The pyrin domain (PYD) is found in the N-terminus of inflammasome sensors and the adaptor protein ASC, enables protein-protein interaction between the inflammasome sensor and the adaptor protein, a critical step in inflammasome activation. So far, functionally characterized phosphorylation sites in this domain for NLRP3 have generally been demonstrated to have an inhibitory effect on inflammasome activation.

Ser5 is located in helix 1 of the human NLRP3 PYD at a protein-protein interaction surface with ASC, inhibits the NLRP3 inflammasome in macrophages. Furthermore, the authors have demonstrated that the two negative charges introduced by phospho-serine were able to disrupt the PYD-PYD interaction between NLRP3 and ASC and thereby prevents the activation of this inflammasome (Stutz et al., 2017). The upstream kinase catalyzing Ser5 phosphorylation is AKT (protein kinase B), and pharmacological inhibition of this enzyme resulted in increased NLRP3 inflammasome activity in vitro and in vivo (Zhao et al., 2020). Interestingly, the same researchers also discovered a crosstalk between protein phosphorylation and ubiquitination. Ser5 phosphorylation stabilizes NLRP3 by reducing its ubiquitination on Lys496, which inhibits its proteasome-mediated degradation by the E3-ligase Trim31. Dephosphorylation of Ser5 by Protein Phosphatase 2 A (PP2A) is essential for NLRP3 inflammasome activation as demonstrated by a drastically reduced NLRP3 activation when PP2A was inhibited or down-regulated (Stutz et al., 2017). An additional layer of fine-tuning NLRP3 inflammasome activation is mediated by Bruton’s tyrosine kinase (BTK) (Mao et al., 2020). BTK is bound to NLRP3 during the priming phase of inflammasome activation and inhibits PP2A-mediated dephosphorylation of Ser5 in the PYD domain of NLRP3, consequently BTK inhibitors have been shown to suppress NLRP3 inflammasome activation and IL-1β secretion (Ito et al., 2015).

Another phosphorylation site in the PYD domain of NLRP3 involved in controlling its protein-protein interaction with ASC is Tyrosine 32. While the upstream kinase is currently unknown, the lipid and protein phosphatase PTEN can directly dephosphorylate Tyr32 (Huang et al., 2020), and in turn facilitated NLRP3-ASC interaction, inflammasome assembly and activation.

The interaction between a NLR and ASC by protein phosphorylation can not only be controlled through phosphorylation and dephosphorylation of the inflammasome sensor, but also at the level of the inflammasome adaptor protein. There are two putative phosphorylation sites located in the PYD domain of ASC: Ser46 and Tyr60. The latter was identified and functionally characterized by Mambwe and colleagues by using the pharmacological protein tyrosine phosphatase (PTPase) inhibitor phenylarsine oxide (PAO), which prevented AIM2 and NLRP3 inflammasome activation in human and mouse macrophages. Moreover, site-directed mutagenesis of ASC tyrosine residue Tyr60 to phenylalanine, a non-phosphorylatable tyrosine mimic led to a significant reduction in inflammasome activity measured by IL-1β release (Mambwe et al., 2019), highlighting the role of Tyr60 phosphorylation for inflammasome assembly and function. However, it remains unclear if Tyr60 phosphorylation has a direct effect on AIM2/NLRP3 protein-protein interaction with ASC.

Subcellular location of ASC is dependent on its phosphorylation status. The protein kinase IKKα phosphorylates murine ASC in the PYD domain at serine 16 (Thr16 in human) and at Ser193 within the CARD domain (human Ser195) (Martin et al., 2014). Phosphorylation at these two sites is important for the nuclear localization of an ASC/IKKα protein complex. Signal 1 of the inflammasome activation leads to PP2A-dependent dephosphorylation of ASC and subsequent release of ASC into the cytosol for inflammasome assembly. Loss of IKKα kinase activity results in markedly enhanced NLRP3, AIM2 and NLRC4 inflammasome activation, with aberrant caspase-1 activation and IL-1β secretion (Martin et al., 2014).

