    Crystal structures of the human DHOase domain with non-substrate ligands revealed a loop-in binding mode distinct from E. coli, identifying a druggable active-site loop.

    Evidence X-ray crystallography at 1.97 Å with 5-fluorouracil and malate, mutagenesis, and fluorescence quenching

    PMID:36671534

    Open questions at the time
    • Drug efficacy of loop targeting not demonstrated in cells
    • Single lab

Open questions

Synthesis pass · forward-looking unresolved questions
  • How the metabolic kinase/allosteric regulatory network, the nuclear-import requirement, and the diverse partner-driven moonlighting functions are integrated within the assembled CAD hexamer in vivo remains unresolved.
  • No full-length hexamer structure linking domains and regulatory sites
  • Mechanism of activation-dependent nuclear import unidentified
  • Whether moonlighting interactions require catalytic activity largely untested

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0140097 catalytic activity, acting on DNA 3 GO:0003677 DNA binding 2 GO:0016740 transferase activity 2 GO:0016787 hydrolase activity 2 GO:0016874 ligase activity 1 GO:0098772 molecular function regulator activity 1
Localization
GO:0000228 nuclear chromosome 1 GO:0005634 nucleus 1 GO:0005829 cytosol 1
Pathway
R-HSA-1430728 Metabolism 2 R-HSA-1640170 Cell Cycle 2 R-HSA-5357801 Programmed Cell Death 2
Partners
ADENOVIRUS PTPAKT2ARCIIAEBOLA NPICADNOD2
Complex memberships
CAD hexamerCAD/DFF40-ICAD/DFF45 complex

Evidence

Reading pass · 26 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1998 Caspase-3 cleaves ICAD (inhibitor of CAD/DFF40) at two specific sites, inactivating its CAD-inhibitory function and thereby releasing active CAD to mediate internucleosomal DNA degradation during apoptosis. Site-directed mutagenesis of both caspase-3 cleavage sites in ICAD blocked DNA degradation in Jurkat cells treated with Fas ligand or staurosporine, even though caspase activity was retained. Site-directed mutagenesis of ICAD caspase-3 cleavage sites; stable transfection of Jurkat cells; apoptosis induction assays Nature High 9422513
2004 Crystal structure of activated CAD/DFF40 reveals it forms a homodimer with a deep active-site crevice suited for cleaving internucleosomal linker DNA. ICAD/DFF45 sequesters the nonfunctional CAD monomer and can also disassemble the active dimer via its middle domain; caspase cleavage of ICAD releases CAD to self-assemble into the active dimer. X-ray crystallography of activated CAD/DFF40; biochemical dimer assembly/disassembly assays Molecular Cell High 15149602
2000 The N-terminal CAD domain of CAD/DFF40 adopts a ubiquitin superfold (one alpha-helix, five beta-strands) and binds tightly to the CAD domain of ICAD (Kd ~55 nM), indicating that this domain-domain interaction is critical for inhibition of DNase activity and for correct folding of CAD. Multi-dimensional NMR spectroscopy; surface plasmon resonance binding assay Journal of Molecular Biology High 10764577
2001 CAD/DFF40 is essential for oligonucleosomal DNA fragmentation and final nuclear condensation/fragmentation during apoptosis, but is dispensable for high-molecular-weight DNA cleavage and early-stage chromatin condensation, demonstrating the existence of one or more separate nucleases responsible for those earlier events. Gene knockout (CAD−/− DT40 chicken cells with full ORF deletion); apoptosis induction assays; DNA gel electrophoresis; nuclear morphology analysis The Journal of Biological Chemistry High 11577114
2001 DFF40/CAD is a Mg2+-dependent endonuclease specific for double-stranded DNA that is optimally active at 50–125 mM K+ (matching apoptotic cytoplasmic ionic conditions), inhibited by Zn2+ and Cu2+, and active over pH 7.0–8.5. At high ionic strength the enzyme introduces single-strand nicks rather than double-strand breaks. In vitro endonuclease activity assays with purified DFF40/CAD across varying ionic conditions, divalent metals, and pH Molecular and Cellular Biochemistry High 11330826
