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Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly.

George J, Motshwene PG, Wang H, Kubarenko AV, Rautanen A, Mills TC, Hill AV, Gay NJ, Weber AN - J. Biol. Chem. (2010)

Bottom Line: Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction.Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1.The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome.

View Article: PubMed Central - PubMed

Affiliation: Division Toll-like receptors and Cancer, German Cancer Research Centre DKFZ, Heidelberg, 69120 Germany.

ABSTRACT
Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

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S34Y and R98C fail to interact with IRAK4. A, the interaction of S34Y and R98C with the IRAK4 DD is reduced. Protein complexes were immunoprecipitated using anti-Myc antibody from HEK293 cells transiently transfected with Myc-MyD88 WT or mutants and Strep-HA-tagged IRAK4 DD, respectively. Precipitates and whole cell lysates (WCL) were analyzed by immunoblot (IB). B, MyD88 S34Y-and R98C-IRAK4 interactions are strongly reduced in the context of MyD88 FL and IRAK4 DD (Panel 1). Similar results were obtained in the DD-DD interactions (Panel 2). Interactions are reduced to a level of no interaction in the context of mutant FL MyD88-FL IRAK4 interactions (Panel 3). Protein A-tagged MyD88 WT and mutants in FL or DD context and Renilla luciferase-tagged IRAK4 FL or DD constructs were transfected into HEK293 cells, and LUMIER measurements were conducted as described under “Experimental Procedures.” One representative of at least two independent experiments is shown. C, S34Y and R98C are unable to assemble into a Myddosome on size exclusion chromatography of mixtures of bacterially purified MyD88 DDs and IRAK4 DDs (added in excess). WT MyD88+IRAK4 (upper graph) mixtures elute in a discrete Myddosome peak. This peak is absent in S34Y (middle graph) and R98C (lower graph) mixtures. Both mutants elute in the void volume, containing higher order oligomers only and no IRAK4 DD. R98C also shows a dimer peak. Individual peak fractions from gel filtration (labeled GF), purified MyD88 DD or IRAK4 DD alone (for size comparison) were analyzed by reducing SDS-PAGE (D) or dynamic light scattering (marked DLS) to measure the size of protein species found in these fractions or samples (E). One representative of two independent experiments is shown respectively.
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Figure 5: S34Y and R98C fail to interact with IRAK4. A, the interaction of S34Y and R98C with the IRAK4 DD is reduced. Protein complexes were immunoprecipitated using anti-Myc antibody from HEK293 cells transiently transfected with Myc-MyD88 WT or mutants and Strep-HA-tagged IRAK4 DD, respectively. Precipitates and whole cell lysates (WCL) were analyzed by immunoblot (IB). B, MyD88 S34Y-and R98C-IRAK4 interactions are strongly reduced in the context of MyD88 FL and IRAK4 DD (Panel 1). Similar results were obtained in the DD-DD interactions (Panel 2). Interactions are reduced to a level of no interaction in the context of mutant FL MyD88-FL IRAK4 interactions (Panel 3). Protein A-tagged MyD88 WT and mutants in FL or DD context and Renilla luciferase-tagged IRAK4 FL or DD constructs were transfected into HEK293 cells, and LUMIER measurements were conducted as described under “Experimental Procedures.” One representative of at least two independent experiments is shown. C, S34Y and R98C are unable to assemble into a Myddosome on size exclusion chromatography of mixtures of bacterially purified MyD88 DDs and IRAK4 DDs (added in excess). WT MyD88+IRAK4 (upper graph) mixtures elute in a discrete Myddosome peak. This peak is absent in S34Y (middle graph) and R98C (lower graph) mixtures. Both mutants elute in the void volume, containing higher order oligomers only and no IRAK4 DD. R98C also shows a dimer peak. Individual peak fractions from gel filtration (labeled GF), purified MyD88 DD or IRAK4 DD alone (for size comparison) were analyzed by reducing SDS-PAGE (D) or dynamic light scattering (marked DLS) to measure the size of protein species found in these fractions or samples (E). One representative of two independent experiments is shown respectively.