Given this evidence that phosphorylation events in this domain negatively impact the interaction between the inflammasome sensor and ASC, one can speculate that phosphorylation events for other NLRPs might have negative effects on their inflammasome activation. Table 1 summarizes the putative phosphorylation site in this domain beyond the NLRP3 and ASC. It would be interesting to experimentally test role of these putative phosphorylation sites in other NLRs on their respective inflammasome activation. Due to its different molecular mechanism of activation, an exception from this hypothesis could be NLRP1 (Mitchell et al., 2019; Sandstrom et al., 2019). NLRP1 is kept in an autoinhibited confirmation and activation requires autoproteolysis, a process termed “functional degradation” (Sandstrom et al., 2019). It would be interesting to see, if the putative phosphorylation site at Thr41 in the PYD or for that matter other phosphorylated amino acid residues could lead to an inflammasome activation. The upstream kinase facilitating this phosphorylation is currently unknown, but it could provide an alternative molecular mechanism to activate the NLRP1 inflammasome.

NLR proteins contain a central NACHT domain, which possess nucleoside-triphosphatase (NTPase) activity. In a recent review, Sandall and colleagues highlight the importance of ATP binding and hydrolysis for inflammasome activation (Sandall et al., 2020).

The NLRP3 ATPase activity is sensitive to phosphorylation at Ser295 by protein kinase PKA, as demonstrated by Mortimer et al. (2016). Interestingly, phosphorylation at Ser295 does not only hinders the ATPase activity, but also induces protein ubiquitylation, which inhibited the inflammasome independently of protein degradation (Guo et al., 2016). However, Zhang et al. (2017) report an opposing effect of NLRP3 Ser295 phosphorylation when phosphorylated by the protein kinase PKD. Under these experimental conditions, the phosphorylation of Ser295 is important for NLRP3 inflammasome activation. Further investigation is needed to fully understand the opposing effects of NLRP3 Ser295 phosphorylation by PKA and PKD. Spatial and temporal effects of protein phosphorylation could offer a possible explanation for the opposite effects of Ser295 phosphorylation. PKD mediated phosphorylation of Ser295 occurs when NLRP3 is localized at mitochondria-associated membranes (MAM) and is required for the release of NLRP3, the subsequent cytosolic localization, ASC recruitment and inflammasome complex formation (Zhang et al., 2017). PKA-mediated phosphorylation occurs in the cytosol and prevents conformational changes crucial for inflammasome complex formation (Mortimer et al., 2016).

Despite the identification of other putative phosphorylation sites in various inflammasome sensor proteins (Table 1), little is known what these events do to the activation status of the respective inflammasome. Given the inhibitory effect of Ser295 phosphorylation for NLRP3 inflammasome activity by interfering with ATPase-dependent structural changes, one can speculate that phosphorylation in the proximity of ATP binding leads to inhibition of inflammasome activity. A distinct motif in the NACHT domain of NLR proteins is the Walker-A “P-loop” motif with the consensus sequence of GxxGxGK [T/S]. The functionally important Walker-A motif binds the phosphate groups of ATP and catalyzes phosphoryl transfer (Kawabori et al., 1978). Phosphorylation of the threonine or serine residue within the Walker-A motif has been detected for two NLR proteins: NLRP3 (Thr233) and NLRP12 (Ser224) (Hornbeck et al., 2012; Hornbeck et al., 2019). Upstream kinases responsible for these two phosphorylation events are yet to be identified but could provide an alternative to modulate NLRP3 or NLRP12 activities.

NLRs (apart from NLRP10) contain leucine-rich repeat (LRR) domain, usually located towards the C-terminus. The role of this domain includes auto-inhibition and ligand sensing. Several phosphorylation sites were identified in various LRR domains and can be found summarized in (Table 1).

The protein-protein interaction between NLRP3 and NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins) has been identified to be important for inflammasome assembly and activation (He et al., 2016; Schmid-Burgk et al., 2016; Shi et al., 2016; Sharif et al., 2019). NLRP3-activating stimuli promoted the NLRP3-NEK7 interaction in a process that is dependent on potassium efflux (He et al., 2016), but the kinase activity of NEK7 is dispensable and just the protein-protein interaction between NLRP3 and the catalytic domain of NEK7 is sufficient for the activation of the NLRP3 inflammasome. NEK7 plays a scaffolding role in the activation of the NLRP3 inflammasome complex by bridging adjacent NLRP3 subunits with bipartite interactions (Sharif et al., 2019). Recently, the phosphorylation of Ser803 (situated in the third LRR domain) in NLRP3 has been demonstrated to inhibit NEK7-NLRP3 interaction (Niu et al., 2021). Phosphomimic substitutions of Ser803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. Full-length, membrane-associated NLRP3 possess a double-ring cage structure, which is facilitated by LRR-LRR and PYD-PYD interactions (Andreeva et al., 2021). Ser803 is located close to positively charged residues from the adjacent LRR (e.g., R1009), and the phosphorylation likely stabilizes the NLRP3 cage structure. The kinase facilitating this PTM has been identified as CSNK1A1, while the phosphatase that could counteract this phosphorylation remains elusive.