2008 DFF40/CAD exclusively cleaves double-stranded DNA and does not cleave single-stranded DNA, RNA, double-stranded RNA, or RNA-DNA heteroduplexes; non-substrate nucleic acids act as competitive inhibitors of dsDNA cleavage, suggesting they bind the enzyme's DNA-binding site. In vitro nuclease assays with synthetic single- and double-stranded ribo- and deoxyribo-oligonucleotides; in vivo apoptosis RNA degradation correlation Apoptosis High 18283539
2006 DFF40/CAD is inhibited by polyanions including RNA, single-stranded DNA, poly-glutamic acid, and heparin (nanomolar potency); these polyanions bind to positively charged surfaces formed by the alpha-4 helices of the DFF40/CAD homodimer, impairing dsDNA binding. In vitro nuclease inhibition assays with purified DFF40/CAD; structural mapping to homodimer surface Apoptosis Medium 16699957
2003 CIIA (CAD inhibitor that interacts with ASK1) binds directly to CAD and inhibits its nuclease activity without affecting caspase-3-mediated ICAD cleavage; CIIA also inhibits ASK1 oligomerization-dependent activation; antisense knockdown of endogenous CIIA promotes DNA fragmentation and apoptosis. Co-immunoprecipitation; in vitro nuclease activity assay; antisense knockdown in L929 cells; apoptosis assays The Journal of Cell Biology Medium 14557248
1986 The dihydroorotase (DHO) domain of CAD is located at the extreme amino terminus of the polypeptide; it contains one tightly bound zinc atom per subunit that participates in catalysis; the isolated 44 kDa domain forms a concentration-dependent dimer–monomer equilibrium; chelating agents inhibit its activity. Controlled elastase proteolysis of CAD; atomic absorption spectrophotometry; 65Zn radiolabeling; hydrodynamic characterization; chelator inhibition assays The Journal of Biological Chemistry High 2871022
1983 The aspartate transcarbamylase (ATCase) domain of CAD is a distinct ~40 kDa structural domain released by early controlled proteolysis; it forms an oligomer of 2–3 subunits; its kinetic parameters (Km, Vmax) closely match those of intact CAD, but the isolated domain is less stable (half-life 10.6 h vs 60.2 h for intact CAD), indicating stabilizing interactions with adjacent domains. Controlled proteolysis; ion-exchange chromatography purification; SDS-PAGE; steady-state kinetics; thermal stability assay The Journal of Biological Chemistry High 6300078
1988 Domain order in CAD is GLNase-CPSase-DHOase-ATCase (confirmed by antibody probing of limited proteolysis fragments, ruling out the previously proposed DHOase-GLNase-CPSase-ATCase order). Phosphorylation by cAMP-dependent protein kinase (PKA) at two sites (site 1 near C-terminal end of CPSase domain, site 2 unmapped) accelerates protease cleavage of CAD and abolishes the protective effect of UTP. Limited proteolysis with elastase/trypsin; immunoblotting with domain-specific antibodies; in vitro PKA phosphorylation; analytical isoelectric focusing of tryptic phosphopeptides European Journal of Biochemistry High 3345746
1988 CAD is predominantly cytosolic in mammalian cells with a punctate distribution suggesting association with subcellular structures, not uniformly soluble; it is largely excluded from the nucleus and not concentrated near mitochondria, indicating that the intermediate dihydroorotate diffuses freely rather than being channeled to the mitochondrial enzyme dihydroorotate dehydrogenase. Differential centrifugation fractionation; immunocytochemical localization (two methods); immunofluorescence microscopy in BHK cells FASEB Journal Medium 2903106
2005 MAP kinase (ERK1/2) phosphorylates CAD at Thr-456 to activate de novo pyrimidine biosynthesis; phospho-Thr-456 CAD is preferentially localized in the nucleus associated with insoluble nuclear substructures including the nuclear matrix. Nuclear import of CAD is not driven by MAP kinase phosphorylation itself. When CAD is excluded from the nucleus (NES-CAD construct), pyrimidine biosynthesis rate, nucleotide pools, and cell growth are each reduced (~21%, 36%, and 60% respectively), demonstrating that nuclear localization is required for optimal pathway activity. Cell fractionation; fluorescence microscopy; phospho-specific antibodies; Thr-456 mutagenesis; NLS-CAD and NES-CAD fluorescent constructs; HPLC nucleotide pool measurement; growth rate assays The Journal of Biological Chemistry High 15890648
