Yeast two-hybrid screening for pyrin-binding protein.
Although certain bacterial toxins induce pyrin inflammasome formation (22), FMF attacks are not always triggered by infection but some other factor including stress and menstrual cycle. Therefore, we hypothesized that some endogenous proteins expressed in neutrophil may interact with pyrin and play an important role as a damage-associated molecular pattern. To identify a molecule that interacts with pyrin, we performed a yeast two-hybrid screening assay using human leukocyte cDNA library as prey. Our use of full-length pyrin (FL-pyrin; aa 1-781) as bait yielded 60 positive colonies (Fig. 1). To exclude false positives as much as possible before subsequent confirmatory binding assays, and to identify pyrin-binding proteins which interact with pyrin via its PRY/SPRY (B30.2) domain where mutations are often observed in patients with FMF, we also performed the screening using the C-terminal region of pyrin molecule (C-pyrin; aa 598–781) as bait. We found 124 positive colonies among which only 9 were isolated from both screenings using FL-pyrin bait and that using C-pyrin bait in common (Fig. 1). The colony-direct PCR and sequence revealed the 9 candidates for genes encoding pyrin-binding molecules, including FTH1, EEF1A1, TPT1, COX5A, CYBA, B2M, ACTB, FLOT1, and LTB (Table 1).
Table 1
The genes obtained commonly from both yeast two-hybrid library screenings using FL- and C-pyrin baits.
Symbol/Gene ID | Full Name | Summary |
FTH1/2945 | ferritin, heavy polypeptide 1 | Iron storage protein |
EEF1A1/1915 | eukaryotic translation elongation factor 1 alpha 1 | Enzymatic delivery of aminoacyl tRNAs to the ribosome |
TPT1/7178 | tumor protein, translationally-controlled 1 | Regulator of cellular growth and proliferation |
COX5A/9377 | cytochrome c oxidase subunit Va | Terminal enzyme of the mitochondrial respiratory chain |
CYBA/1535 | cytochrome b-245, alpha polypeptide | Primary component of the microbicidal oxidase system of phagocytes |
B2M/567 | beta-2 microglobulin | Component of human major histocompatibility classⅠ |
ACTB/60 | actin beta | Cytoskeleton |
FLOT1/10211 | flotillin 1 | Vesicle trafficking and cell morphology |
LTB/4050 | lymphotoxin beta | Inducer of the inflammatory response system |
β2MG interacts with pyrin.
β2MG protein is a component of major histocompatibility complex (MHC) class I molecules, which are present on the plasma membrane of all nucleated cells. In neutrophils, β2MG molecules are expressed not only on the cell surface as an MHC class I component but also inside the cells (23). Two-thirds of β2MG expression amount in neutrophils is localized in gelatinase granules or specific granules although the physiological role of this intragranular β2MG is still unknown (23). As we identified the B2M gene, which encodes β2MG protein, as the candidate for genes encoding pyrin-binding molecules by the yeast two-hybrid screening, we focused on β2MG and tried to confirm the interaction with pyrin by immunoprecipitation assay. We subcloned the full-length B2M cDNA into pcDNA3.1(-) vectors with V5 tag in the N-terminus and co-transfected them with FLAG-tagged pyrin expression vector into Lenti-X 293T cells. The co-immunoprecipitation analysis using anti-FLAG Ab revealed that V5-tagged β2MG was co-precipitated with FLAG-tagged pyrin (Fig. 2A). Additionally, the reciprocal immunoprecipitation assay using anti-V5 Ab showed that FLAG-pyrin was co-precipitated with V5-β2MG (Fig. 2B). To further confirm the pyrin-β2MG interaction, the expression vectors for FLAG-pyrin and V5-β2MG were co-transfected into HEK293 cells and the localization of these fusion proteins was examined by immunofluorescence staining. As shown in Fig. 2C, the overexpressed V5-β2MG distributed homogeneously in the cytoplasm and did not show any specific structure when FLAG-pyrin was not overexpressed. On the other hand, V5-β2MG co-localized with pyrin in the speck-like structures in the perinuclear position in more than 90% of the cells in which both FLAG-pyrin and V5-β2MG were overexpressed.
To confirm the localization of endogenous pyrin and β2MG, we examined human neutrophils isolated from the peripheral blood of healthy donors and stained with anti-pyrin and anti-β2MG Abs. β2MG staining was not co-localized with pyrin staining in neutrophils without any stimulation (Fig. 3, upper panels). Because interaction between neutrophils and MSU crystals has been shown to elicit various responses including IL-1β secretion (24), we next investigated their localization in the stimulated neutrophils. Interestingly, β2MG was co-localized with pyrin in neutrophils stimulated with LPS and MSU in the speck-like structures (Fig. 3, lower panels), suggesting that the pyrin-β2MG interaction is induced by neutrophil activation.