Phosphorylation at Ser891/895 in mouse macrophages (corresponding to Ser894/898 in human) enables the binding of the E3-ligase TrCP1 in (Ubiquitination chapter), which in turns catalyzes the proteasomal degradation of NLRP3 via ubiquitination at Lys380 (Wang D. et al., 2021). While the upstream kinase remains to be identified, the NLRP3 can be stabilized by the Hippo pathway protein YAP, which blocks the TrCP1/NLRP3 interaction.

NLRP3 has been shown to be phosphorylated at Tyr861 by an unknown kinase, allowing its interaction with the autophagy machinery Sequestosome 1 (SQSTM1) to target to the phagophore, and thereby control inflammasome activation (Spalinger et al., 2016). Protein tyrosine phosphatase non-receptor 22 (PTPN22) counteracts this mechanism in an ASC-dependent manner to dephosphorylates pTyr861, allowing NLRP3 inflammasome assembly (Spalinger et al., 2017).

The C-terminal HIN-200 domain binds to dsDNA through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone (Jin et al., 2012). AIM2 is kept in an autoinhibited confirmation through PYD and HIN-200 domain protein-protein interaction, which is liberated by dsDNA binding (Jin et al., 2012). The PYD domain is then released to engage with the downstream inflammasome adaptor protein ASC through homotypic PYD-PYD interactions.

Three phosphorylation sites in the HIN-200 domain of AIM2 have been identified (Table 1): Thr254, Thr257, and Thr262 (Hornbeck et al., 2012; Hornbeck et al., 2019). One could speculate that the negative charges introduced by phosphorylation could interfere with the dsDNA binding to the HIN-200 domain of AIM2. If this hypothesis is true, the upstream kinase responsible for these phosphorylation events, once identified, could provide an attractive therapeutic target for diseases of aberrant AIM2 activation from self-dsDNA, a key driver of inflammatory and autoimmune diseases (Man et al., 2016).

The CARD domain, through protein-protein interactions, enables the formation of inflammasome complexes and unsurprisingly, protein phosphorylation has been identified as a mechanism controlling this process. Two phosphorylated tyrosine residues (Tyr137 and Tyr146) in ASC have been functionally characterized. When Mambwe and colleagues established that the pharmacological PTPase inhibitor PAO had the profound inhibitory effect on the AIM2 and NLRP3 inflammasomes respectively, in human and murine macrophages (Mambwe et al., 2019). They identified the CARD domain-located tyrosine Tyr137 to be important for AIM2 and NLRP3 inflammasomes assembly and function. In their study, the authors built on previous studies, which identified Tyr146 phosphorylation as crucial signal controlling ASC function (Hara et al., 2013; Chung et al., 2016; Darweesh et al., 2019; Gavrilin et al., 2021). Several kinases have been associated with directly or indirectly controlling the phosphorylation status: Syk, Jnk (Hara et al., 2013), Pyk2 (Chung et al., 2016), protein kinase R (PKR) (Darweesh et al., 2019) and cAbl (Gavrilin et al., 2021). This demonstrates that ASC regulation through tyrosine phosphorylation is a very multifaceted system, which might involve many cells type stimulation- and kinase redundancy-dependent layers of complexity.

Very little is known how Caspase activity is regulated by CARD domain phosphorylation despite the identification of several putative phosphorylation sites (Table 1). One could speculate that Caspase-1 or Caspase-4 protein-protein interaction with ASC could be affected by phosphorylation in the respective CARD domains, however this needs to be experimentally tested.

Caspase-1 and Caspase-4 are directly inflammasome-associated proteases in human cells, whereas Caspase-11, is the Caspase-4 orthologue in mice. All three caspases have in common that the proteolytic cleavage of their respective substrates is catalyzed by their peptidase domains. Several phosphorylation sites have been found in this domain (Table 1), but so far only one has been functionally characterized.

