RIPK2 Knockout Cell Lysate

We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-B appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management.

Receptor interacting protein 2 (RIP2) is an intracellular kinase and key signaling partner for the pattern recognition receptors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2). As such, RIP2 represents an attractive target to probe the role of these pathways in disease. In an effort to design potent and selective inhibitors of RIP2 we established a crystallographic system and determined the structure of the RIP2 kinase domain in an apo form and also in complex with multiple inhibitors including AMP-PCP (beta,gamma-Methyleneadenosine 5'-triphosphate, a non-hydrolysable adenosine triphosphate mimic) and structurally diverse ATP competitive chemotypes identified via a high-throughput screening campaign. These structures represent the first set of diverse RIP2-inhibitor co-crystal structures and demonstrate that the protein possesses the ability to adopt multiple DFG-in as well as DFG-out and C-helix out conformations. These structures reveal key protein-inhibitor structural insights and serve as the foundation for establishing a robust structure-based drug design effort to identify both potent and highly selective inhibitors of RIP2 kinase. (C) 2015 Elsevier Ltd. All rights reserved.