Mentions: MyD88 homo-oligomerization has been proposed to nucleate Myddosome assembly (17). Because S34Y and R98C impact on this step, our modeling results prompted us to determine whether the association of the MyD88 variants with downstream signaling components of the IRAK family is altered. We investigated the association of FL and DD MyD88 WT and the mutants with FL and DD IRAK4 (residues 1–106) by co-immunoprecipitation and LUMIER. We initially generated an N-terminal Strep-HA-tagged construct of IRAK4 solely comprising the DD, a construct known to be sufficient for MyD88 interactions (16, 17). Fig. 5A shows that all FL MyD88 mutants were still able to interact with IRAK4 DDs (an interaction found to be highly dependent on the presence of EDTA; data not shown). However, the band intensities implied reduced affinities of S34Y and R98C for IRAK4 DDs. This was confirmed using LUMIER (Fig. 5B, Panel 1). We also analyzed the association of MyD88 DD-only constructs with IRAK4 DDs. In agreement with the previous experiment, the DDs of S34Y and R98C showed more than a 10-fold reduced binding but nevertheless weak binding to the IRAK4 DD (Fig. 5B, Panel 2). We sought to confirm this finding using the FL constructs of both MyD88 and IRAK4. As the association of IRAK4 with MyD88 is a transient event, it cannot be captured by co-immunoprecipitation (30). In view of this, we conducted LUMIER experiments, and this showed that the interaction of MyD88 WT FL with IRAK4 FL was comparable with MyD88 FL or DD interactions with IRAK4 DDs. By contrast, no interaction of IRAK4 FL could be detected with the S34Y and R98C mutants (Fig. 5B, Panel 3), implying that the presence of the kinase domain influences the assembly of the Myddosome in vitro. To confirm these findings by an alternative method, we purified MyD88 WT, S34Y, and R98C DDs from bacteria (16) and tested their ability to interact with purified IRAK4 DD. We analyzed the elution profiles of mixtures of the MyD88 mutants in the presence of a molar excess of IRAK4 DDs by size exclusion chromatography (Fig. 5C). Individual fractions or purified reference proteins were analyzed by SDS-PAGE (Fig. 5D). IRAK4 is recruited into a characteristic higher Mr Myddosome peak by WT MyD88 (elution volume Ve = 11.95 ml corresponding to 165 kDa, lane 5) but not by S34Y or R98C. In these mixtures, the IRAK4 DD instead eluted exclusively on its own (Ve = 18 ml, corresponding to an IRAK4 DD monomer). S34Y (lane 3) and R98C DDs (not shown) were present in the void volume (Ve = 8.38 ml), but the peaks did not contain any IRAK4 DDs. In the case of R98C, a second peak (Ve = 14.7 ml, lane 2) eluted at a position characteristic of a MyD88 WT dimer (not shown, see Ref. 16). Individual peaks or samples (as indicated in Fig. 5, C and D) were also analyzed by dynamic light scattering, which provides an assessment of the size of complexes formed by MyD88 mutants and/or IRAK4 DDs. As found previously (16), MyD88 WT DD formed two species, one corresponding to a MyD88 DD dimer (with a hydrodynamic diameter, d(H) of 8.6 nm), the other to a higher oligomer (d(H) of 135 nm; Fig. 5E). By contrast, the Myddosome complex fraction (Ve = 11.95 ml, lane 5 in Fig. 5D) is detected as one predominant species of d(H) 13 nm (16). Void volume fractions of S34Y and R98C contained mainly higher order oligomers only, suggesting they that they have an enhanced tendency to aggregate (d(H) 110.02 and 64.05 nm, respectively). This is especially evident with the S34Y DD, which does not appear to form a stable dimeric species and is highly resistant to proteolysis from its GST tag (Fig. 5D, cf. lane 1 and 2 with lane 3), indicating that the cleavage site is sequestered by aggregation. R98C DD aggregation appears partially reversible, as the R98C DD void volume fraction also gave a minor signal corresponding to a dimer (d(H) 7.43 nm). Although there are likely to be subtle differences between MyD88 and IRAK4 DDs from bacteria and in mammalian cells, our data conclusively illustrate the failure of MyD88 S34Y and R98C to associate with IRAK4 and identify this as the defect in signal transduction.


Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly.