2007 CAD Thr-456 phosphorylation by MAP kinase (ERK1/2) activates pyrimidine biosynthesis; PKA phosphorylation of Ser-1406 antagonizes this by blocking ERK-mediated Thr-456 phosphorylation (and vice versa). Both MAP kinase and PKA form stable complexes with CAD, and the mutual antagonism results from steric interference by bound kinases. PKC (delta and epsilon isoforms) phosphorylates CAD at Ser-1873 and promotes MAP kinase phosphorylation of Thr-456 in a PKC-dependent manner; deletion of Ser-1873 abolishes PMA-induced Thr-456 phosphorylation. In vitro kinase assays with purified CAD and PKC isoforms; LC/MS phosphosite identification; PKC activator (PMA) and inhibitor treatments; Co-immunoprecipitation of kinase-CAD complexes; deletion mutagenesis Molecular and Cellular Biochemistry / Frontiers in Bioscience High 17206380 17485345
2022 Oncogenic β-catenin transcriptionally upregulates AKT2, which then phosphorylates CAD at Ser-1406 and Ser-1859 to potentiate de novo pyrimidine synthesis, driving hepatocellular carcinoma proliferation. Inhibition of the β-catenin/AKT2/CAD axis preferentially suppressed β-catenin mutant cell proliferation and tumor formation. Untargeted metabolomics; reporter gene assays; phospho-site identification by mass spectrometry; siRNA knockdown; mouse liver tumor models; pharmacological inhibition Proceedings of the National Academy of Sciences USA High 36122209
2023 CAD de novo pyrimidine synthesis is allosterically regulated during the cell cycle by UTP binding to two Animalia-specific loops in the CAD allosteric domain; these loops sense and bind UTP (a CAD allosteric inhibitor), and a signal transmission loop controls formation of the substrate channel to regulate CAD activity. Mutation scanning established that allostery is the primary mechanism of cell-cycle-phase-dependent CAD activity change. Cell synchronization + metabolomics; systematic mutational scanning; structural modeling based on mitochondrial CPS homologue; in cellulo CAD activity assays Nature Metabolism High 36747088
1998 Adenovirus precursor terminal protein (pTP) physically binds to CAD at active viral DNA replication sites on the nuclear matrix; pTP-CAD complexes are immunoprecipitable from cell extracts and from in vitro-translated proteins; both pTP and CAD co-localize in nuclear foci that also contain sites of active DNA replication (BrdU incorporation). Far-Western blotting; amino acid sequencing; co-immunoprecipitation from cell extracts; in vitro translation co-IP; confocal co-localization immunofluorescence Journal of Virology Medium 9525610
2020 Ebola virus nucleoprotein NP directly interacts with CAD and recruits it into viral inclusion bodies; this recruitment depends on the glutaminase (GLN) domain of CAD. siRNA knockdown of CAD impairs both EBOV genome replication and transcription; substrate rescue experiments demonstrate that CAD's pyrimidine synthesis function is required for these processes. Co-immunoprecipitation; co-immunofluorescence; siRNA knockdown; reverse genetics life-cycle modelling; substrate (uridine) rescue experiments; GLN-domain deletion constructs Cells Medium 32370067
2012 CAD (the pyrimidine biosynthesis enzyme) binds directly to NOD2 and inhibits NOD2-dependent NF-κB and p38 MAPK activation as well as intracellular killing of Salmonella; reduction of CAD expression or pharmacological CAD inhibition enhances NOD2 antibacterial signaling function, identifying CAD as a negative regulator of NOD2. Immunoprecipitation-coupled mass spectrometry identification of NOD2 interactome; co-immunoprecipitation; reporter gene assays; gentamicin protection assay; siRNA knockdown; CAD inhibitor treatment Gastroenterology Medium 22387394
2011 CAD (pyrimidine biosynthesis enzyme) physically interacts with the androgen receptor (AR) in prostate tumor cells and promotes AR nuclear translocation and transcriptional activity, revealing a non-metabolic function of CAD in nuclear receptor signaling. AR immunopurification from xenograft tumors; co-immunoprecipitation in prostate cell lines; AR transcriptional reporter assays; nuclear localization imaging FASEB Journal Medium 21982950
