5. Results
5.5. NLRP3 ∆ exon 5 does not interact with NEK7
While the stochastic single-cell NLPR3 isoform expression and NLRP3 activity provide a physiological relevance for NLRP3 splicing, the molecular mechanistic reason for the NLRP3 ∆ exon 5 inactivity remained to be determined. NEK7 binding was demonstrated as a pre-requisite for NLRP3 activation 97–99. To test whether NLRP3 ∆ exon 5 was inactive due to the loss of the NEK7 interaction, NLRP3-NEK7 Co-IPs were performed from iMos using the mCitrine tag to pull down either NLRP3 full-length or NLRP3 ∆ exon 5. While NLRP3 full-length co-precipitated NEK7, NEK7 could not be detected in the Co-IP of NLRP3 ∆ exon 5 (Figure 5-18 A). In the literature, NEK7 is postulated to bind NLRP3 progressively upon activation 99. However, here the interaction could be observed independent of NLRP3 activation.
Yet the same assay was also performed after NLRP3 activation with nigericin. As a further control, treatment with the NLRP3 inhibitor CRID3 was included (Figure 5-18 B). Regardless of the activation status NLRP3-NEK7 interaction could be demonstrated, but only for the NLRP3 full-length variant.
Figure 5-18 NLRP3 full-length, but not NLRP3 ∆ exon 5 interacts with NEK7
A Co-immunoprecipitation (IP) from iMos stably expressing the respective NLRP3-mCitrine variants. IP was performed in GFP-trap plates. B as A, but from LPS-primed and nigericin-activated iMos with and without treatment with the NLRP3 inhibitor CRID3. WCL – whole cell lysate.
Two hypotheses why NLRP3 ∆ exon 5 is inactive and why it does not interact with NEK7 are conceivable: Either, due to the overall shortened LRR, or because exon 5 is critical for the NEK7 interaction. To distinguish between these two hypotheses, a NLRP3 variant containing no exon 5 but a doubled exon 6 was created (Figure 5-19 A). This was possible due to the high degree of conservation of the LRR exons. As before, stable iMo cell lines were created and IL-1β secretion was analyzed after NLRP3, AIM2 and NLRC4 activation (Figure 5-19 B). While all cell lines secreted IL-1β upon AIM2 or NLRC4 activation, NLRP3 ∆ exon 5 and 2x exon 6 could both not be activated with NLRP3 stimuli. All cell lines responded to priming with LPS as assessed by TNF secretion (Figure 5-19 C). These results suggest that not the shortened overall length of NLRP3 ∆ exon 5 LRR but rather the specific absence of exon 5 caused the NLRP3 inactivity.
Assuming that exon 5 acts as the interaction surface for NEK7 binding, is can be assumed that the surface exposed amino acids are especially important. Therefore, a NLRP3 hybrid variant of NLRP3 full-length and 2x exon 6 was generated. All amino acids defining the NLRP3 exon 5 surface according to the structural model were mutated to their respective analogues from exon 6 (Figure 5-20 A). IL-1β secretion after inflammasome activation was measured, demonstrating the inactivity of NLRP3 lacking the exon 5 surface, while all cells were capable of IL-1β secretion after AIM2 or NLRC4 activation (Figure 5-20 B). The TNF response as a surrogate for priming was similar for all cell lines under all conditions (Figure 5-20 C).
Figure 5-19 NLRP3 ∆ exon 5 is not inactive due to its shortened LRR
A Models of the NLRP3 LRRs based on the crystal structure of human ribonuclease inhibitor. Shown are the LRR model structures and schematics of NLRP3 full-length and NLRP3 ∆ exon 5, as well as an artificially created LRR: NLRP3 LRR lacking exon 5 but carrying a duplicate exon 6. R. Brinkschulte generated the ribbon models. B Stable iMo cell lines expressing the respective NLRP3 variants were created as described above. IL-1β secretion was analyzed after activation of the NLRP3, AIM2 or NLRC4 inflammasome. C TNF secretion after LPS treatment. B and C mean and SD of technical triplicates N=1.
