Palmitoylation facilitates inflammation through suppressing NOD2 degradation mediated by the selective autophagy receptor SQSTM1

ABSTRACT

The intracellular pattern recognition receptor NOD2 senses bacterial peptidoglycan to drive proinflammatory and antimicrobial responses. Dysregulation of NOD2 signaling confers susceptibility to several immunological and inflammatory diseases. Although palmitoylation of NOD2 is required for its membrane recruitment and activation, whether palmitoylation can modulate the stability of NOD2 to orchestrate inflammation remains unclear. Recently, we have revealed that S-palmitoylation restricts SQSTM1-mediated selective macroautophagic/autophagic degradation of NOD2, and identified a gain-of-function R444C variant of NOD2 short isoform (NOD2s^R444C) in autoinflammatory disease, which induces excessive inflammation through its enhanced S-palmitoylation level and decreased autophagic degradation.

KEYWORDS:

• Inflammation
• NOD2
• S-palmitoylation
• selective autophagy
• SQSTM1

NOD2 (nucleotide binding oligomerization domain containing 2) senses conserved motifs in bacterial peptidoglycan and activates innate immune signaling pathways that drive proinflammatory and antimicrobial responses. It is recognized that dysregulation of NOD2 signaling is involved in multiple inflammatory disorders. Recently, S-palmitoylation is reported to play a crucial role in mediating the membrane localization and ligand-induced signaling of NOD2. To gain insight into further regulatory functions of S-palmitoylation in NOD2 signaling, we applied 2-bromopalmitate (2BP), a general S-palmitoylation inhibitor, to reduce the S-palmitoylation of NOD2 and observed the decrease of NOD2 protein levels in both dose-dependent and time-dependent manners [1]. Overexpression of ZDHHC5 promotes the protein stability of NOD2, whereas knockdown of ZDHHC5 significantly decreases NOD2 abundance. Likewise, the degradation rates of S-palmitoylation-deficient NOD2 mutants are remarkably higher than that of wild-type (WT) NOD2. Additionally, NOD2-induced NFKB/NF-κB activation is decreased by 2BP treatment or mutation of S-palmitoylation sites on NOD2, whereas ZDHHC5 overexpression enhances the activation of NOD2 signaling. Moreover, the degradation of NOD2 following 2BP treatment can be blocked by autophagy and autolysosome inhibitors, but not a proteasome inhibitor. The 2BP-induced NOD2 degradation is abrogated in BECN1 or ATG5 knockout (KO) cells, in which autophagy is largely impaired, indicating that S-palmitoylation suppression results in the autophagic degradation of NOD2.

Selective autophagy requires cargo receptors that link cargoes to expanding phagophore membranes. NOD2 mainly interacts with SQSTM1 rather than other cargo receptors, and the NOD2-SQSTM1 association is enhanced under rapamycin-induced autophagy conditions. Additionally, cycloheximide-chase results showed the degradation of NOD2 almost disappears in SQSTM1 KO cells. The protein levels of NOD2 are decreased with the treatment of 2BP in WT cells, but not in SQSTM1 KO cells, indicating that the autophagic degradation of NOD2 is mediated by SQSTM1. Overexpression of SQSTM1 leads to NOD2 degradation, but this phenomenon is partially rescued in the presence of ectopic ZDHHC5. Inhibition of NOD2 S-palmitoylation by 2BP or mutation of S-palmitoylation sites enhances NOD2-SQSTM1 interaction. Furthermore, ZDHHC5 overexpression significantly inhibits the colocalization of NOD2 and SQSTM1, whereas restricting palmitoylation markedly enhances their colocalization.

