title: Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2.
abstract: NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52246, USA.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Ver Heul
authors.forename: C Andrew
authors.initials: CA
authors.lastname: Fowler
authors.forename: S
authors.initials: S
authors.lastname: Ramaswamy
authors.forename: Robert C
authors.initials: RC
authors.lastname: Piper
mesh_terms: D000595:Amino Acid Sequence; D000071183:Autophagy-Related Proteins; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D053474:Nod1 Signaling Adaptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053473:Nod2 Signaling Adaptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C518959:ATG16L1 protein, human; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D053474:Nod1 Signaling Adaptor Protein; D053473:Nod2 Signaling Adaptor Protein; D025801:Ubiquitin; C506253:RIPK2 protein, human; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2