Abstract
Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.
Highlights
IntroductionNucleotide-binding oligomerization domain-containing protein (Nod) 2 is a nuclear factor- (NF-) κB-activating intracellular pattern recognition receptor, which was identified as a susceptibility gene product of Crohn’s disease, an inflammatory bowel disease [1,2,3].Nod was reported to be oligomerized with adaptor protein RICK (RIP2/RIPK2) and IKK complexes, which can activate NF-κB by muramyl dipeptide (N-AcetylmuramylL-Alanyl-D-Isoglutamine: MDP), one of the components of bacterial cell-wall peptidoglycan, and is utilized as an immune-stimulatory adjuvant for vaccination and for developing antibodies [4,5,6,7,8].The Scientific World JournalPrimer name S1-Nod F Nod2-T1(F) R Nod2-T1(CARDs) R S1-RICK F RICK-T1(F) R S1-RICK(CARD) F attB1-S1 attB2-T1 Nod2-R334W F Nod2-R334W R Nod2-N670K F Nod2-N670K R Primer sequence5-CCACCCACCACCACCAATGGGGGAAGAGGGTGGTTCAG-3 5-TCCAGCACTAGCTCCAGAAAGCAAGAGTCTGGTGTCCCT-3 5-TCCAGCACTAGCTCCAGACTTGCATGTGGCAGCTTCCA-3 5-CCACCCACCACCACCAATGAACGGGGAGGCCATCTG-3 5-TCCAGCACTAGCTCCAGACATGCTTTTATTTTGAAGTAAATTTA-3 5-CCACCCACCACCACCAATGCTGCAGCCTGGTATAGCCC-3 5-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCACCCACCACCACCAATG-3 5-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCCAGCACTAGCTCCAGA-3 5-TTCCCATTCAGCTGCTGGCAGCTGCAGTGC-3 5-GCACTGCAGCTGCCAGCAGCTGAATGGGAA-3 5-GCCGAGCCGCACAAACTTCAGATCACAGCA-3 5-TGCTGTGATCTGAAGTTTGTGCGGCTCGGC-3Underline indicates S1 or T1 sequence
Nod2 was reported to be oligomerized with adaptor protein RICK (RIP2/RIPK2) and IKK complexes, which can activate nuclear factor- (NF-)κB by muramyl dipeptide (N-AcetylmuramylL-Alanyl-D-Isoglutamine: MDP), one of the components of bacterial cell-wall peptidoglycan, and is utilized as an immune-stimulatory adjuvant for vaccination and for developing antibodies [4,5,6,7,8]
It was discovered that an autoinflammatory disease, Blau syndrome (BS)/early-onset sarcoidosis (EOS), was caused by a point mutation of Nod2, encoding a constitutively active form, resulting in NF-κB activation [9,10,11]
Summary
Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is a nuclear factor- (NF-) κB-activating intracellular pattern recognition receptor, which was identified as a susceptibility gene product of Crohn’s disease, an inflammatory bowel disease [1,2,3].Nod was reported to be oligomerized with adaptor protein RICK (RIP2/RIPK2) and IKK complexes, which can activate NF-κB by muramyl dipeptide (N-AcetylmuramylL-Alanyl-D-Isoglutamine: MDP), one of the components of bacterial cell-wall peptidoglycan, and is utilized as an immune-stimulatory adjuvant for vaccination and for developing antibodies [4,5,6,7,8].The Scientific World JournalPrimer name S1-Nod F Nod2-T1(F) R Nod2-T1(CARDs) R S1-RICK F RICK-T1(F) R S1-RICK(CARD) F attB1-S1 attB2-T1 Nod2-R334W F Nod2-R334W R Nod2-N670K F Nod2-N670K R Primer sequence5-CCACCCACCACCACCAATGGGGGAAGAGGGTGGTTCAG-3 5-TCCAGCACTAGCTCCAGAAAGCAAGAGTCTGGTGTCCCT-3 5-TCCAGCACTAGCTCCAGACTTGCATGTGGCAGCTTCCA-3 5-CCACCCACCACCACCAATGAACGGGGAGGCCATCTG-3 5-TCCAGCACTAGCTCCAGACATGCTTTTATTTTGAAGTAAATTTA-3 5-CCACCCACCACCACCAATGCTGCAGCCTGGTATAGCCC-3 5-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCACCCACCACCACCAATG-3 5-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCCAGCACTAGCTCCAGA-3 5-TTCCCATTCAGCTGCTGGCAGCTGCAGTGC-3 5-GCACTGCAGCTGCCAGCAGCTGAATGGGAA-3 5-GCCGAGCCGCACAAACTTCAGATCACAGCA-3 5-TGCTGTGATCTGAAGTTTGTGCGGCTCGGC-3Underline indicates S1 or T1 sequence. Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is a nuclear factor- (NF-) κB-activating intracellular pattern recognition receptor, which was identified as a susceptibility gene product of Crohn’s disease, an inflammatory bowel disease [1,2,3]. Nod was reported to be oligomerized with adaptor protein RICK (RIP2/RIPK2) and IKK complexes, which can activate NF-κB by muramyl dipeptide (N-AcetylmuramylL-Alanyl-D-Isoglutamine: MDP), one of the components of bacterial cell-wall peptidoglycan, and is utilized as an immune-stimulatory adjuvant for vaccination and for developing antibodies [4,5,6,7,8]. Italic indicates mutation codons for specific amino acids. Other granulomatous inflammatory diseases were reported to be associated with Nod2 [13, 14]. Nod has been thought to be involved in granulomatous disorders [15] and may be an attractive drug target for treatment of these chronic inflammatory granulomatous diseases
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