In this paper we report the identification of the E3 ubiquitin ligase TRIM27 as a new interaction partner for NOD2. Mapping studies indicated that NOD2 binds via its NBD domain to the TRIM27 B30.2/PRY-SPRY domain. The B30.2 domain is composed of the ~61 aa PRY and the ~140 aa SPRY domain and is most commonly found at the C-terminus of TRIM proteins 
. The SPRY domain is evolutionarily more ancient as it is found in animals, plants and fungi, whereas the B30.2 domain is only found in vertebrates with an adaptive immune system 
. In humans, B30.2/PRY-SPRY domains are only present at the C-terminus in TRIM proteins and in butyrophilin-related transmembrane glycoproteins (BTNs), which are receptors of the immunoglobulin superfamily 
. Of note, the TRIM27 binding site for NOD2 correlates with the findings for other TRIM proteins: TRIM21 binds its interaction partners IRF3 and IRF8 via the B30.2/PRY-SPRY domain 
and TRIM25 binds RIG-I via the C-terminal SPRY domain 
. TRIM20 (also known as pyrin), regulates caspase-1 activation and IL-1β production by interacting with caspase-1 via its PRY-SPRY and with ASC via its PYD domain, respectively 
. Mutations in the human TRIM20 PRY-SPRY domain are associated with inherited Familial Mediterranean Fever (FMF). TRIM20 knock-in mice expressing a TRIM20 with the mutant human PRY-SPRY domain reflect the inflammatory phenotype found in patients by forming spontaneous ASC-dependent, NLRP3-independent inflammasomes 
. This clearly links the PRY-SPRY domain in TRIM20 to immune functions. Another intriguing example underscoring the involvement of PRY-SPRY domains in NLR regulation is present in zebrafish (Danio rerio
). One class of NLR proteins in D. rerio
comprises molecules harboring a PRY-SPRY domain fused to the canonical NLR sequence 
. Of note, PRY-SPRY domains from D. rerio
NLRs are closely related to zebrafish TRIMs, indicating domain shuffling during evolution 
. This suggests that TRIM and NLR proteins have been tied together in evolution to function in innate immune responses.
TRIM proteins are involved in the regulation of PRR- and IFN-signaling pathways, typically by acting as ubiquitin ligases. TRIM-mediated ubiquitination events have been documented to both enhance immune response as shown i.e. for TRIM8, TRIM21, TRIM23, TRIM25 and TRIM56 
or inhibit them as shown for TRIM21 and TRIM30-α 
. We show here that also TRIM27 is involved in regulating PRR-signaling by negatively regulating NOD2. We observed that NOD2 is ubiquitinated by K48-linked ubiquitin chains and that this ubiquitination is enhanced by TRIM27 overexpression, whereas overexpression of an E3 ligase-deficient mutant of TRIM27 (TRIM27 E3) reduces NOD2 ubiquitination ().
K48-linked ubiquitination usually targets proteins to degradation 
, which is partly mediated by TRIM proteins in PRR- and IFN-signaling pathways: Mouse TRIM30-α promotes ubiquitination and degradation – although not by proteasomal but rather by lysosomal degradation – of TAB2 and TAB3 thereby inhibiting TLR-induced NF-κB activation 
, whereas TRIM8 decreases SOCS-1 protein levels leading to an inhibition of IFN-γ-signaling repression by SOCS-1 
. Ubiquitinated NOD2 is also targeted to proteasomal degradation, as inhibition of the 26S proteasome with bortezomib led to NOD2 protein stability and accumulation of ubiquitinated NOD2 (). Of note, NOD2 protein stability is effectively dependent on TRIM27. This is evidenced by the finding that knock-down of endogenous TRIM27 as well as overexpression of TRIM27 E3 inhibited NOD2 degradation (). The latter indicates that TRIM27 E3 acts as dominant negative over WT TRIM27 and endogenous TRIM27. We were surprised that overexpression of TRIM27 did not correlate with reduced NOD2 protein stability. Our interpretation is that this might be due to the activity of endogenous TRIM27 that might be sufficient to target even ectopically expressed NOD2 efficiently for degradation or that degradation at the proteasome is rate-limiting for overexpressed NOD2. TRIM27 has also been shown to confer SUMO E3 ligase activity 
. However, we did not detect SUMOylated NOD2, suggesting that NOD2 might not be a substrate for TRIM27-mediated SUMOylation (data not shown). In most cases, proteins are ubiquitinated in the cytosol and are degraded in this compartment. However, also evidence for nuclear ubiquitination and protein degradation exists 
. Based on our results showing that only a small fraction of NOD2 seems to shuttle to the nucleus () we suggest that NOD2 might be ubiquitinated in the cytoplasm.