β2MG binds to the hotspot of FMF-associated mutations of pyrin.
Because the B2M gene was identified not only by yeast two-hybrid screening using FL-pyrin bait but also using C-pyrin bait, we expected that β2MG can bind to the PRY/SPRY domain of pyrin. To further investigate the binding site for β2MG in pyrin molecule, we constructed the vectors which express FLAG-tagged pyrin lacking PRY/SPRY domain corresponding to exon 10 (ΔE10 pyrin) and pyrin PRY/SPRY domain (E10 pyrin) and transfected them with V5-β2MG expression vector into Lenti-X 293T cells. Although FLAG-E10 pyrin co-precipitated V5-β2MG, FLAG-ΔE10 pyrin did not (Fig. 4A). The reciprocal immunoprecipitation assay using anti-V5 Ab showed that FLAG-E10 pyrin was co-precipitated with V5-β2MG (Fig. 4B). Consistent with the data, immunofluorescence staining revealed that the overexpressed V5-β2MG was not colocalized with FLAG-ΔE10 pyrin but with FLAG-E10 in speck-like structures (Fig. 4C). These results indicate that pyrin interacts with β2MG via its PRY/SPRY domain.
FMF-associated mutations accumulate in the middle portion of the pyrin PRY/SPRY domain (Fig. 5A). Among them, M680I, M694V, M694I, and V726A are listed as the minimum set of clearly pathogenic sequence variants recommended to screen in the genetic diagnostic guidelines developed by Shinar et al. (25). To elucidate the relationship between these mutations and β2MG binding, we constructed expression vectors for FLAG-tagged pyrin deletion mutants, pyrin E10-1 (aa 598–659), pyrin E10-2 (aa 660–727) and pyrin E10-3 (aa 728–781). These expression vectors were co-transfected with V5-tagged β2MG into Lenti-X 293T cells and subjected to co-immunoprecipitation analysis. As shown in Fig. 5B, all FLAG-tagged E10 pyrin mutants could co-precipitate V5-β2MG. However, FLAG-tagged pyrin E10-2, which includes the hotspot of FMF-associated mutations, co-precipitated V5-β2MG much more strongly as compared to other E10 mutants, suggesting the important role of mutations in the pyrin-β2MG interaction.
β2MG-pyrin interaction recruits PSTPIP1 leading to the assembly of pyrin inflammasome.
PSTPIP1, which was identified as a genetic cause of another autoinflammatory disorder, PAPA syndrome, has been shown to interact with the B-box/coiled-coil domain of pyrin (26). Furthermore, PSTPIP binding has been shown to activate pyrin by unmasking the pyrin domain, promoting ASC-mediated oligomerization and inflammasome formation (14). Thus, we next investigated whether pyrin-β2MG interaction affects pyrin-ASC and pyrin-PSTPIP1 interactions. As shown in Fig. 6A, when HEK293 cells overexpressing FLAG-tagged pyrin and V5-tagged ASC were subjected to immunofluorescence staining, FLAG-pyrin and V5-ASC were co-localized in the cytoplasm with the speck formation (arrows in the light field images), indicating pyrin-ASC interaction. To clarify the effect of pyrin-β2MG interaction on pyrin-ASC interaction, FLAG-tagged pyrin, and V5-tagged ASC expression vectors were transfected into Lenti-X 293T cells with or without V5-tagged β2MG expression vector. The co-immunoprecipitation analysis showed that pyrin did not interact with ASC (Fig. 6B) and β2MG overexpression did not induce pyrin-ASC interaction (Fig. 6C).
We next examined the effect of pyrin-β2MG interaction on pyrin-PSTPIP1 interaction. First, we investigated the interaction between pyrin, PSTPIP1, and ASC in the absence of β2MG overexpression by the co-immunoprecipitation analysis using FLAG-pyrin, V5-ASC expression vectors together with V5-tagged wild-type PSTPIP1 or one of the most common PAPA-associated PSTPIP1 mutation, A230T. FLAG-pyrin co-precipitated A230T PSTPIP1 drastically as compared to the wild-type PSTPIP1 (Fig. 6D), consistent with the previous study showing that A230T mutation induces its hyperphosphorylation and increases the affinity to pyrin (26). Additionally, the pyrin-ASC interaction was also promoted by the mutation, indicating that the PSTPIP1 mutation increases the pyrin inflammasome formation (Fig. 6D). Then, FLAG-pyrin and V5-PSTPIP1 expression vectors were transfected into Lenti-X 293T cells with or without a V5-β2MG expression vector. Co-immunoprecipitation analysis revealed that β2MG overexpression drastically promoted the pyrin-PSTPIP1 interaction (Fig. 6E).