2017 Executioner caspase activation of CAD/DFF40 endonuclease is essential for TRAIL-induced mutagenesis and for vincristine-induced mutagenesis in surviving cells; this identifies a non-apoptotic, pro-mutagenic role for caspase-activated CAD. Genetic knockout/inhibition of executioner caspases and CAD; mutation frequency assays in surviving cells after TRAIL or vincristine treatment Cell Death & Disease Medium 28981092
2022 CAD activity persists in memory CD8+ T cells after initial activation (sustained phosphorylation in mTORC1-dependent manner) and supports ongoing de novo pyrimidine synthesis required for pre-rRNA synthesis and ribosomal biogenesis; CAD inhibition reduces ribosomal protein levels and diminishes the speed and magnitude of cytokine production upon rechallenge, while CAD overexpression enhances recall responses. CAD activity/phosphorylation assays in resting vs. activated T cells; metabolomics; CAD inhibitor treatment; CAD overexpression; pre-rRNA and ribosomal protein quantification; cytokine production assays Science Immunology Medium 35622902
2023 Crystal structures of the dihydroorotase domain of human CAD (huDHOase) complexed with 5-fluorouracil (1.97 Å) and with malate reveal that, unlike the E. coli DHOase, huDHOase binds non-substrate ligands in a loop-in mode at the active site; site-directed mutagenesis and fluorescence quenching confirmed the binding interaction, identifying the dynamic active-site loop as a drug-targeting site. X-ray crystallography (1.97 Å); site-directed mutagenesis; fluorescence quenching binding assay Biomolecules High 36671534
2021 Afatinib (EGFR-TKI) directly targets CAD, the key de novo pyrimidine biosynthesis enzyme, suppressing CD8+ T lymphocyte proliferation; this mechanism underlies afatinib-mediated immunosuppression distinct from its EGFR kinase inhibition. Biochemical target identification of afatinib binding to CAD; CD8+ T cell proliferation assays with afatinib; in vivo Lewis lung carcinoma and MC38 tumor models; tumor-infiltrating lymphocyte quantification Cancer Research Medium 33771897
2013 The ATCase domain of human CAD forms homotrimers in solution and crystallizes in space group P6322 (2.1 Å resolution), establishing the structural basis for CAD hexamer formation through ATCase trimer-of-trimers interactions. Recombinant domain expression in bacteria; gel filtration (homotrimer confirmation); X-ray crystallography (2.1 Å) Acta Crystallographica Section F Medium 24316846
2020 CRISPR/Cas9 knockout of CAD in human cells confirmed that CAD enzymatic activity is required for cell survival in the absence of exogenous uridine; a complementation assay using transient transfection of recombinant CAD into knockout cells was validated as a reliable pathogenicity test for CAD missense variants. CRISPR/Cas9 gene knockout; uridine-dependent growth complementation assay; transient transfection of variant CAD constructs Genetics in Medicine High 32461667

Source papers

Stage 0 corpus · 100 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1998 Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 1355 9422513
2005 Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. Journal of cellular biochemistry 166 15723341
2016 CAD mutations and uridine-responsive epileptic encephalopathy. Brain : a journal of neurology 94 28007989
2004 Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway. Molecular cell 90 15149602
2001 CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis. The Journal of biological chemistry 87 11577114
2008 Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus. Applied microbiology and biotechnology 84 18584171
2013 Identification of CAD candidate genes in GWAS loci and their expression in vascular cells. Journal of lipid research 82 23667179
2000 The DFF40/CAD endonuclease and its role in apoptosis. Acta biochimica Polonica 82 11996094
2007 The CENP-A NAC/CAD kinetochore complex controls chromosome congression and spindle bipolarity. The EMBO journal 77 18007590
1986 The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics. The Journal of biological chemistry 72 2871022
1986 Structure of DNA formed in the first step of CAD gene amplification. The EMBO journal 72 3023056
1981 The cloning and reintroduction into animal cells of a functional CAD gene, a dominant amplifiable genetic marker. Cell 69 6277500
1982 Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells. Molecular and cellular biology 68 6125880
