Natural loss-of-function mutation of myeloid differentiation protein 88 disrupts its ability to form Myddosomes

Abstract

Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knock-out mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. Unlike wild-type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. We observed that IRAK4 co-localizes with MyD88 in these aggregates, and thus these foci appear to be "Myddosomes." The MyD88 S34Y loss-of-function mutant demonstrates how proper cellular localization of MyD88 to the Myddosome is a feature required for MyD88 function.

FIGURE 1.

Transient expression of MyD88 S34Y is unable to activate NF-κB and IRF-5 dependent ISRE reporters. A, schematic of MyD88 domain structure depicting the position of the various nonsynonymous SNPs is shown. B, HEK293T cells were transfected with HA-tagged versions of either WT MyD88 or the different variants of MyD88, and expression levels were checked using anti-HA antibody. C, HEK293T cells were transfected with the indicated MyD88 construct and either NF-κB luciferase (left) or ISRE-luciferase + IRF5 (right). Twenty-four hours later, luciferase activity was measured in cell lysates. Renilla luciferase values were used to normalize for transfection efficiency. Results are reported as a mean of triplicate determinations ± S.D. (error bars). The graphs are a representative of three (left) or two (right) independent experiments. DD, death domain; WB, Western blotting.

FIGURE 2.

MyD88-deficient macrophages expressing S34Y have impaired cytokine production. A, MyD88-deficient macrophages were infected with an IRES-GFP-expressing retrovirus MSCV2.2, carrying HA-tagged versions of either WT MyD88 or the mutant S34Y. The positive cells were sorted based on GFP expression, and the expression levels of the proteins were checked using an anti-HA antibody. B, cells were stimulated overnight with the indicated TLR ligands, and the levels of TNF-α in the culture supernatants were analyzed by ELISA. Results are reported as a mean of triplicate determinations ± S.D. (error bars). The graph is a representative of three independent experiments. C, same supernatants (only LPS stimulation) were analyzed for IFN-β levels by ELISA. Results are reported as a mean of triplicate determinations ± S.D. The graph is a representative of three independent experiments.

FIGURE 3.

IκB degradation and MAPK (p38) activation are impaired in macrophages that express MyD88 S34Y. A, NF-κB activation was examined by assessing IκB degradation. Cells were plated in a 6-well plate and stimulated with either 10 nm PAM₂CSK₄ or 20 μm R848 for the indicated time intervals. Cell lysates were made, run on a gel, transferred to nitrocellulose membrane, and blotted (IB) with an anti-IκB antibody. GAPDH levels served as an internal loading control. B, same cell lysates were also analyzed for p38 MAPK activation by examining phosphorylation of p38 using anti-pp38 antibody. Total p38 MAPK levels acted as an internal loading control.

FIGURE 4.

Macrophages expressing S34Y are unable to degrade IRAK1 effectively. IRAK1 degradation was examined in the reconstituted cell lines as a marker of an active signal complex formation. Cells were plated in a 6-well plate and stimulated with either 10 nm PAM₂CSK₄ or 20 μm R848 for the indicated time intervals. Cell lysates were made, run on a gel, transferred to nitrocellulose membrane, and blotted (IB) with an anti-IRAK1 antibody. GAPDH levels served as an internal loading control.

FIGURE 5.

Unlike WT MyD88, S34Y does not form oligomeric aggregates. A, citrine-tagged WT MyD88 or S34Y (green) was transfected into HEK293T cells. The next day, live cells were imaged using standard confocal procedures. B, HEK293T cells were transiently transfected with the indicated plasmids. FRET between cerulean- and citrine-tagged WT MyD88 or cerulean- and citrine-tagged S34Y was calculated by measuring SE fluorescence using FRET SE wizard on the Leica SP2 confocal laser-scanning microscope. The FRET efficiency is shown as a color-coded scale of values between 0 and 100%.

FIGURE 6.

S34Y does not traffic to the cell membrane. A, macrophages expressing either citrine-tagged MyD88 WT or S34Y were imaged using standard confocal microscopy. The cells were either left untreated (left panel) or stimulated with LPS (100 ng/ml) for 45 min (right panel). B, HEK293T cells were transfected with cerulean-tagged Mal either by itself or in conjunction with citrine-tagged WT MyD88 or S34Y. The next day the cells were imaged using standard confocal microscopy procedures. C, HEK293T cells were transiently transfected with the indicated plasmids. FRET between Mal and WT MyD88 or S34Y was calculated by measuring SE fluorescence using FRET SE wizard on the Leica SP2 confocal laser-scanning microscope. The FRET efficiency is shown as a color-coded scale of values between 0 and 100%.

FIGURE 7.

S34Y fails to assemble into a Myddosome. A, cerulean-tagged IRAK4 (red) was transfected either by itself (i) or in conjunction with citrine-tagged WT MyD88 (ii) or S34Y (iii) (green) into HEK293T cells. The next day, the cells were imaged using sequential scanning, and co-localization of the two proteins was assessed. These images are representative of at least three independent experiments. B, HEK293T cells were transiently transfected with the indicated plasmids. FRET between IRAK4 and WT MyD88 or S34Y was calculated by measuring SE fluorescence using FRET SE wizard on the Leica SP2 confocal laser-scanning microscope. The FRET efficiency is shown as a color-coded scale of values between 0 and 100%. C, FRET efficiency for both the samples (IRAK4-WT MyD88 and IRAK4-S34Y) was obtained by calculating the average efficiency of different regions in each FRET image. This experiment is a representative of two independent experiments.