title: The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells.
abstract: OBJECTIVE
  Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn's disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies.
  
  
  METHODS
  Carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors.
  
  
  RESULTS
  CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD.
  
  
  CONCLUSIONS
  The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Richmond
authors.forename: Amrita
authors.initials: A
authors.lastname: Kabi
authors.forename: Craig R
authors.initials: CR
authors.lastname: Homer
authors.forename: Noemí
authors.initials: N
authors.lastname: Marina-García
authors.forename: Kourtney P
authors.initials: KP
authors.lastname: Nickerson
authors.forename: Alexey I
authors.initials: AI
authors.lastname: Nesvizhskii
authors.forename: Arun
authors.initials: A
authors.lastname: Sreekumar
authors.forename: Arul M
authors.initials: AM
authors.lastname: Chinnaiyan
authors.forename: Gabriel
authors.initials: G
authors.lastname: Nuñez
authors.forename: Christine
authors.initials: C
authors.lastname: McDonald
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000502:physiology* / Q000627:therapeutic use; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000037:antagonists & inhibitors / Q000502:physiology* / Q000627:therapeutic use; D002460:Cell Line; D002478:Cells, Cultured; D003424:Crohn Disease / Q000188:drug therapy / Q000276:immunology* / Q000382:microbiology; D003851:Deoxyribonucleases / Q000502:physiology*; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000502:physiology* / Q000627:therapeutic use; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007413:Intestinal Mucosa / Q000276:immunology / Q000382:microbiology*; D013058:Mass Spectrometry; D016328:NF-kappa B / Q000502:physiology; D053473:Nod2 Signaling Adaptor Protein / Q000276:immunology* / Q000502:physiology; D012475:Salmonella / Q000254:growth & development / Q000276:immunology; D015398:Signal Transduction
chemical_list: C033626:CAD trifunctional enzyme; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D053473:Nod2 Signaling Adaptor Protein; D001221:Aspartate Carbamoyltransferase; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)