Dysfunctional NOD2 variants are involved in multiple inflammatory disorders. However, the regulatory mechanisms are still incompletely understood. Through screening, we found the R444C variant of the short isoform of NOD2 (NOD2s^R444C), which is a previously ignored variant reported in autoinflammatory diseases, can significantly enhance NFKB activation, but the NOD2l^R471C variant (which has the same amino acid mutation in NOD2s) does not affect the NOD2-mediated NFKB activation. The excessive NFKB activation induced by the NOD2s^R444C variant was further confirmed in THP-1-derived macrophages (DMs). NOD2s^R444C THP-1-DMs display enhanced muramyl dipeptide-induced phosphorylation of IκB kinase (IKK) complex and NFKBIA/IKBA and increased expression levels of pro-inflammatory cytokines compared to that of WT NOD2s THP-1-DMs, whereas a similar amount of phosphorylation of the IKK complex, p-NFKBIA/IKBA and pro-inflammatory cytokines are detected in WT NOD2l and NOD2l^R471C THP-1-DMs.

To figure out the detailed mechanism of the NOD2s^R444C variant in promoting inflammatory responses, we determined the rates of protein degradation and found that the R444C mutation increases NOD2s stability, but NOD2l^R471C shows the same degradation rate as WT NOD2l. Because S-palmitoylation attenuates the SQSTM1-mediated autophagic degradation of NOD2, we next checked whether the NOD2s^R444C variant maintains its stability through enhancing its S-palmitoylation level. Indeed, the R444C mutation increases the S-palmitoylation of NOD2s, whereas there is no obvious change of S-palmitoylation between the NOD2l^R471C variant and WT NOD2l. Moreover, the interaction of ZDHHC5 and NOD2s^R444C is much stronger than that of WT NOD2s, whereas the interaction of NOD2s^R444C and SQSTM1 is much weaker when compared to that of WT NOD2s (Figure 1). However, NOD2l^R471C and WT NOD2l exhibit the same binding abilities with both ZDHHC5 and SQSTM1. Knockdown of ZDHHC5 in THP-1-DMs markedly decreases the S-palmitoylation of both WT NOD2s and the NOD2s^R444C variant, increases their interaction with SQSTM1 and eliminates the excessive inflammation activation caused by the NOD2s^R444C variant. Conversely, knockdown of SQSTM1 increases the transcription and secretion of IL1B/IL-1β, IL6 and TNF/TNF-α in WT NOD2s and NOD2s^R444C THP-1-DMs to the same level.

Figure 1. A proposed working model to illustrate how the NOD2s^R444C variant contributes to excessive inflammation. Upon muramyl dipeptide (MDP) stimulation, NOD2 undergoes self-oligomerization and recruits RIPK2 to trigger NFKB activation and proinflammatory cytokine production. Normally, palmitoylation of NOD2s by ZDHHC5 promotes the membrane recruitment of NOD2 and restricts the SQSTM1-mediated autophagic degradation of NOD2. In patients with the NOD2s^R444C variant, palmitoylation of NOD2s is largely increased through its enhanced protein interaction with ZDHHC5. Increased NOD2 palmitoylation might reduce the threshold of NOD2 activation, leading to excessive NOD2-mediated inflammation and the risk of developing inflammatory diseases.

In conclusion, our study indicates that S-palmitoylation of NOD2 attenuates its binding ability to the cargo receptor SQSTM1, hence inhibiting the autophagic degradation of NOD2. We also demonstrate that the gain-of-function NOD2s^R444C variant possesses a higher level of S-palmitoylation by stronger binding ability to ZDHHC5, thus restricting its SQSTM1-mediated autophagic degradation. These findings may promote the understanding of the regulatory role of S-palmitoylation in controlling NOD2 stability through the crosstalk with autophagy, and provide insights into the association between dysfunctional S-palmitoylation and the occurrence of inflammatory diseases.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (92042303, 31870862, 32000544, 81873869, 31970700 and 32170876), Guangdong Basic and Applied Basic Research Foundation (2020B1515120090), the Natural Science Foundation of Guangdong Province (No. 2020A1515110193) and Guangzhou Women and Children’s Medical Centre project grant (0160001).