Receptor-interacting protein kinase 2 (RIPK2) and nucleotide-binding oligomerization domain (NOD) cell signaling inhibitors based on a 3,5-diphenyl-2-aminopyridine scaffold.

Receptor-interacting protein kinase 2 (RIPK2) is a key mediator of nucleotide-binding oligomerization domain (NOD) cell signaling that has been implicated in various chronic inflammatory conditions. A new class of RIPK2 kinase/NOD signaling inhibitors based on a 3,5-diphenyl-2-aminopyridine scaffold was developed. Several co-crystal structures of RIPK2•inhibitor complexes were analyzed to provide insights into inhibitor selectivity versus the structurally related activin receptor-like kinase 2 (ALK2) demonstrating that the inhibitor sits deeper in the hydrophobic binding pocket of RIPK2 perturbing the orientation of the DFG motif. In addition, the structure-activity relationship study revealed that in addition to anchoring to the hinge and DFG via the 2-aminopyridine and 3-phenylsulfonamide, respectively, appropriate occupancy of the region between the gatekeeper and the αC-helix provided by substituents in the 4- and 5-positions of the 3-phenylsulfonamide were necessary to achieve potent NOD cell signaling inhibition. For example, compound 18t (e.g. CSLP37) displayed potent biochemical RIPK2 kinase inhibition (IC50 = 16 ± 5 nM), >20-fold selectivity versus ALK2 and potent NOD cell signaling inhibition (IC50 = 26 ± 4 nM) in the HEKBlue assay. Finally, in vitro ADME and pharmacokinetic characterization of 18t further supports the prospects of the 3,5-diphenyl-2-aminopyridine scaffold for the generation of in vivo pharmacology probes of RIPK2 kinase and NOD cell signaling functions.

How legumes recognize rhizobia.

Legume plants have developed the capacity to establish symbiotic interactions with soil bacteria (known as rhizobia) that can convert N2 to molecular forms that are incorporated into the plant metabolism. The first step of this relationship is the recognition of bacteria by the plant, which allows to distinguish potentially harmful species from symbiotic partners. The main molecular determinant of this symbiotic interaction is the Nod Factor, a diffusible lipochitooligosaccharide molecule produced by rhizobia and perceived by LysM receptor kinases; however, other important molecules involved in the specific recognition have emerged over the years. Secreted exopolysaccharides and the lipopolysaccharides present in the bacterial cell wall have been proposed to act as signaling molecules, triggering the expression of specific genes related to the symbiotic process. In this review we will briefly discuss how transcriptomic analysis are helping to understand how multiple signaling pathways, triggered by the perception of different molecules produced by rhizobia, control the genetic programs of root nodule organogenesis and bacterial infection. This knowledge can help to understand how legumes have evolved to recognize and establish complex ecological relationships with particular species and strains of rhizobia, adjusting gene expression in response to identity determinants of bacteria.

NOD-like receptor cooperativity in effector-triggered immunity.

Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are basic elements of innate immunity in plants and animals. Whereas animal NLRs react to conserved microbe- or damage-associated molecular patterns, plant NLRs intercept the actions of diverse pathogen virulence factors (effectors). In this review, we discuss recent genetic and molecular evidence for functional NLR pairs, and discuss the significance of NLR self-association and heteromeric NLR assemblies in the triggering of downstream signaling pathways. We highlight the versatility and impact of cooperating NLR pairs that combine pathogen sensing with the initiation of defense signaling in both plant and animal immunity. We propose that different NLR receptor molecular configurations provide opportunities for fine-tuning resistance pathways and enhancing the host's pathogen recognition spectrum to keep pace with rapidly evolving microbial populations.

LRRsearch: An asynchronous server-based application for the prediction of leucine-rich repeat motifs and an integrative database of NOD-like receptors.