2021 Pyrimidine Biosynthetic Enzyme CAD: Its Function, Regulation, and Diagnostic Potential. International journal of molecular sciences 67 34638594
2015 Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer. British journal of cancer 64 26633560
1985 Abundant nuclear ribonucleoprotein form of CAD RNA. Molecular and cellular biology 55 3990684
2013 The expanding roles of the Sd(a)/Cad carbohydrate antigen and its cognate glycosyltransferase B4GALNT2. Biochimica et biophysica acta 50 24112972
2001 Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus. Journal of neurochemistry 50 11752060
1983 The isolation and characterization of the aspartate transcarbamylase domain of the multifunctional protein, CAD. The Journal of biological chemistry 50 6300078
1989 Evolution and stability of chromosomal DNA coamplified with the CAD gene. Molecular and cellular biology 48 2569669
2001 Ionic and cofactor requirements for the activity of the apoptotic endonuclease DFF40/CAD. Molecular and cellular biochemistry 42 11330826
2019 CAD, A Multienzymatic Protein at the Head of de Novo Pyrimidine Biosynthesis. Sub-cellular biochemistry 41 31939163
2018 Low-cost, self-made CAD/CAM-guiding system for mandibular reconstruction. Surgical oncology 41 29937172
2019 Biocompatibility of Polymer and Ceramic CAD/CAM Materials with Human Gingival Fibroblasts (HGFs). Polymers 40 31484458
2001 High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6. Proceedings of the National Academy of Sciences of the United States of America 40 11717437
1995 Identification of an origin of bidirectional DNA replication in the ubiquitously expressed mammalian CAD gene. Molecular and cellular biology 40 7623808
2010 Secondary prevention of CAD with ACE inhibitors: a struggle between life and death of the endothelium. Cardiovascular drugs and therapy 39 20577898
2003 Identification of a novel antiapoptotic protein that antagonizes ASK1 and CAD activities. The Journal of cell biology 39 14557248
1988 Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine-biosynthetic protein CAD. European journal of biochemistry 38 3345746
1998 Adenovirus preterminal protein binds to the CAD enzyme at active sites of viral DNA replication on the nuclear matrix. Journal of virology 37 9525610
2021 Deciphering CAD: Structure and function of a mega-enzymatic pyrimidine factory in health and disease. Protein science : a publication of the Protein Society 36 34288185
2022 Oncogenic β-catenin stimulation of AKT2-CAD-mediated pyrimidine synthesis is targetable vulnerability in liver cancer. Proceedings of the National Academy of Sciences of the United States of America 35 36122209
2005 Nuclear localization and mitogen-activated protein kinase phosphorylation of the multifunctional protein CAD. The Journal of biological chemistry 34 15890648
2017 Executioner caspases and CAD are essential for mutagenesis induced by TRAIL or vincristine. Cell death & disease 33 28981092
2000 Structure of the CAD domain of caspase-activated DNase and interaction with the CAD domain of its inhibitor. Journal of molecular biology 31 10764577
2021 Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD. Cancer research 30 33771897
2020 Bio-mechanical characterization of a CAD/CAM PMMA resin for digital removable prostheses. Dental materials : official publication of the Academy of Dental Materials 30 33257084
2023 Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle. Nature metabolism 29 36747088
2012 The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells. Gastroenterology 28 22387394
2023 The Effects of Thermocycling on the Physical Properties and Biocompatibilities of Various CAD/CAM Restorative Materials. Pharmaceutics 27 37631336
2022 Fibrotic Signaling in Cardiac Fibroblasts and Vascular Smooth Muscle Cells: The Dual Roles of Fibrosis in HFpEF and CAD. Cells 27 35626694
2021 CAD increases the long noncoding RNA PUNISHER in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling. Molecular therapy. Nucleic acids 27 34484864
2020 Expanding the clinical and genetic spectrum of CAD deficiency: an epileptic encephalopathy treatable with uridine supplementation. Genetics in medicine : official journal of the American College of Medical Genetics 27 32820246