Caspase-1 is phosphorylated by the protein kinase p21-activated kinase 1 (PAK1) at Ser376 upon Helicobacter pylori LPS treatment of THP-1 cells (Basak et al., 2005). The LPS-induced Caspase-1 activation was abrogated in cells transfected with Caspase-1 (Ser376Ala) mutant. This demonstrates that this PTM is important for Caspase-1 activation and given its conservation as Thr352 in Caspase-4, it would be interesting to study if Caspase-4 activity upon inflammasome stimulation could be regulated via a similar phosphorylation-dependent mechanism.

Many phosphorylation sites are situated in between the distinct protein domains of NLRs (see Table 1). The NLRP3 inflammasome has been studied extensively and some of the interdomain phosphorylation sites have been functionally characterized.

BTK kinase activity inhibits PP2A function and thereby lead to inhibition of NLRP3 de-phosphorylation of Ser5 of NLRP3 in PYD domain preventing its oligomerization (Mao et al., 2020). Recently, four tyrosine residues have been identified by Bittner et al. (2021) as direct BTK phosphorylation upon in vitro and in vivo inflammasome activation. These sites are Tyr136, Tyr140, Tyr143, and Tyr164, that are situated in the polybasic linker between PYD and NACHT domains, which is critical for interaction with negatively charged phosphatidylinositol phosphates (PIPs) at organelles, such as the Golgi. Ablation of BTK kinase activity or mutation of the respective tyrosine residues decreased the formation of cytosolic NLRP3 oligomers, complexes with ASC and IL-1β release (Bittner et al., 2021). This study confirms previous studies demonstrating a physical interaction of BTK with NLRP3 and ASC as well as BTK inhibition by kinase inhibitors or in knock-out mice blocks NLRP3 inflammasome activation (Ito et al., 2015; Liu X. et al., 2017). The BTK-mediated tyrosine phosphorylation at the above-mentioned residues is introducing negative charges, which could weaken the interaction between NLRP3 and phospholipids at the Golgi and the relevance of such a disruption has been proven previously (Chen and Chen, 2018). The contradicting effects of BTK kinase-mediated phosphorylation events (Ser5 vs. Tyr136, Tyr140, Tyr143, and Tyr164) for NLRP3 inflammasome activity highlight again the temporal and spatial control of the NLRP3 inflammasome through protein phosphorylation/dephosphorylation.

Phosphorylation of mouse NLRP3 at Ser198 (mouse Ser194) is catalyzed by c-Jun N-terminal kinase 1 (JNK1) in response to chronic LPS treatment or other TLR ligands (Song et al., 2017), in a transcription-independent manner. This phosphorylation even is crucial for inflammasome activation by promoting NLRP3 self-association. In addition, phosphorylation of Ser198 enables inflammasome activation by promoting NLRP3 deubiquitylation by BRCC3, a component of the cytosolic BRISC complex (Py et al., 2013). This crosstalk between phosphorylation and ubiquitylation was further proven by Ren and colleagues, who identified ABRO1, a subunit of the BRISC deubiquitylate complex, to be necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1 (Ren et al., 2019).

As discussed for Ser803 phosphorylation (LRR domain), the interaction between NEK7 and NLRP3 is crucial for its inflammasome activation, the NLRP3-NEK7 binding induces NLRP3 deubiquitylation by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3 (He et al., 2016; Niu et al., 2021). While Ser803 phosphorylation is catalyzed by the protein kinase CSNK1A1, recently Dufies and colleagues identified Thr659 as an important site for NLRP3 and NEK7 protein-protein interaction (Dufies et al., 2021). In an Escherichia coli infection model system, PAK1 phosphorylates Thr659, which in turn facilitates NLRP3-NEK7 interactions, inflammasome formation and IL-1β cytokine maturation. PAK1 appears to be key regulator in the anti-bacterial host-responses by phosphorylating Thr659 on NLRP3 and Ser376 of Caspase-1.