To assess whether β2MG promotes ASC recruitment, we have overexpressed V5-β2MG together with pyrin, PSTPIP1, and ASC in the cells, and investigated the change of ASC recruitment by the β2MG-mediated enhancement of pyrin-PSTPIP1 interaction. As a result, β2MG overexpression promoted ASC recruitment (Fig. 6F). The ability is PSTPIP1-dependent because the induction of ASC recruitment was not observed without PSTPIP1 overexpression (Fig. 6C). These results suggest that β2MG functions as a pyrin-binding protein inducing PSTPIP1-mediated pyrin inflammasome formation.
Caspase-1 p20 subunit inhibits the association of β2MG with pyrin.
The activation of caspase-1 is caused by the autocleavage of pro-caspase-1, which leads to the formation of active caspase-1 p10/p20 tetramer (27). It is a pivotal process for IL-1β and IL-18 production after the assembly of the pyrin inflammasome. On the other hand, p20 and p10 have been shown to interact with the PRY/SPRY domain of pyrin directly, resulting in the inhibitory effect on IL-1β production (18). Thus, we hypothesized that the binding of β2MG and caspase-1 to pyrin may compete on the pyrin PRY/SPRY domain. We constructed the expression vectors for V5-tagged caspase-1 p20 and transfected them into the Lenti-X 293T cells with the expression vectors for FLAG-tagged ΔE10 or E10 pyrin. Co-immunoprecipitation analysis revealed that caspase-1 p20 interacts with the PRY/SPRY domain of pyrin as shown in the previous report (Fig. 7A) (18). To investigate the relationship between the FMF-associated pyrin mutations and caspase-1 binding, we co-transfected the vectors for FLAG-tagged pyrin deletion mutants, pyrin E10-1, E10-2 or E10-3 with V5-tagged p20 into Lenti-X 293T cells, and subjected them to co-immunoprecipitation analysis. As is the case with β2MG, FLAG-tagged E10-2, which includes the hotspot of FMF-associated mutations, co-precipitated V5-p20 best among the pyrin deletion mutants (Fig. 7B). To elucidate whether caspase-1 has an inhibitory effect on pyrin-β2MG interaction, FLAG-tagged pyrin and V5-tagged β2MG expression vectors were co-transfected into Lenti-X 293T cells with or without V5-tagged caspase-1 p20 expression vector. Co-immunoprecipitation analysis revealed that overexpression of caspase-1 p20 inhibits the interaction between pyrin and β2MG (Fig. 7C). These results suggest that p20 acts as an inhibitor for β2MG-mediated promotion of pyrin inflammasome formation and suppresses the excess inflammation via a negative feedback mechanism.
Pyrin M694V mutation changes the balance of affinities toβ2MG and caspase-1 p20.
Homozygous M694V mutation can cause severer forms of FMF and AA amyloidosis in higher prevalence (28). To elucidate whether M694V mutation affects β2MG affinity for pyrin, we constructed expression vectors for FLAG-tagged full-length pyrin and E10-2 fragment with M694V mutation and compared the β2MG affinity between for M694V-mutated pyrin or E10-2 fragment and for the wild type by co-immunoprecipitation assay. As shown in Fig. 8A and 8B, the M694V mutation did not make any impact on the β2MG affinity in the absence of caspase-1 p20 overexpression. Because our data showed that caspase-1 p20 inhibits the association of β2MG with pyrin, we next investigated the effect of M694V mutation on inhibition of pyrin-β2MG interaction by p20. Unlike wild-type pyrin, M694V-mutated pyrin co-precipitated β2MG even in the presence of p20, suggesting that pyrin M694V mutation weakened negative feedback on PSTPIP1-mediated pyrin inflammasome formation by caspase-1 p20 (Fig. 8C).
Finally, we examined whether pyrin M694V mutation affects PSTPIP1 recruitment by co-immunoprecipitation analysis. As a result, M694V-mutation itself did not affect the pyrin-PSTPIP1 interaction in the presence of β2MG (Fig. 8D). These findings suggest that pyrin M694V mutation leads to the overproduction of pyrin inflammasome not by promoting β2MG-induced PSTPIP1 recruitment but by reduction of the inhibitory effect of caspase-1 p20 on pyrin-β2MG interaction.