2020 CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation. Cell proliferation 27 33179842
2018 The Sda and Cad glycan antigens and their glycosyltransferase, β1,4GalNAcT-II, in xenotransplantation. Xenotransplantation 27 29430727
1999 Structure and promoter analysis of murine CAD and ICAD genes. Cell death and differentiation 27 10467348
2012 PINK1 overexpression protects against C2-ceramide-induced CAD cell death through the PI3K/AKT pathway. Journal of molecular neuroscience : MN 26 22212487
2022 Circ-E-Cad encodes a protein that promotes the proliferation and migration of gastric cancer via the TGF-β/Smad/C-E-Cad/PI3K/AKT pathway. Molecular carcinogenesis 25 36453704
2021 The Connection between Czc and Cad Systems Involved in Cadmium Resistance in Pseudomonas putida. International journal of molecular sciences 25 34575861
2020 The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription. Cells 25 32370067
2020 Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy. Genetics in medicine : official journal of the American College of Medical Genetics 24 32461667
2015 Genetic basis of dyslipidemia in disease precipitation of coronary artery disease (CAD) associated type 2 diabetes mellitus (T2DM). Diabetes/metabolism research and reviews 24 25470794
2014 Manipulating cinnamyl alcohol dehydrogenase (CAD) expression in flax affects fibre composition and properties. BMC plant biology 24 24552628
2007 Studies of the central nervous system-derived CAD cell line, a suitable model for intraneuronal transport studies? Journal of neuroscience research 24 17335077
2008 The cad locus of Enterobacteriaceae: more than just lysine decarboxylation. Anaerobe 23 18572426
2019 A missense mutation (p.Tyr452Cys) in the CAD gene compromises reproductive success in French Normande cattle. Journal of dairy science 22 31056337
1987 Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification. Molecular and cellular biology 22 3600632
2021 Vitamin D Receptor (VDR) Gene Polymorphisms and Risk of Coronary Artery Disease (CAD): Systematic Review and Meta-analysis. Biochemical genetics 20 33590380
2020 The association between Matrix Metallo-proteinases-9 (MMP-9) gene family polymorphisms and risk of Coronary Artery Disease (CAD): a systematic review and meta-analysis. BMC cardiovascular disorders 20 32429880
2020 Association of ESR1 (rs2234693 and rs9340799), CETP (rs708272), MTHFR (rs1801133 and rs2274976) and MS (rs185087) polymorphisms with Coronary Artery Disease (CAD). BMC cardiovascular disorders 20 32682401
2016 An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells. Neuro-oncology 20 26755073
2001 Correlation between C677T MTHFR gene polymorphism, plasma homocysteine levels and the incidence of CAD. American journal of cardiovascular drugs : drugs, devices, and other interventions 20 14728017
1988 Intracellular location of the multidomain protein CAD in mammalian cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20 2903106
2021 The Art of Designing DNA Nanostructures with CAD Software. Molecules (Basel, Switzerland) 19 33920889
2020 Response of two gingival cell lines to CAD/CAM composite blocks. Dental materials : official publication of the Academy of Dental Materials 19 32561116
2012 IL-6 gene polymorphisms and CAD risk: a meta-analysis. Molecular biology reports 19 23242654
2011 Identification of CAD as an androgen receptor interactant and an early marker of prostate tumor recurrence. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 18 21982950
2008 The major apoptotic endonuclease DFF40/CAD is a deoxyribose-specific and double-strand-specific enzyme. Apoptosis : an international journal on programmed cell death 18 18283539
2007 Protein kinase C modulates the up-regulation of the pyrimidine biosynthetic complex, CAD, by MAP kinase. Frontiers in bioscience : a journal and virtual library 18 17485345
2021 Computational Analysis of Gly482Ser Single-Nucleotide Polymorphism in PPARGC1A Gene Associated with CAD, NAFLD, T2DM, Obesity, Hypertension, and Metabolic Diseases. PPAR research 17 34394332
2021 Clinical effects with customized brackets and CAD/CAM technology: a prospective controlled study. Progress in orthodontics 17 34866163
