Structural Biochemistry/NOD-like Receptors (NLR)

The nucleotide binding domain and leucine-rich-repeat-containing-proteins or NLR family are a type of cytoplasmic pattern recognition receptors (PRR) responsible for initial proinflammatory and antiviral responses. 22 members of the human NLR protein family have been reported and 2 main subfamilies within the NLR family can be distinguished, NLRC and NLRP. Other members of the NLR family include NLRA or CIITA (class II major histocompatibility complex transactivator), NLRB or NAIP (NLR family apoptosis inhibitory protein), and NLRX.



Structure
All NLR proteins contain a conserved NOD motif and up to 2 other characteristic domains. An N-terminal effector binding domain is responsible for signal transduction and activation of inflammatory response. Examples of amino-terminal domains in NLRs are the acidic transactivation domain, caspase activation and recruitment domain (CARD), pyrin domain (PYD), TOLL/interleukin-1 receptor (TIR) or baculoviral inhibitor of apoptosis repeat (BIR) domain. The central NOD or NACHT domain is a conserved, intermediary NTPase which shares similarities with NB-ARC motif of the apoptotic mediator APAF1. A C-terminal leucine-rich repeat (LRR) domain is responsible for ligand sensing and modulation of NLR activity. PAMPs (pathogen-associated molecular pattern) or DAMPs (danger-associated molecular pattern) detected by the C-terminal LRR motif causes conformational rearrangement of the NLR, which triggers oligomerization. The newly exposed N-terminal effector domain induces the recruitment and activation of CARD and/or PYD-containing effector molecules which enhances spatial proximity and further oligomerization. NLRC proteins such as NOD1 and NOD2 are distinguished by NACHT-LRR-CARD domains whereas NACHT-LRR-PYD domains are characteristic of the NLRP family. PYD domains in particular drive caspase activation and pro-inflammatory cytokine processing in NLRP proteins. Oligomerization of NACHT domains upon ligand sensing is thought to be critical for activation and formation of high molecular weight inflammasome complexes.

Receptor-Ligand Interaction
In the NLRC family, NOD1 and NOD2 act as intracellular microbial sensors that identify and bind peptidoglycan fragments released from bacterial cell walls. NOD1 recognizes N-acetyl glucosamine-N-acetyl muramic acid disaccharide linked to an tripeptide with an N-terminal meso-diaminopimelate (mDAP). This component is characteristic of most Gram-negative bacteria. For NOD2, broad range recognition of muramyl dipeptide (MDP), which is found in both Gram-negative and Gram-positive bacterial peptidoglycan, allows for detection during cell wall synthesis or degradation of bacterial components after lysozymal activity. In the NLRP family, a wider repertoire of PAMPs and DAMPs such as microbial toxins, cytosolic dsDNA, and uric acid may activate inflammatory signaling. PYD domains in NLRP proteins are shown to associate with apoptosis associated speck-like protein (ASC), which is an adaptor molecule that interacts with CARD of pro-caspase-1 and leads to the formation of the inflammasome. The presence of N-terminal PYRINN-PAAD-DAPIN domain (PYD) and C-terminal CARD on ASC adaptor molecule facilitate signaling with NLR protein members such as homotypic PYD-PYD or CARD-CARD interactions in order to stimulate and/or regulate caspase-1, NF-κB activation, and secretion of IL-1β and IL-18.

Signaling Cascade and the Inflammasome
NOD1 activation may result in apoptosis through a number of protein-protein interactions. Notably, NOD1 has been shown to bind to pro-caspase-9, leading to caspase-mediated cell death. NOD1, via CARD may interact with the CSN6 component of the COP9 signalosome, which may synergize in the apoptotic pathway. However, NOD1 has also been shown to interact with both receptor-interacting serine-threonine protein kinase 2 (RIP2) and pro-caspase-1 as well for the enhancement of pro-IL-1β processing. NOD2 has been shown to be associated with mitochondrial antiviral signaling protein (MAVS) for the induction of type I interferons. Both NLRC proteins NOD1 and NOD2 interact with RIP2 through CARD-CARD interactions to induce nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling. RIP2 interacts with regulatory NF-κB subunit NEMO/IKKγ, triggering IκB phosphorylation and NF-κB activation. NOD2, on the other hand, also interacts with TGF-β-activated kinase 1 and GRIM-19 which allows for the activation of IFN-β and NF-κB, subsequently upregulating production of chemokines and antimicrobial peptides. The inflammasome is a signal platform that is significant in the NLR family and its main purpose is for the processing and maturation of proinflammatory cytokines, IL-1β and IL-18. It is a multiprotein oligomer comprised of NLRP proteins, ASC adaptor molecule, caspase-1 and in some cases, caspase-5. Recruitment of the inflammasome complex permits binding from ASC molecule to caspase-1 p45 precursor, pro-caspase-1, which is autocatalytically cleaved into p10 and p20 subunits. Caspase-1 is then assembled into an active form consisting of p10/p20 heterodimers. Ultimately, caspase-1 is involved in inflammatory signaling through proteolytic cleavage of pro-IL-1B and pro-IL-18 into biologically active IL-1βp17 and IL-18p18, respectively. Several known inflammasomes exist composed of varying NLR and PYHIN proteins such as NLRP1, NLRP3, NLRC4 and AIM2. NLRP1 inflammasome consists of ASC adaptor protein, caspase-1 and caspase-5. NLRP2/3 inflammasome is composed of ASC adaptor protein, NLRP2, NLRP3, CARDINAL and caspase-1. NLRP3 inflammasome is the most characterized and studied inflammasome model to date. In particular, 3 models for NLRP3 activation are currently debated. One model suggests that extracellular ATP acts as an agonist for P2X7 receptor which triggers K+ efflux and pannexin-1 mediated membrane pore formation. This is thought to allow the entry of extracellular factors for direct NLRP3 activation as well as NLRP3 detection in K+ efflux and loss of membrane integrity. The second model implies that lysosomal destabilization initiated by the presence of DAMPs may cause rupture and release of lysosome content into the cytosol. Detection of lysosome components such as lysosomal protease cathepsin B may prompt activation as a direct NLRP3 ligand. The third model for NLRP3 activation suggests production of reactive oxygen species or ROS may be caused by NLRP3 agonists and detection of ROS via thioredoxin-interacting protein (TXNIP), a ROS-sensitive NLRP3 ligand may induce activation.

Regulation
The effects of inflammation and apoptosis must be regulated in the event of NLR stimulation in order to be prevent adverse effects in local tissue and systemic applications. Certain NLRs are complexed with ubiquitin ligase-associated protein SGT1 (suppressor of G2 allele of SKP1) and HSP90 (heat shock protein 90 kDa) which keep the receptors in an inactive but signal receptive state. Also, NLRP12 acts to silence NF-κB and MAPK activation by inhibiting the phosphorylation of interleukin-1 receptor-associated kinase 1 (IRAK-1). Anti-apoptotic proteins Bcl-2 and Bcl-XL have been shown to bind and inhibit NLRP1 through the prevention of ATP binding to NLRP and inhibition of oligomerization via Bcl-XL.