A key inflammasome sensors responsible for the immune response to bacterial infections is the NLRC4 inflammasome (Amer et al., 2006; Franchi et al., 2006; Miao et al., 2006). NLRC4 is phosphorylated at Ser533 phosphorylation (Qu et al., 2012), and mutation of serine 533 to alanine (Ser533Ala) did not activate Caspase-1 and pyroptosis in response to Salmonella typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. The phosphorylation site Ser533 is conserved among the NLRC4 proteins from different species, which suggests that it is potentially important for the structure or function of NLRC4. A screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate kinase upstream of Ser533 phosphorylation (Hornbeck et al., 2019) and, PKCδ phosphorylated NLRC4 S533 in vitro, whilst immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro (Hornbeck et al., 2019). However, more recent studies discovered a more complex NLRC4 biology in vivo. NLRC4 Ser533Ala knock-in mice were generated by two groups independently and used in S. Tryphimurium in vivo infection models and in ex vivo mechanistic studies using bone marrow derived macrophages (BMDMs). Both studies demonstrated that the phosphorylation at Ser533 had no to very subtle effect for NLRC4 inflammasome activation and the response to S. Tryphimurium infection. While Qu and colleagues observed a delayed, but not completely blocked response and a potential crosstalk with the NLRP3 inflammasome (Qu et al., 2012), however, by using a Nlrc4-deficient mouse line, mice with Ser533Asp phosphomimic or Ser533Ala non-phosphorylatable NLRC4, Tenthorey and colleagues did not observe a requirement for Ser533 phosphorylation in NLRC4 inflammasome function nor any involvement of NLRP3 inflammasome (Tenthorey et al., 2020). A possible explanation for the divergent results could be different experimental conditions, such as animal housing, different cell culture conditions for BMDMs.

A second kinase with the ability to phosphorylate NLRC4 at Ser533 is LRRK2 (Liu W. et al., 2017). LRRK2 has been found to form a complex with NLRC4 in the macrophages, and the formation of the LRRK2-NLRC4 complex led to the phosphorylation of NLRC4 at Ser533 (Liu W. et al., 2017). Importantly, the kinase activity of LRRK2 is required for optimal NLRC4 inflammasome activation, whereas LRRK2 deficiency reduced Caspase-1 activation and IL-1β secretion in response to NLRC4 inflammasome activators in macrophages (Liu W. et al., 2017). Future studies are required to investigate the temporal and spatial control of NLRC4 Ser533 phosphorylation and redundancies/synergies between the two kinases PKCδ and LRRK2 in vitro and in vivo models.

Taken together, phosphorylation of inflammasome components occur at various stages of activation or inhibition of these multimeric protein complexes. From controlling the priming step (e.g., NLRP3 Ser198), to inflammasome complex formation (e.g., NLRP3 Ser803), the subcellular localization (e.g., NLRP3 Tyr136, Tyr140, Tyr143 and Tyr164) and to protein stability (e.g., NLRP3 Ser5). The validated residues that are phosphorylated in inflammasome components so far are represented in (Figures 2A,B). Future studies are needed to provide a deeper understand of:

1) upstream kinases and corresponding protein phosphatases

In 2011 Liu et al. (2016) described tripartite motif 11 (TRIM11) as a key negative regulator of the AIM2 inflammasome. TRIM11 is an E3-ligase that induces its auto-polyubiquitination at Lys458 after binding to AIM2 and promotes AIM2 degradation. More recently, USP21 has been identified as a DUB that deubiquitylates AIM2 and induce its stability (Hong et al., 2021). The authors also demonstrated that USP21 is essential in AIM2 inflammasome assembly and absence of USP21 does not affect binding of AIM2 to DNA but impairs AIM2-ASC assembly (Hong et al., 2021).

Moreover, Lys39 and Lys235 were identified as a new ubiquitylation sites in a mass spectrometry screening (PhosphositePlus). The identification of E3-ligase(s), which mediates ubiquitylation of Lys39 and Lys235 could shed light on the AIM2 inflammasome regulation (Hornbeck et al., 2012; Hornbeck et al., 2019).

Finally, the E3-ligase HUWE1 has been described as an interactor of AIM2. The BH3 domain of HUWE1 was important for its interaction with the HIN domain of AIM2. This interaction leads to the Lys27-linked polyubiquitination of AIM2 leading to the assembly of this inflammasome (Guo et al., 2020).

ASC is post-translationally modified by phosphorylation and ubiquitylation, although for the latter fewer information are available. Guan et al. (2015) described that TNFR-associated factor 3 (TRAF3) was the E3-ligase responsible for ASC ubiquitylation at Lys174, and they demonstrated that the process is essential in ASC speck formation (Guan et al., 2015). Activation of the AIM2 inflammasome with poly (dA:dT) results in Lys63-linked polyubiquitination of ASC in macrophages mediated by p62 (Shi et al., 2012). Whereas the linear ubiquitylation of ASC can be mediated by LUBAC ubiquitin complex to mediates NLRP3/ASC assembly (Rodgers et al., 2014). More recently, Zhang and colleagues discovered the modification of ASC at Lys55 by Peli1 (Zhang et al., 2021). In this case the ubiquitin chain formed is Lys-63 facilitates ASC/NLRP3 interaction and ASC oligomerization and inflammasome activation. Moreover, TRAF6 have been linked to ASC ubiquitylation (Chiu et al., 2016).