2023 Beyond genetics: Deciphering the impact of missense variants in CAD deficiency. Journal of inherited metabolic disease 16 37540500
2022 Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge. Science immunology 16 35622902
2019 Prediction and management of CAD risk based on genetic stratification. Trends in cardiovascular medicine 16 31543237
2010 Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells. Pharmacological research 16 21145398
2006 The apoptotic endonuclease DFF40/CAD is inhibited by RNA, heparin and other polyanions. Apoptosis : an international journal on programmed cell death 16 16699957
1988 Chromosome aberrations associated with CAD gene amplification in Chinese hamster cultured cells. Mutation research 16 2896299
1982 Properties of dispersed, highly repeated DNA within and near the hamster CAD gene. Molecular and cellular biology 16 6180303
2021 Engineering the Cad pathway in Escherichia coli to produce glutarate from L-lysine. Applied microbiology and biotechnology 15 33907891
1987 Transcriptional regulation of the human CAD gene during myeloid differentiation. Molecular and cellular biology 15 2885743
2020 Exploring Metabolic Consequences of CPS1 and CAD Dysregulation in Hepatocellular Carcinoma by Network Reconstruction. Journal of hepatocellular carcinoma 14 32021853
2012 CD10 and E-cad expression in urinary bladder urothelial and squamous cell carcinoma. Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer 14 23339695
2007 Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade. Molecular and cellular biochemistry 14 17206380
2021 PCSK9 genetic (rs11591147) and epigenetic (DNA methylation) modifications associated with PCSK9 expression and serum proteins in CAD patients. The journal of gene medicine 13 33885177
2020 Computer-Aided Design and Manufacturing (CAD/CAM) for Bioprinting. Methods in molecular biology (Clifton, N.J.) 13 32207104
2018 Association of rs662799 in APOA5 with CAD in Chinese Han population. BMC cardiovascular disorders 13 29310573
2017 PON1 L55M and Q192R gene polymorphisms and CAD risks in patients with hyperlipidemia : Clinical study of possible associations. Herz 12 28835968
2013 Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD. Acta crystallographica. Section F, Structural biology and crystallization communications 12 24316846
2008 Endosomal colocalization of melanocortin-3 receptor and beta-arrestins in CAD cells with altered modification of AKT/PKB. Neuropeptides 12 18291523
2007 Extracellular cAMP inhibits D1 dopamine receptor expression in CAD catecholaminergic cells via A2a adenosine receptors. Journal of neurochemistry 12 17254022
2022 A Deep Learning-Based Computer Aided Detection (CAD) System for Difficult-to-Detect Brain Metastases. International journal of radiation oncology, biology, physics 11 36289038
2021 CAD Genes: Genome-Wide Identification, Evolution, and Their Contribution to Lignin Biosynthesis in Pear (Pyrus bretschneideri). Plants (Basel, Switzerland) 11 34371647
2003 Adrenergic differentiation and SSR2a receptor expression in CAD-cells cultured in serum-free medium. Neurochemistry international 11 12441163
2024 Elevated circulating LncRNA NORAD fosters endothelial cell growth and averts ferroptosis by modulating the miR-106a/CCND1 axis in CAD patients. Scientific reports 10 39414920
2023 Complexed Crystal Structure of the Dihydroorotase Domain of Human CAD Protein with the Anticancer Drug 5-Fluorouracil. Biomolecules 10 36671534
2021 The role of the DFF40/CAD endonuclease in genomic stability. Apoptosis : an international journal on programmed cell death 10 33387146
2021 Homo- and Hetero-Dimers of CAD Enzymes Regulate Lignification and Abiotic Stress Response in Moso Bamboo. International journal of molecular sciences 10 34884720
2020 Identification of Potential Biomarkers for CAD Using Integrated Expression and Methylation Data. Frontiers in genetics 10 33033488
2005 Immunohistochemical characterisation of differentiated CAD cells: expression of peptides and chromogranins. Histochemistry and cell biology 10 16007468
2022 Identification and Functional Analysis of CAD Gene Family in Pomegranate (Punica granatum). Genes 9 36672766

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