Introducing as many mutations, even in a highly conserved repeat unit, may cause an unspecific loss of functionality, caused by mis-folding of the protein. Therefore, a rescue mutation strategy was chosen: Based on the inactive 2x exon 6 NLRP3 variant, a surface rescue for the residues of exon 5 was generated (Figure 5-21 A).
Due to the high level of conservation of the LRR exons, the overall physico-chemical characteristics of the hybrid isoforms are similar to the wildtype (wt) isoforms (see appendix, Table 12-2). Stable iMo cell lines were primed and activated as before. As expected, all cell lines secreted IL-1β after AIM2 and NLRC4 activation, but only the NLRP3 full-length and NLRP3 2x exon 6 surface rescue variant were responsive to
Figure 5-20 The NLRP3 exon 5 surface is relevant for the activity
An exon 5 hybrid variant, in which all surface amino acids of the structural model were mutated to their analogue from exon 6, was created and a stable cell line expressing this NLRP3 variant was generated.
A IL-1β secretion was analyzed after activation of the NLRP3, AIM2 or NLRC4 inflammasome. B TNF secretion after LPS treatment. A and B mean and SEM of 5 independent experiments.
NLRP3 activators (Figure 5-21 B), while all of them were equally primed (Figure 5-21 C).
Taken together, these experiments provide evidence that the surface of exon 5 is needed for the activation of NLRP3.
Given that NLRP3 ∆ exon 5 was not capable of interacting with NEK7 and that the specific surface of NLRP3 exon 5 is needed for activation, the exon 5 replacement mutants from above were used to investigate the NEK7 interaction. NLRP3 Co-IPs for the interaction with NEK7 were performed from iMo lysates using the mCitrine tag to pull down NLRP3 (Figure 5-21 D). Although the interaction between NLRP3 2x exon 6 surface rescue and NEK7 was weaker than between NLRP3 full-length and NEK7, a clear increase in interaction was detectable compared to the ∆ exon 5 and 2x exon 6 NLRP3 variants. It has to be taken into account that in these cell lines human NLRP3 interacts with mouse NEK7, which may be sufficient to allow for NLRP3 activation, but may be more sensitive to minor structural differences at the interaction side than a human-human interaction pair. Therefore, the experiments were repeated in 293T cells, which were transiently transfected to express the
different NLRP3 isoforms before mCitrine pull-downs were performed. Under these conditions, a solid interaction between NLRP3 2x exon 6 surface rescue and NEK7 could be detected, while ∆ exon 5 and 2x exon 6 NLRP3 variants did not interact with NEK7 (Figure 5-21 E).
Taken together, these experiments map the interaction site of NEK7 to exon 5 of NLRP3 and thereby explain why NLRP3 ∆ exon 5 is inactive.
Figure 5-21 The NLRP3 exon 5 surface is essential for the interaction with NEK7
A Schematic and ribbon model of an artificial NLRP3 hybrid LRR based on the crystal structure of human ribonuclease inhibitor. Surface residues of exon 5 are shown in red. The model was generated by R. Brinkschulte. The LRR lacks exon 5 but carries a duplicate exon 6 in which all surface amino acids of exon 5 were rescued. NLRP3-deficient iMos were reconstituted with full-length or the hybrid NLRP3-mCitrine variants as before. B IL-1β secretion was analyzed after activation of the NLRP3, AIM2 or NLRC4 inflammasome. C TNF secretion after LPS treatment. B and C mean and SEM of 5 independent experiments. D Co-immunoprecipitation (IP) from iMos stably expressing the respective NLRP3-mCitrine variants. IP was performed in GFP-trap plates. Representative of 2 independent experiments. E As D, but from HEK 293T cells transiently transfected to express the respective NLRP3-mCitrine variants. Representative of 3 independent experiments.