The leucine-rich repeat (LRR) motifs of the nucleotide-binding oligomerization domain like receptors (NLRs) play key roles in recognizing and binding various pathogen associated molecular patterns (PAMPs) resulting in the activation of downstream signaling and innate immunity. Therefore, identification of LRR motifs is very important to study ligand-receptor interaction. To date, available resources pose restrictions including both false negative and false positive prediction of LRR motifs from the primary protein sequence as their algorithms are relied either only on sequence based comparison or alignment techniques or are over biased for a particular LRR containing protein family. Therefore, to minimize the error (≤5%) and to identify a maximum number of LRR motifs in the wide range of proteins, we have developed "LRRsearch" web-server using position specific scoring matrix (PSSM) of 11 residue LRR-HCS (highly conserved segment) which are frequently observed motifs in the most divergent classes of LRR containing proteins. A data library of 421 proteins, distributed among five known NLR families has also been integrated with the "LRRsearch" for the rich user experience. The access to the "LRRsearch" program is freely available at http://www.lrrsearch.com/.

NOD proteins: regulators of inflammation in health and disease.

Entry of bacteria into host cells is an important virulence mechanism. Through peptidoglycan recognition, the nucleotide-binding oligomerization domain (NOD) proteins NOD1 and NOD2 enable detection of intracellular bacteria and promote their clearance through initiation of a pro-inflammatory transcriptional programme and other host defence pathways, including autophagy. Recent findings have expanded the scope of the cellular compartments monitored by NOD1 and NOD2 and have elucidated the signalling pathways that are triggered downstream of NOD activation. In vivo, NOD1 and NOD2 have complex roles, both during bacterial infection and at homeostasis. The association of alleles that encode constitutively active or constitutively inactive forms of NOD2 with different diseases highlights this complexity and indicates that a balanced level of NOD signalling is crucial for the maintenance of immune homeostasis.

Activation of nucleotide-binding oligomerization domain 1 (NOD1) receptor signaling in Labeo rohita by iE-DAP and identification of ligand-binding key motifs in NOD1 by molecular modeling and docking.

The nucleotide-binding oligomerization domain 1 (NOD1) receptor recognizes various pattern-associated structures of microbes through its leucine-rich repeat (LRR) domain and activates signaling cascades to induce innate immunity. This report describes the activation of NOD1 receptor signaling by gamma-D-glutamyl-meso-diaminopimelic acid (or γ-D-Glu-mDAP [iE-DAP]) in a commercially important fish species, rohu (Labeo rohita). It also described critical motifs in the NOD1-LRR domain that could be involved in binding iE-DAP, lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C). The activation of NOD1 receptor signaling was studied by injecting iE-DAP, and analysis of tissue samples for NOD1 and receptor-interacting serine/threonine kinase (RICK) expression was done by quantitative real-time polymerase chain reaction (qRT-PCR) assay. To identify ligand-binding motifs in NOD1, the 3D model of NOD1-LRR was generated, followed by a 6-ns molecular dynamics simulation. Molecular docking of LPS with NOD1-LRR was executed at the Hex and PatchDock servers, and iE-DAP and poly I:C in the AutoDock 4.2, FlexX 2.1, Glide 5.5, and GOLD 4.1 programs. The results of qRT-PCR revealed significant (p < 0.05) upregulation of NOD1 and RICK expression. Molecular docking revealed that the amino acid residues at LRR1-2, LRR3-7, and LRR8-9 could be involved in poly I:C, LPS, and iE-DAP binding, respectively. In fish, this is the first report describing the 3D structure of NOD1-LRR and its critical ligand-binding motifs.

Defining the origins of the NOD-like receptor system at the base of animal evolution.

Distinguishing self from nonself and the onset of defense effector mechanisms upon recognition of pathogens are essential for the survival of all life forms in the animal kingdom. The family of nucleotide -binding and oligomeriszation domain-like receptors (NLRs) was first identified in vertebrates and comprises a group of pivotal sensor protein of the innate immune system for microbial cell wall components or danger signals. Here, we provide first evidence that early diverging metazoans have large and complex NLR repertoires. The cnidarian NACHT/NB-ARC genes include novel combinations of domains, and the number of one specific type (NB-ARC and tetratricopeptide repeat containing) in Hydra is particularly large. We characterize the transcript structure and expression patterns of a selected HyNLR, HyNLR type 1 and describe putative interaction partners. In a heterologous expression system, we show induced proximity recruitment of an effector caspase (HyDD-Caspase) to the HyNLR type 1 protein upon oligomerization indicating a potential role of caspase activation downstream of NLR activation in Hydra. These results add substantially to our understanding of the ancestral innate immune repertoire as well as providing the first insights into putative cytoplasmic defense mechanisms at the base of animal evolution.