On the other hand, USP50 has been described as a DUB able to deubiquitylate Lys-63 ubiquitin chains from ASC (Lee et al., 2017). Apart of these examples, not much more is found in the literature about ASC ubiquitylation, although some PTM sites are described on PhosphositePlus database because of the massive screening studies (all summarized in Table 2).

Despite its tremendous importance in the inflammasome complex no ubiquitin PTMs have been demonstrated on Caspase-1 protein. On the PhosphositePlus database several lysines in the Caspase-1 sequence have been postulated to be ubiquitylated, including Lys37, Lys44, Lys53, Lys134, Lys148, Lys158, Lys204, Lys268, Lys274, Lys278, Lys319, Lys320, and Lys325 (see Table 2). All of them are a result of massive mass spectrometry analysis including the more recent one carried out by Akimov et al. (2018), but so far, no biochemical experiments have been published to prove the ubiquitylation of these sites or potential functional effects. Further investigations are needed to demonstrate the regulation of Caspase-1 by ubiquitin PTMs. On the other hand, several publications indicate the potential role of multiple DUBs in Caspase-1 activity regulation (Lopez-Castejon et al., 2013), but no direct ubiquitylation of Caspase-1 has been proven to date.

Reported as an inflammatory caspase, (Martinon and Tschopp, 2007), the only reported ubiquitin modifications or residues of Caspase-4 can be found on PhosphositePlus. Yet again, validation of Lys87, Lys107, and Lys129 is required to understand the significance of this discovery (Table 2).

Interleukin-1α and -β are key molecules regulated by inflammasome effector proteins. Several years ago, IL-1α and -β were reported to be ubiquitylated and degraded via proteasome (Ainscough et al., 2014). More recently, a precursor of IL-1β has been demonstrated to be ubiquitylated at Lys133 consisting of mixed Ly11, 48 and 63 poly-Ub chains, promoting its proteasomal degradation. Consequently, Lys133Arg mutants have increased level of IL-1b percussor, the mutation abrogated IL-1β processing and secretion (Vijayaraj et al., 2021).

Inflammatory caspases cleave the pore-forming protein called gasdermin D (GSDMD) to active GSDMD amino (N)-terminus to bind to the cell membrane to induce a highly inflammatory form of programmed cell death called pyroptosis (Kayagaki et al., 2015). PhosphositePlus has reported several potential lysine residues ubiquitylated in GSDMD (Wagner et al., 2011; Udeshi et al., 2013) (Table 2). Two of these sites Lys203 and Lys204 has been reported to be ubiquitylated by SYVN1, an E3-ligase that mediates K27 polyubiquitylation of GSDMD and promotes its activation (Shi et al., 2022).

Other authors have recently described that Shigella ubiquitin ligase IpaH7.8 is able to ubiquitylate GSDMD and promotes its degradation via proteasome in humans but not in rodents (Luchetti et al., 2021).

Another paper also refers to the ubiquitylation of GSDMD mediated by an environmental toxicant, sodium arsenite (NaAsO2). Their data showed reduced GSDMD Lys48 and Lys63 polyubiquitylation inhibiting its degradation via proteasome to promote pyroptosis. These findings could have an impact on prevention and treatment of type 2 diabetes (Zhu et al., 2022).

The validated residues that are ubiquitylated in inflammasome components so far are represented in (Figures 2A,B). Future studies are needed to provide a deeper understanding of:

1) upstream E3-ligases and corresponding DUBs

Lysine acetylation is a PTM mediated by lysine acetyltransferases (KATs). This is a reversible process catalysed by lysine deacetylases (KDACs) (Gil et al., 2017) (Figure 1). Inflammasome-induced ASC and NLRP3 colocalization by microtubule re-arrangement via acetylation of α-tubulin due to sirtuin 2 (SIRT2) deacetylase inactivation was reported (Misawa et al., 2013), but acetylation of inflammasome components were not assessed in this paper. The role of microtubules in NLRP3 inflammasome assembly has been challenged, although activation of pyrin inflammasome appears critically dependent on this process (Park et al., 2016; Van Gorp et al., 2016). A recent study demonstrated that acetylation of NLRP3 at Lys21, Lys22, and Lys24 (in mouse, Lys23, Lys24, and Lys26 in human NLRP3) promotes activation of the NLRP3 inflammasome, which is controlled by the NAD-dependent protein deacetylase sirtuin-2 (SIRT2). LPS and ATP were shown to promote the acetylation level on NLRP3, and in turn increase the production of IL-1β, which was abrogated by Lys21/22/24Arg mutant. The role of SIRT2-NLRP3 axis has also been implicated in the aging-associated chronic inflammation and insulin resistance (He et al., 2020). SIRT3 deficiency leads to significantly impaired NLRC4 inflammasome activation, ASC oligomerization, and pyroptosis both in vitro and in vivo. SIRT3 interacts with and deacetylates NLRC4 at Lys71 or Lys272 to promote its activation (Guan et al., 2021). However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome.

ADP-ribosylation is a reversible process that mediate the addition of one or more ADP-ribose to a protein (Cohen and Chang, 2018) (Figure 1). Many pathogen-derived molecules have been reported to assemble the NLRP3 inflammasome complex. It was shown that a Mycoplasma pneumonia-derived toxin and designated community-acquired respiratory distress syndrome (CARDS) toxin, which interact with and activates NLRP3 inflammasome, dependent on its ADP-ribosyltransferase activity. Upon stimulation by these toxins, NLRP3 was found to be ADP-ribosylated, suggesting that ADP-ribosylation of NLRP3 is a functional PTM for its positive regulation, but further investigation is required to understand the molecular basis of inflammasome activation (Bose et al., 2014).

Nitrosylation is a chemical process that add a NO (nitric oxide) group to a thiol (S-H) group of a protein (Mannick and Schonhoff, 2004) (Figure 1). Endogenous nitrous oxide (NO) has been shown to regulate NLRP3 inflammasome activation by either IFN-β pre-treatment or chronic LPS stimulation. Moreover, S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, markedly inhibited NLRP3 inflammasome activation, S-nitrosylation of NLRP3 was detected in macrophages treated with SNAP, and this modification may account for the NO mediated mechanism controlling inflammasome activation. Further investigation is required to understand the mechanism by which nitrosylation regulate NLRP3 inflammasome activation is not understood (Hernandez-Cuellar et al., 2012).

Prenylation is a post-translational modification involving transfer of farnesyl or geranylgeranyl lipid groups on characteristic C-terminal CAAX motifs of proteins (Figure 1). Prenylation of small GTPases of Rab, Ras and Rho families is important for their cell membrane anchorage and for a variety of critical cell processes, including membrane trafficking, integrin signaling and apoptosis (Wang and Casey, 2016). A link between inflammasome activation and defective protein prenylation leading to cytosolic accumulation of unprenylated Rab and other small GTPases was identified as the underlying cause of auto-inflammatory disease resulting from recessive hippomorphic mutation in the mevalonate kinase gene. Defective activity of mevalonate kinase, a key component of mevalonate-cholesterol biosynthesis pathway, leading to decreased production of isoprenoid lipids and lack of protein prenylation (Mandey et al., 2006; Munoz et al., 2017). Studies from two different laboratories suggested that loss of K-Ras (Akula et al., 2016) or Rho GTPase (Park et al., 2016) prenylation led to pyrin inflammasome activation in murine macrophages. In contrast, a recent study, using human monocytic cell line (THP-1) or primary PBMC, has implicated NLRP3 inflammasome as the key driver of IL-1β release in cells with defective protein prenylation (Skinner et al., 2019). The discrepancies between these studies may be explained by different cell types used in these studies and emphasizes the importance of using relevant cell type for dissecting and elucidating signaling networks involved in the pathogenesis of human diseases.

Citrullination of proteins is a process in which arginine residue of a protein is converted to citrulline by deamination, which is catalyzed by peptidyl arginine deiminases (PADs) (Figure 1). This process coverts positively charged arginine to citrulline, which leads to increase in the hydrophobicity of proteins, changes in protein folding, and thereby affecting their structure and function. These citrullinated proteins are thought to act as neoantigens for the immune system to mount an immune response to produce antibodies against them causing auto-immune disorders [reviewed in (Ciesielski et al., 2022)]. However, the cellular and molecular basis of protein citrullination in autoimmune disease pathogenesis is not fully understood. The PADs have been suggested to play a role in NETosis, a process implicated in several autoimmune diseases including RA and lupus. Recently, PAD4 was reported to promote NETosis by regulating NLRP3 inflammasome activation through post-transcriptional regulation of ASC and NLRP3 levels (Munzer et al., 2021). PAD2 and PAD4 have also been shown to play a role in NLRP3 inflammasome activation in macrophages (Mishra et al., 2019). Future studies are warranted to elucidate the link between protein citrullination and NLRP3 inflammasome activation.