George J, Motshwene PG, Wang H, Kubarenko AV, Rautanen A, Mills TC, Hill AV, Gay NJ, Weber AN - J. Biol. Chem. (2010)

S34Y and R98C fail to interact with IRAK4. A, the interaction of S34Y and R98C with the IRAK4 DD is reduced. Protein complexes were immunoprecipitated using anti-Myc antibody from HEK293 cells transiently transfected with Myc-MyD88 WT or mutants and Strep-HA-tagged IRAK4 DD, respectively. Precipitates and whole cell lysates (WCL) were analyzed by immunoblot (IB). B, MyD88 S34Y-and R98C-IRAK4 interactions are strongly reduced in the context of MyD88 FL and IRAK4 DD (Panel 1). Similar results were obtained in the DD-DD interactions (Panel 2). Interactions are reduced to a level of no interaction in the context of mutant FL MyD88-FL IRAK4 interactions (Panel 3). Protein A-tagged MyD88 WT and mutants in FL or DD context and Renilla luciferase-tagged IRAK4 FL or DD constructs were transfected into HEK293 cells, and LUMIER measurements were conducted as described under “Experimental Procedures.” One representative of at least two independent experiments is shown. C, S34Y and R98C are unable to assemble into a Myddosome on size exclusion chromatography of mixtures of bacterially purified MyD88 DDs and IRAK4 DDs (added in excess). WT MyD88+IRAK4 (upper graph) mixtures elute in a discrete Myddosome peak. This peak is absent in S34Y (middle graph) and R98C (lower graph) mixtures. Both mutants elute in the void volume, containing higher order oligomers only and no IRAK4 DD. R98C also shows a dimer peak. Individual peak fractions from gel filtration (labeled GF), purified MyD88 DD or IRAK4 DD alone (for size comparison) were analyzed by reducing SDS-PAGE (D) or dynamic light scattering (marked DLS) to measure the size of protein species found in these fractions or samples (E). One representative of two independent experiments is shown respectively.
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Figure 5: S34Y and R98C fail to interact with IRAK4. A, the interaction of S34Y and R98C with the IRAK4 DD is reduced. Protein complexes were immunoprecipitated using anti-Myc antibody from HEK293 cells transiently transfected with Myc-MyD88 WT or mutants and Strep-HA-tagged IRAK4 DD, respectively. Precipitates and whole cell lysates (WCL) were analyzed by immunoblot (IB). B, MyD88 S34Y-and R98C-IRAK4 interactions are strongly reduced in the context of MyD88 FL and IRAK4 DD (Panel 1). Similar results were obtained in the DD-DD interactions (Panel 2). Interactions are reduced to a level of no interaction in the context of mutant FL MyD88-FL IRAK4 interactions (Panel 3). Protein A-tagged MyD88 WT and mutants in FL or DD context and Renilla luciferase-tagged IRAK4 FL or DD constructs were transfected into HEK293 cells, and LUMIER measurements were conducted as described under “Experimental Procedures.” One representative of at least two independent experiments is shown. C, S34Y and R98C are unable to assemble into a Myddosome on size exclusion chromatography of mixtures of bacterially purified MyD88 DDs and IRAK4 DDs (added in excess). WT MyD88+IRAK4 (upper graph) mixtures elute in a discrete Myddosome peak. This peak is absent in S34Y (middle graph) and R98C (lower graph) mixtures. Both mutants elute in the void volume, containing higher order oligomers only and no IRAK4 DD. R98C also shows a dimer peak. Individual peak fractions from gel filtration (labeled GF), purified MyD88 DD or IRAK4 DD alone (for size comparison) were analyzed by reducing SDS-PAGE (D) or dynamic light scattering (marked DLS) to measure the size of protein species found in these fractions or samples (E). One representative of two independent experiments is shown respectively.