NLR sensors meet at the SGT1-HSP90 crossroad.

The NLR (nucleotide-binding domain and leucine-rich repeat containing) proteins provide pathogen-sensing systems that are conserved in both plants and animals. They can be activated directly or indirectly by pathogen-derived molecules through mechanisms that remain largely elusive. Studies in plants revealed that the molecular chaperone, HSP90, and its co-chaperones, SGT1 and RAR1, are major stabilizing factors for NLR proteins. More recent work indicates that SGT1 and HSP90 are also required for the function of NLR proteins in mammals, underscoring the evolutionary conservation of innate immune system regulatory mechanisms. Comparative analyses of plant and mammalian NLR proteins, together with recent insights provided by the structure of SGT1-HSP90 complex, have begun to uncover the mechanisms by which immune NLR sensors are regulated.

Activating immunity: lessons from the TLRs and NLRs.

The Toll-like receptors and NOD-like receptors are key families in the innate immune response. The specific detection of activating ligand facilitates receptor interactions, the formation of multiprotein signalling complexes and initiation of signal transduction cascades. This process can trigger the upregulation of proinflammatory mediators, apoptosis, and modulation of other immune defences. Recently, significant advances have been made in the identification of new activating ligands and the determination of the molecular basis of ligand recognition within these receptor families. Understanding these processes provides information essential to the development of new vaccine adjuvants and the treatment of infectious diseases, inflammatory disorders and, potentially, cancer.

Toll-like receptors and NOD-like receptors: domain architecture and cellular signalling.

The innate immune system forms the first line of defense against pathogens. The Toll-like receptors and the Nod-like receptors are at the forefront of both extracellular and intracellular pathogen recognition. They recognize the most conserved structures of microbes and initiate the response to infection. In addition to the microbial stimuli, they are now also being implicated in the recognition of danger-associated stimuli, making them pivotal in disorders unrelated to microbial pathogenesis. Toll-like receptors and the Nod-like receptors share commonalities in structure, ligands and downstream signalling but they differ in their localization, and extent of influence on a wide variety of cellular processes including apoptosis. Here we discuss the common ligand recognition and signalling modules in both these classes of receptors.

Understanding diversity of human innate immunity receptors: analysis of surface features of leucine-rich repeat domains in NLRs and TLRs.

BACKGROUND: The human innate immune system uses a system of extracellular Toll-like receptors (TLRs) and intracellular Nod-like receptors (NLRs) to match the appropriate level of immune response to the level of threat from the current environment. Almost all NLRs and TLRs have a domain consisting of multiple leucine-rich repeats (LRRs), which is believed to be involved in ligand binding. LRRs, found also in thousands of other proteins, form a well-defined "horseshoe"-shaped structural scaffold that can be used for a variety of functions, from binding specific ligands to performing a general structural role. The specific functional roles of LRR domains in NLRs and TLRs are thus defined by their detailed surface features. While experimental crystal structures of four human TLRs have been solved, no structure data are available for NLRs. RESULTS: We report a quantitative, comparative analysis of the surface features of LRR domains in human NLRs and TLRs, using predicted three-dimensional structures for NLRs. Specifically, we calculated amino acid hydrophobicity, charge, and glycosylation distributions within LRR domain surfaces and assessed their similarity by clustering. Despite differences in structural and genomic organization, comparison of LRR surface features in NLRs and TLRs allowed us to hypothesize about their possible functional similarities. We find agreement between predicted surface similarities and similar functional roles in NLRs and TLRs with known agonists, and suggest possible binding partners for uncharacterized NLRs. CONCLUSION: Despite its low resolution, our approach permits comparison of molecular surface features in the absence of crystal structure data. Our results illustrate diversity of surface features of innate immunity receptors and provide hints for function of NLRs whose specific role in innate immunity is yet unknown.

