INTRODUCTION:

NOD-like receptors
Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are pattern-recognition
receptors similar to toll-like receptors (TLRs). While TLRs are transmembrane receptors, NLRs are
cytoplasmic receptors that play a crucial role in the innate immune response by recognizing pathogen-
associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Based on
their N-terminal domain, NLRs are divided into four subfamilies: NLRA, NLRB, NLRC, and NLRP.
NLRs can also be divided into four broad functional categories: inflammasome assembly, signalling
transduction, transcription activation, and autophagy.
NOD1 and NOD2
NOD1 (CARD4) and NOD2 (CARD15) are two members of the growing family of Nod-like receptors
characterized by a nucleotide-oligomerization domain and ligand-recognizing leucine-rich repeats.
They are intracellular pattern-recognition molecules involved in the recognition of peptidoglycan
(PGN). They detect specific motifs within the PGN. NOD1 senses the iE-DAP dipeptide which is found
in PGN of all Gram-negative and certain Gram-positive bacteria. Whereas, NOD2 recognizes the
muramyl dipeptide (MDP) structure found in almost all bacteria. Both sensors signal via the
serine/threonine RIP2 (RICK, CARDIAK) kinase which interacts with IKK leading to the activation of
NF-κB and the production of inflammatory cytokines such as TNF-α and IL-6.

c
Fig 1: Schematic structure of Lys -PGN (found in Gram-positive bacteria) and DAP -PGN (found in
Gram-negative bacteria)

Inflammasomes
Inflammasomes are intracellular cytosolic protein complexes that are assembled in response to PAMPs
and DAMPs and function to cleave the inactive precursor forms of an IL-1β, IL-18, and IL-33 (members
of IL-1 family of cytokines) into their pro-inflammatory active forms.

IMMUNOLOGICAL FUNCTION
The PRRs include the Toll-like receptor (TLR) family present at the cell surface or within phagosomes
and the cytosolic nucleotide-binding and olimerization domain (NOD)-like receptor family (NLR).
Recognition of PAMPs by PRRs results in intracellular signaling cascades that leads to:
1. induction of the production of inflammatory cytokines
2. upregulation of costimulatory molecules and activation of the antimicrobial defense
STRUCTURE
NLR

There are 23 NLR family members in humans. NLRs are expressed in many cell types including
immune cells and epithelial cells, although certain NLR family members are expressed primarily in
phagocytes including macrophages and neutrophils. NLRs are multi-domain proteins composed of a
variable N-terminal effector region consisting of caspase recruitment domain (CARD), pyrin domain
(PYD), acidic domain, or baculovirus inhibitor repeats (BIRs), a centrally located NOD that is critical
for activation, and C-terminal leucine-rich repeats (LRRs) that senses PAMPs. The NOD domain (also
referred as the NACHT cassette) is closely related to the oligomerization module found in the AAA+
family of adenosine triphosphatases (ATPases).The N-terminal domain of the NLRs mediates signalling
through its interaction with downstream factors. CARD and PYD domains mediate homophilic
interactions and belong to the death domain-fold superfamily, which are involved in several cellular
processes including apoptosis and inflammation.

Fig 2: Structure of NLR families

Inflammasomes
NLRP1
NLRP1 contains at its N-terminal a pyrin domain (PYD) and at its C-terminus an FIIND motif and
a CARD, which distinguishes it from the other inflammasomes. Upon activation, the C-terminal CARD
homotypically interacts with the CARD of procaspase-1 or procaspase-5, while its N-terminal PYD
homotypically interacts with the PYD of adaptor protein ASC, whose CARD can then recruit another
pro-caspase-1. The overall recruitment and cleavage of procaspase-1 can then activate all downstream
caspase-1 pathways.
NLRP3
NLRP3 contains a PYD domain like NLRP1 and thus activates caspase-1 the same way, using its PYD
to recruit ASC. It forms only one oligomer per cell, and its oligomer is made of seven NLRP3 molecules.
It is known to be the biggest inflammasome of all, covering about 2 um in diameter.
NAIP/NLRC4
NLRC4 (also known as IPAF) is the only known subset of the NLRC family to form an inflammasome
and contains only a CARD domain in addition to the NOD and LRR, which it uses to recruit the adaptor
protein ASC or pro-caspase-1 directly. In contrast to other inflammasomes, NLRC4 applies NAIPs
(NLR family apoptosis inhibitory proteins) as upstream receptors.
The Adaptor ASC
Apoptosis-associated speck like protein containing a caspase recruitment domain (ASC or Pycard)
plays a key role in activation of the inflammasome. ASC helps recruit caspase-1 to associate with NLRs
in the inflammasome complex via its CARD domain.
AIM2
AIM2 is a 343 amino acid non-NLR family protein with pyrin (DAPIN) and a HIN-200 domains, the
former of which is activated in AIM2 by dsDNA.
PYRIN
Pyrin is composed of a PYD domain, two B-boxes, a coiled-coil domain as well as a B30.2 domain at
its C-terminus, whereby the latter is only present in human pyrin.