Mentions: MyD88 homo-oligomerization has been proposed to nucleate Myddosome assembly (17). Because S34Y and R98C impact on this step, our modeling results prompted us to determine whether the association of the MyD88 variants with downstream signaling components of the IRAK family is altered. We investigated the association of FL and DD MyD88 WT and the mutants with FL and DD IRAK4 (residues 1–106) by co-immunoprecipitation and LUMIER. We initially generated an N-terminal Strep-HA-tagged construct of IRAK4 solely comprising the DD, a construct known to be sufficient for MyD88 interactions (16, 17). Fig. 5A shows that all FL MyD88 mutants were still able to interact with IRAK4 DDs (an interaction found to be highly dependent on the presence of EDTA; data not shown). However, the band intensities implied reduced affinities of S34Y and R98C for IRAK4 DDs. This was confirmed using LUMIER (Fig. 5B, Panel 1). We also analyzed the association of MyD88 DD-only constructs with IRAK4 DDs. In agreement with the previous experiment, the DDs of S34Y and R98C showed more than a 10-fold reduced binding but nevertheless weak binding to the IRAK4 DD (Fig. 5B, Panel 2). We sought to confirm this finding using the FL constructs of both MyD88 and IRAK4. As the association of IRAK4 with MyD88 is a transient event, it cannot be captured by co-immunoprecipitation (30). In view of this, we conducted LUMIER experiments, and this showed that the interaction of MyD88 WT FL with IRAK4 FL was comparable with MyD88 FL or DD interactions with IRAK4 DDs. By contrast, no interaction of IRAK4 FL could be detected with the S34Y and R98C mutants (Fig. 5B, Panel 3), implying that the presence of the kinase domain influences the assembly of the Myddosome in vitro. To confirm these findings by an alternative method, we purified MyD88 WT, S34Y, and R98C DDs from bacteria (16) and tested their ability to interact with purified IRAK4 DD. We analyzed the elution profiles of mixtures of the MyD88 mutants in the presence of a molar excess of IRAK4 DDs by size exclusion chromatography (Fig. 5C). Individual fractions or purified reference proteins were analyzed by SDS-PAGE (Fig. 5D). IRAK4 is recruited into a characteristic higher Mr Myddosome peak by WT MyD88 (elution volume Ve = 11.95 ml corresponding to 165 kDa, lane 5) but not by S34Y or R98C. In these mixtures, the IRAK4 DD instead eluted exclusively on its own (Ve = 18 ml, corresponding to an IRAK4 DD monomer). S34Y (lane 3) and R98C DDs (not shown) were present in the void volume (Ve = 8.38 ml), but the peaks did not contain any IRAK4 DDs. In the case of R98C, a second peak (Ve = 14.7 ml, lane 2) eluted at a position characteristic of a MyD88 WT dimer (not shown, see Ref. 16). Individual peaks or samples (as indicated in Fig. 5, C and D) were also analyzed by dynamic light scattering, which provides an assessment of the size of complexes formed by MyD88 mutants and/or IRAK4 DDs. As found previously (16), MyD88 WT DD formed two species, one corresponding to a MyD88 DD dimer (with a hydrodynamic diameter, d(H) of 8.6 nm), the other to a higher oligomer (d(H) of 135 nm; Fig. 5E). By contrast, the Myddosome complex fraction (Ve = 11.95 ml, lane 5 in Fig. 5D) is detected as one predominant species of d(H) 13 nm (16). Void volume fractions of S34Y and R98C contained mainly higher order oligomers only, suggesting they that they have an enhanced tendency to aggregate (d(H) 110.02 and 64.05 nm, respectively). This is especially evident with the S34Y DD, which does not appear to form a stable dimeric species and is highly resistant to proteolysis from its GST tag (Fig. 5D, cf. lane 1 and 2 with lane 3), indicating that the cleavage site is sequestered by aggregation. R98C DD aggregation appears partially reversible, as the R98C DD void volume fraction also gave a minor signal corresponding to a dimer (d(H) 7.43 nm). Although there are likely to be subtle differences between MyD88 and IRAK4 DDs from bacteria and in mammalian cells, our data conclusively illustrate the failure of MyD88 S34Y and R98C to associate with IRAK4 and identify this as the defect in signal transduction.

Bottom Line: Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction.Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1.The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome.

View Article: PubMed Central - PubMed

Affiliation: Division Toll-like receptors and Cancer, German Cancer Research Centre DKFZ, Heidelberg, 69120 Germany.

ABSTRACT
Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

Show MeSH
Related in: MedlinePlus