Regulation of PRRs by ubiquitination and proteasomal degradation is a common theme for PRRs in animals and plants: Triad3A is known to control TLR4 and TLR9 signaling 
and plant FLS2 has been shown to be ubiquitinated and degraded in a negative feedback-loop by PUB12/13 
. Our data suggest that NOD2 signaling is regulated in a similar manner. Indeed, gene reporter assays in HEK293T cells showed that TRIM27 overexpression specifically reduces NOD2-mediated MDP-induced NF-κB activation in a dose-dependent manner, whereas TNF- and IKK-β-induced NF-κB activation is not affected. The negative regulating effects of TRIM27 on NOD2 signaling are not necessarily explained by NOD2 degradation but might also be due to circumvention of adaptor protein recruitment to the NOD2 complex. However, TRIM27 did not inhibit NOD2-RIP2 interaction (Figure S4A
). In contrast to our findings, Zha et al. reported that TRIM27 interacts with IKK-α, IKK-β, IKK-ε and negatively regulates NF-κB, IFN-β and ISRE activation induced by these kinases in a RING-domain-independent manner 
. The discrepancies between the two data sets remain unclear and need further independent investigation.
In contrast to most TRIM proteins, TRIM27 expression is not altered in response to IFNs 
. Among the TRIM proteins induced by type I and II IFNs, many have been shown to confer antiviral activity, although IFN-inducibility is not a prerequisite for this 
. Constitutively expressed TRIM proteins have also been shown to trigger antiviral immune responses. TRIM27 overexpression, however, did not influence Sendai virus-induced IFN-β activation (Figure S4B
). We therefore assume that TRIM27 might predominantly function in other pathways including NOD2 signaling.
NOD2 has been reported to be localized in the cytosol and at the cell membrane at steady-state conditions. Unexpectedly, we found that ectopically expressed NOD2, in contrast to NOD1, can shuttle to the nucleus (), dependently on a functional Walker A motif, i.e. ATPase activity. Although, a strong bipartite nuclear localization signal (NLS) in the NOD2 sequence could not be identified. Due to the low expression of endogenous NOD2, we were not able to establish the sub-cellular localization of endogenous NOD2. So although nuclear shuttling was verified for both NOD2 and epitope-tagged versions of NOD2 ectopically expressed in different cells by both cell biological and biochemical techniques, we cannot formally exclude that this is a particularity of ectopically expressed NOD2. Of note, also two other members of the human NLR family are known to shuttle to the nucleus. CIITA, one of the founding members of the NLR family, has long been recognized to regulate MHC class II gene expression by acting as a scaffold for DNA-binding transcription factor assembly in the nucleus 
. Recently, also NLRC5 has been shown to localize to the nucleus and to regulate MHC class I expression 
. Of note, nuclear translocation and functionality are also shown for several NLR-related plant R proteins. For tobacco N, barley MLA, arabidopsis RPS4 and potato RX R proteins nuclear localization is essential for defense activation 
. For these it has been suggested that pathogen recognition takes place in the cytoplasm, whereas they act in the nucleus to induce transcriptional reprogramming to activate immune responses 
. This is exemplified by the tobacco N protein, which confers resistance to Tobacco mosaic virus (TMV). Here MAP kinase activation in the cytosol is necessary for N-mediated resistance in addition to its nuclear functions 
. In analogy, it is tempting to speculate if NOD2 might not only recognize pathogens and activate MAP kinases and NF-κB in the cytosol, but likely possess additional nuclear functions to mediate and control immune responses. TRIM27 is known to be recruited to specific subnuclear compartments 
and was suggested as a transcriptional repressor 
. This raises the intriguing possibility that TRIM27, in addition to its role in NOD2 regulation, might be functionally involved in orchestrating NOD2 nuclear functions. Future research might help to address these open questions.
We and others recently demonstrated that some mutations in the NBD domain of NOD2 can result in enhanced NF-κB activation, whereas the corresponding mutations do not increase the activity of NOD1 
. This suggests that NOD2 is more prone for autoactivation compared to NOD1, requesting tighter control mechanisms to act on NOD2 to prevent unwanted auto-inflammatory responses. This is in line with the findings that I) more negative regulators are reported for NOD2 than for NOD1 
, II) there are more disease-associated mutations found in NOD2 than in NOD1 
, and III) NOD1 is expressed at basal levels in most tissues whereas NOD2 expression is more restricted and can be induced by pathogen stimuli 
. Here, we add another difference between NOD1 and NOD2 regulation and show that NOD2, but not NOD1, is controlled by ubiquitination and subsequent proteasomal degradation dependent on TRIM27 E3 ligase activity. We identified TRIM27 as a negative regulator of NOD2-mediated inflammatory responses and detected enhanced TRIM27 expression in the colon of Crohn's disease patients. Clinically, this makes TRIM27 an interesting new target for NOD2-associated diseases. It is conceivable that targeting TRIM27 might be advantageous when NOD2 activity is altered, such as in Crohn's disease.