Fig. 1. Three domains of NOD2. The CARD domain interacts with
RIP2. The NBD domain is responsible for the oligomerization of NOD. The
LRR domain recognizes MDP.
2.2 The MDP dependent activation of NOD2
The activation of NOD2 signaling pathways can be divided into two
categories: MDP dependent and MDP independent. The MDP dependent
pathways have been extensively investigated. Cells phagocytose bacteria
to form phagosome, which fuses with lysosome and further matures into
phagolysosomes, where the bacteria are digested into PGN, the major
component of bacteria cell wall[15]. The basic unit of PGN, MDP,
should be phosphorylated by N-acetylglucosamine kinase (NAGK) before it
is recognized by NOD2[16]. In macrophages and dendritic cells, the
SLC15 family endosomal peptide transporter protein 1 PHT1 (SLC15A4)
enables transmembrane transport of MDP, recruiting NOD2. And endosomes
serve as a signaling platform for triggering the innate immune
response[3,17–19]. Disrupting endosomal acidic chemicals inhibits
NOD2-induced NF-κB activation, which may be due to the dependence of
ligand transport on proton gradients as the energy source[20]. When
bacteria are not invading the cytoplasmic region of the host, it can
also cross the plasma membrane and localize to the cytoplasm through the
plasma membrane transporter protein Human peptide transporter 1 hPepT1
(SLC15A2), which is expressed in intestinal epithelial cells[21,22].
Heat shock protein 70 (HSP70) is a positive regulator of NOD2 signaling,
which prolongs the half-life of NOD2 and enhances NF-κB activity
[23].
Although studies initially described NOD2 as a cytoplasmic receptor, it
has been shown that membrane targeting of NOD2 is required for NF-κB
activation[24]. This was illustrated by the CD-related NOD2
loss-of-function mutation 3020insC, which, despite being fully capable
of binding MDP, fails to bind to the membrane and therefore cannot
detect bacterial invasion [25,26]. Besides the anchorage of NOD2 to
membranes by cytoskeletal components (vimentin) and binding proteins
(FRMPD2, FERM and PDZ structural domain 2) or to endosomes by endosomal
proteins (SLC15A)[27,28,18], studies have shown that palmitoylation
of NOD1/2 oligomerization is needed not only for steady-state membrane
binding but also for MDP-induced signaling pathways. During this
process, the presence of Zinc
Finger DHHC-Type
Palmitoyltransferase 5
(ZDHHC5) and its enzymatic
activity are essential for proper recruitment of NOD1/2 to the site of
bacterial entry and the formation of phagosomes[29]. But how ZDHHC5
is attracted to bacterial entry sites is still unknown[29].
2.3 The MDP independent activation of NOD2
In contrast to MDP dependent activation, the mechanism of
MDP-independent activation of NOD2 is ambiguous. It has been shown that
viruses can activate NOD2[30,31], such as
respiratory syncytial virus (RSV)
and Middle East respiratory
syndrome coronavirus (MERS-CoV). Once viral ssRNA is recognized, NOD2
can enhance IFN-β expression through activation of IRF3 by
mitochondrial antiviral signaling
protein (MAVS)[30]. Keestra-Gounder et al. also proposed a link
between endoplasmic reticulum
(ER) stress and the increase of IL-6 secretion via NOD1/NOD2 signaling,
with MDP non-dependence[32]. Accumulation of unfolded or misfolded
proteins, as well as viral or bacterial infections, can induce an
unfolded protein response (UPR) in the endoplasmic reticulum, which is a
protective response that limits cellular damage caused by ER
stress[33]. UPR involves three transmembrane receptors, the
protein kinase RNA-like
endoplasmic reticulum kinase (PERK),
activating transcription factor 6
(ATF6), and inositol-requiring
enzyme 1 (IRE1α). In mice, intraperitoneal injection ofBifidobacterium abortus triggered the type IV secretion system,
which secreted the effector protein system effector protein VceC, and
induced ER stress. IRE1α recruited Tumor necrosis factor
receptor-associated factor 2 (TRAF2) to the ER membrane and induced
NOD1/NOD2 activation to promote IL-6 production via the NF-κB
pathway[32] (Figure 2) .
The cytoskeletal protein vimentin performs cellular functions by
interacting with the LRR structural domain in NOD2, which is recruited
into the cell membrane, affecting NF-κB activation, autophagy, and
bacterial processing[34]. The interaction of the cytoskeletal
regulator RAC1 and ARHGEF7 (also known as PAK3BP) negatively regulates
NOD2 activation. And the downregulation of the expression of both
proteins enhances MDP-driven NOD2 activation[35,36]. In line with
this, in bone marrow monocytes and intestinal epithelial cells , the
disruption of the actin cytoskeleton by cytochrome D also increases
NOD1- and NOD2-mediated NF-κB activation even in the absence of
peptidoglycan, suggesting that cytoskeletal alterations can trigger the
activation of NOD2[36].
2.4 Signaling pathways following NOD2 activation
Activation of NOD2 mainly initiates downstream gene expression through
two signaling pathways, the NF-κB and MAPK signaling pathways(Figure 2) . In response to ligand binding, RIP2 recruited by
NOD2 is extensively and selectively ubiquitinated, in which
methionine 1-linked (M1)
ubiquitin binds the IκB kinase
complex (IKK) complex (IKKβ, IKKα, and NEMO). And the E3 ubiquitin
ligases X-linked inhibitor of apoptosis protein (XIAP), cellular
inhibitor of apoptosis protein 1 (CIAP1) , CIAP2, TNFR-associated factor
2 (TRAF2), TRAF5, and the linear
ubiquitin assembly complex (LUBAC) are also involved in this
process[37–40]. Indeed, the RING domain in XIAP recruits LUBAC, the
trimeric complex contributing to the linear ubiquitination modification,
to RIP2, which is essential for the NOD2-dependent response[40]. In
line with this observation, mutations (Leu207Pro, Val198Met) in the BIR2
domain of XIAP lead to Ub ligase inactivation and impaired
NOD2-dependent immune signaling[41]. Besides, several
deubiquitinating enzymes (DUBs) negatively regulate the activation of
NOD2, including CYLD, A20 and OTULIN[42,43]. Interferon regulatory
factor 4 (IRF4) works as a negative regulator by inhibiting RIP2
ubiquitination in human DCs and thereby reducing NOD2-dependent NF-κB
activation[44]. Next the activated IKK complex acts as an IκB kinase
to phosphorylate IκB protein, followed by auto-ubiquitination and
proteasomal degradation, releasing the NF-κB complex, which is
translocated into the nucleus to promote cytokine expression[45].
In parallel, NOD2 signaling via lysine 63-linked (K63) ubiquitin
initiates the recruitment of the TAK1-TAB2-TAB3 complex[46],
enabling the activation of
extracellular signal-regulated
kinase 1 (ERK1), ERK2,
JUN
N-terminal kinase (JNK), and p38,
which regulate the transcription factor AP-1 through MAPK
phosphorylation[47]. The transcription factors NF-κB and AP-1 alone
or in combination induce cytokine expression in the nucleus[48–50].