Very little is known how gain- or loss-of function mutations affect PTMs of inflammasome components. A few examples how mutations identified in CAPS patients’ crosstalk with PTMs of NLRP3 have been published:

PKA phosphorylation of NLRP3 Ser295 inhibited the NLRP3 ATPase activity, which is required for assembly of NLRP3-ASC complexes, resulting in NLRP3 inflammasome inhibition (Mortimer et al., 2016). A cluster of mutations in NLRP3-encoding residues in proximity to Ser295 have been linked to CAPS. NLRP3-Ser295Ala phenocopied the human CAPS mutants, most notably Asp305Gly (Mortimer et al., 2016). For example, the Ser295Ala and Asp305Gly mutants displayed ATPase activity after PKA incubation in contrast to with wild-type NLRP3. It would be interesting to see, if Ser295 phosphorylation is abolished in patients carrying one or more mutations from this cluster.

The Tyr861Cys mutation in NLRP3 has been reported in patients with CINCA. Phosphorylation of this site has been identified as negative regulator of NLRP3 (Spalinger et al., 2016). Mechanistic studies with the Tyr861Phe mutation of NLRP3, which like Tyr861Cys, can no longer be phosphorylated displayed an elevated level of inflammasome activation compared with wild-type NLRP3 (Spalinger et al., 2016).

The INFEVERS (INternet periodic FEVERS) database is a registry of hereditary auto-inflammatory disorders mutations (Sarrauste de Menthiere et al., 2003). Mutations found for NLRP1, NLRP3, NLRP7, NLRP12, and NLRC4 have been collected. As of April 2022, 256 sequence variants for NLRP3 have been registered and unsurprisingly, many are in close proximity of PTMs. A PTM-focused proteomics analysis of macrophages from CAPS patients could provide valuable mechanistic insights how sequence variations in NLRP3 crosstalk with activating or inhibiting PTMs.

While end products of inflammasomes such as circulating ASC specks and mediators such as IL-1β and IL-18 are strong signals of inflammasome activity, they don’t provide any mechanistic insights where and which of the many NLRs are activated. Furthermore, antibody-based methods used to detect IL-1β and IL-18 might not be able to distinguish between the cleaved and fully active proteins and their uncleaved, less active precursors. Therefore, inflammasome activity might not correlate well with the expression levels of these two mediators in vitro and in vivo.

Post-translational modifications can regulate all aspects of inflammasome biology, from activation and inhibition, subcellular localization, their interactome and function. Monitoring PTM status could allow an assessment of inflammasomes activation status and of the upstream catalyzing enzymes. In the future, a well validated PTM has the potential to enable a precision medicine approach to identify a specific patient population that would benefit from selective inhibition of one respective inflammasome, if a pan inflammasome inhibition is not desirable. Likewise, a well characterized PTM might be an option for a target engagement biomarker for an inflammasome inhibitor.

PTMs have the potential to be used to control inflammasome activation in a specific manner. For instance, modulation of protein ubiquitylation could modulate protein stability, whereas some phosphorylation sites have been implicated in the subcellular localization (Guo et al., 2020; Bittner et al., 2021).

An intriguing, yet unproven application of well characterized inflammasome PTMs could be as diagnostic tools. Because CAPS is an extremely rare disease and patients show a heterogeneous multi-system clinical presentation, a significant delay exists between symptom onset and definitive diagnosis (Welzel and Kuemmerle-Deschner, 2021). Screening for and identification of a PTM associated with constitutively NLRP3 inflammasome activation in CAPS patients might in the future enable earlier diagnosis of their disease and thereby enable earlier therapeutic intervention to avoid morbidity and reduce mortality.

All PTMs are catalyzed by upstream enzymes, which could be drug targets by their own rights due to involvement in diseases not driven by excessive inflammasome activity. For some of these enzymes, a direct measurement of their activity, either in pre-clinical drug development or as target engagement or patient identification biomarker might prove to be too challenging. If a well characterized inflammasome PTM is indeed robustly predictive of upstream enzyme activity, this PTM could be tested as a more accessible, surrogate biomarker for the upstream drug target.