The Nod-like receptor (NLR) family: a tale of similarities and differences.

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs possess a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.

Signal transduction pathways used by NLR-type innate immune receptors.

Proteins from the nucleotide-binding domain, LRR containing (NLR) family are involved in sensing bacterial invasion and danger signals in mammalian cells. Activation of these molecules leads to inflammatory responses which help clearance of invading pathogens. Recent data now shed light on the signal transduction pathways used by NLR proteins. This review summarizes advances in our understanding of signalling through NLRs with special emphasis on the Nod1 and Nod2 pathways.

NLR, the nucleotide-binding domain leucine-rich repeat containing gene family.

The NLR (nucleotide-binding domain leucine-rich repeat containing) family is found in plants and animals, and serves as crucial regulators of inflammatory and innate immune response, though its functions are likely to extend greatly beyond innate immunity, and even beyond the immune system. This review discusses recent findings regarding the function of NLR proteins in the control of IL-1, NF-kappaB, and host response to pathogens including distinct forms of cell death. The review also covers recent advances regarding the biochemical nature of NLRs, its regulation by intracellular nucleotides and extracellular ATP, by the chaperone protein HSP90, and the ubiquitin ligase-associated protein SGT1. Its role in inflammation is linked to the formation of biochemical complexes such as the inflammasome, and its roles in cell death might be linked to the proposed formation of pyroptosome and necrosome.

CARD6 is interferon inducible but not involved in nucleotide-binding oligomerization domain protein signaling leading to NF-kappaB activation.

We have previously reported the cloning and characterization of CARD6, a caspase recruitment domain (CARD)-containing protein that is structurally related to the interferon (IFN)-inducible GTPases. CARD6 associates with microtubules and with receptor-interacting protein 2 (RIP2). RIP2 mediates NF-kappaB activation induced by the intracellular nucleotide-binding oligomerization domain (NOD) receptors that sense bacterial peptidoglycan. Here we report that the expression of CARD6 and RIP2 in bone marrow-derived macrophages is rapidly induced by beta IFN and gamma IFN. This IFN-induced upregulation of CARD6 is suppressed by lipopolysaccharide (LPS), in contrast to LPS's enhancement of IFN-induced RIP2 upregulation. We generated CARD6-deficient (CARD6(-/-)) mice and carried out extensive analyses of signaling pathways mediating innate and adaptive immune responses, including the NOD pathways, but did not detect any abnormalities. Moreover, CARD6(-/-) mice were just as susceptible as wild-type mice to infection by Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Candida albicans, lymphocytic choriomeningitis virus, or mouse adenovirus type 1. Thus, although structural and in vitro analyses strongly suggest an important role for CARD6 in immune defense, the physiological function of CARD6 remains obscure.

From inflammasomes to fevers, crystals and hypertension: how basic research explains inflammatory diseases.

Pattern-recognition receptors, such as Toll-like receptors and NOD-like receptors (NLRs), are able through the recognition of pathogen-associated molecular patterns and danger-associated molecular patterns to sense microbe-dependent and microbe-independent danger and thereby initiate innate immune responses. In some autoinflammatory conditions, abnormalities in NLR signaling pathways are involved in pathogenesis, as exemplified by NOD2 mutations associated with Crohn's disease. Some other NLRs are components of the inflammasome, a caspase-1- and prointerleukin-1beta-activating complex. Clinical and experimental studies are beginning to reveal the central role of the inflammasome in innate immunity. Here, we focus on monogenic hereditary inflammatory diseases, such as Muckle-Wells syndrome, which are associated with mutations in proteins that modulate the activity of the inflammasome, and on some multifactorial disorders, such as Type 2 diabetes and hypertension.