title: L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1.
abstract: The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322. Electronic address: hlaroui@emory.edu.
authors.forename: Hamed
authors.initials: H
authors.lastname: Laroui
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322.
authors.forename: Yutao
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Emory University, Atlanta, Georgia 30322.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Narui
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Ingersoll
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Ayyadurai
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322.
authors.forename: Moiz A
authors.initials: MA
authors.lastname: Charania
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, California 90032.
authors.forename: Feimeng
authors.initials: F
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098.
authors.forename: Binghe
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Emory University, Atlanta, Georgia 30322.
authors.forename: Khalid
authors.initials: K
authors.lastname: Salaita
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322.
authors.forename: Shanthi V
authors.initials: SV
authors.lastname: Sitaraman
authors.affiliation: Department of Medicine, Division of Digestive Diseases, Emory University, Atlanta, Georgia 30322; Veterans Affairs Medical Center, Decatur, Georgia 30033.
authors.forename: Didier
authors.initials: D
authors.lastname: Merlin
mesh_terms: D001703:Biophysics / Q000379:methods; D018938:Caco-2 Cells; D003960:Diaminopimelic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D007249:Inflammation; D007930:Leucine / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods; D053474:Nod1 Signaling Adaptor Protein / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C555437:L-Ala-gamma-D-Glu-meso-diaminopimelic acid; D053474:Nod1 Signaling Adaptor Protein; D009711:Nucleotides; D009842:Oligopeptides; D010455:Peptides; D034741:RNA, Small Interfering; D003960:Diaminopimelic Acid; D007930:Leucine