Fig 3: Structure of inflammasome

SIGNALLING PATHWAY

Fig 4: NLR and Inflammasome mediated signalling

NLR signalling
(A) NF-κB and AP-1 pathways. Bacterial PG-derived peptides γ-D-glutamyl-m-diaminopimelic acid
(iE-DAP) and MDP are recognized by the cytosolic receptors NOD1 and NOD2. These ligands bind to
NOD1 or NOD2 through the LRR domain of these molecules. This interaction initiates the activation
of NOD1 and NOD2 due to the induction of a complex conformational change that results in protein
oligomerization and further interaction with downstream effectors. NOD1 or NOD2 assembly recruits
RIP2 through CARD–CARD interactions, resulting in RIP2 ubiquitination by IAPs and recruitment of
LUBAC complex by XIAP, with further binding of the TAB1/TAK1 complex. TAK1 gets activated
through autophosphorylation and stimulates downstream IKK complex, including Lys63-linked
polyubiquitination of NEMO (IKKγ), the regulatory subunit of the IKK complex, which also consists
of the catalytic subunits IKK1 (IKKα) and IKK2 (IKKβ). This event is followed by IKK2
phosphorylation, which further phosphorylates the NF-κB inhibitor IκBα. IκBα is then ubiquitinated by
the SCF/β-TrCP complex and further degraded by 26S proteasome. The degradation of IκBα releases
NF-κB dimers to translocate into the nucleus, where they up-regulate target genes involved in host
defence and apoptosis. NOD oligomerization and further RIP2 activation also recruits TAB/TAK1
complexes to mediate the phosphorylation of MAPKs, such as JNK, ERK and p38 MAPK, through the
upstream activation of MKKs. These kinases translocate to the nucleus and then phosphorylate AP-1
transcription factors (c-fos, c-Jun, ATF and JDP family members) to mediate expression of target genes
containing a TRE (TPA DNA-response element).
(B) MAVS/IRF pathway. Activation of both NOD1 and NOD2 by bacterial products induces receptor
oligomerization and RIP2 recruitment, which in turn binds TRAF3 and induces TBK1/IKKϵ activation
through a mechanism that is not completely understood. This is followed by IRF transcription factor
dimerization and activation, resulting in binding to and induction of type I IFN genes. Similarly, virus-
derived single-stranded RNA binds NOD2 and induces its association with mitochondrial receptor
MAVS, resulting in the activation of IRF3 transcription factors and induction of type I IFNs. TRAF3
also directly binds MAVS but its precise role requires further investigation.
Inflammasome signalling:
Host cells detect and respond to the cellular disturbance from the T3SS-dependent infection of EPEC
and EHEC. The sensing of bacterial presence begins when PRRs on the cell membrane recognize PAMPs
(for example. LPS by TLR4). This engagement initiates the activation of NF-κB signalling and allows the
production of inflammasomal components, including NLRP3 and Pro-IL-1β. Moreover, the detection
of PAMPs also triggers the deubiquitination of NLRP3, readying for the sensor protein to assemble the
signature complex of NLRP3/ASC/Caspase-1. In the case of NLRP3, the complex formation also
requires a secondary stimuli from the infection (for example, the disruption of membrane integrity by
Ehx-toxin or the presence of bacterial-derived DNA:RNA hybrids). NLRC4 initiates the complex
assembly after Naip proteins binds to respective ligands. Ultimately, pro-IL-1β is processed to a
secretion-competent form. Currently, only a limited numbers of effectors known to directly target
components of the inflammasome complex; they are NleA, NleB, and NleF. NleA directly interacts with
NLRP3 to interrupt ubiquitin modification of NLRP3; NleB may negatively influence FADD/Caspase-8,
which enhances NLRP3-inflammasome activity; NleF may potentially disrupt NLRP3-inflammasome by
inhibiting Caspase-4 and Caspase-8.
IMMUNOLOGICAL DISORDERS
REFERENCES
1. Modulation of Inflammasome Signaling Pathway by Enteropathogenic and Enterohemorrhagic
Escherichia coli. Hilo Yen*, Masaki Karino and Toru Tobe, published in Front. Cell. Infect.
Microbiol: 26 August 2016

2. Review article: Toll-like Receptors and NOD-like Receptors in Innate Immune Defense during
Pathogenic Infection Hyo Sun Jin1,2, Jeong-Kyu Park1 and Eun-Kyeong Jo1,2*

3. Review article: Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory
diseases. Ricardo G. Correa, Snezana Milutinovic, John C. Reed Bioscience Reports CT 15, 2012

4. NOD-Like Receptors in Infection, Immunity, and Diseases. Young Keun Kim,1 Jeon-Soo
Shin,2,3,4 and Moon H. Nahm 5,6 Yonsei Med J. 2016 Jan 1; 57(1): 5–14. Published online 2015
Nov 30.

5. Function of Nod-like Receptors in Microbial Recognition and Host Defense. Luigi

Franchi, Neil Warner, Kyle Viani, and Gabriel Nuñez. Immunol Rev. 2009 Jan; 227(1): 106–128.

6. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/nod-like-
receptors
7. https://www.nature.com/subjects/nod-like-receptors
8. https://www.invivogen.com/review-nlr
9. Kuby 7th edition