Redox sensitivity of the MyD88 immune signaling adapter

Redox sensitivity of the MyD88 immune signaling adapter

Free Radical Biology and Medicine 101 (2016) 93–101 Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: ww...

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Free Radical Biology and Medicine 101 (2016) 93–101

Contents lists available at ScienceDirect

Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Redox sensitivity of the MyD88 immune signaling adapter ⁎

Benjamin Stottmeier, Tobias P. Dick

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Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

A R T I C L E I N F O

A BS T RAC T

Keywords: MyD88 Thiol oxidation Hydrogen peroxide NF-κB Redox signaling

The transcription factor nuclear factor-κB (NF-κB) mediates expression of key genes involved in innate immunity and inflammation. NF-κB activation has been repeatedly reported to be modulated by hydrogen peroxide (H2O2). Here, we show that the NF-κB-activating signaling adapter myeloid differentiation primary response gene 88 (MyD88) is highly sensitive to oxidation by H2O2 and may be redox-regulated in its function, thus facilitating an influence of H2O2 on the NF-κB signaling pathway. Upon oxidation, MyD88 forms distinct disulfide-linked conjugates which are reduced by the MyD88-interacting oxidoreductase nucleoredoxin (Nrx). MyD88 cysteine residues functionally modulate MyD88-dependent NF-κB activation, suggesting a link between MyD88 thiol oxidation state and immune signaling.

1. Introduction Intracellular signal transduction and H2O2 production can be closely linked. Various extracellular signals, including peptide growth factors [1,2], cytokines [3,4] and agonists of heterotrimeric G proteincoupled receptors [5,6], trigger an elevation of intracellular H2O2 levels upon activation of their specific membrane receptors. H2O2 production is suggested to remain localized to the activated receptor complexes due to the specific activation of co-localized NADPH oxidases (NOX) [7]. NOX enzymes transfer electrons from NADPH to oxygen to generate superoxide, which is then dismutated into H2O2 [8]. H2O2 has been found to act as an intracellular messenger, regulating signal transduction downstream of the activated receptor complexes. H2O2 influences signal transduction by modulating the function of redoxsensitive signaling proteins through reversible oxidation of cysteine residues, which includes the formation of intra- and intermolecular disulfide bonds. A number of signaling proteins have been identified to be redox-regulated, including protein kinases such as Ask1 [9], protein phosphatases such as PTP1B [10] and transcription factors such as STAT3 [11]. The transcription factor NF-κB is an important regulator of innate immunity and inflammation. NF-κB controls the expression of proinflammatory cytokines and other effector proteins involved in the immune response. Activation of NF-κB signaling by cytokines such as IL-1ß and TNF-α or bacterial components such as lipopolysaccharide (LPS) coincides with NOX-dependent H2O2 production [12–14]. H2O2 may oxidize unidentified components of the NF-κB signaling pathway

and thereby influence NF-κB activity [15]. Efforts have been made to characterize possible redox-regulated proteins in the NF-κB signaling pathway. Suggested protein targets for oxidation-dependent regulation of NF-κB activity include NF-κB itself [16] and members of the IκB kinase family [17], which target the NF-κB inhibitor IκBα for degradation. Nevertheless, also proteins of the initial receptor-bound signaling complex have been suggested as possible targets [18]. Yet, specific targets remain unidentified. Recently, the oxidoreductase nucleoredoxin (Nrx) was identified as a potential regulator of NF-κB signaling, acting downstream of the LPS-sensing Toll-like receptor 4 (TLR4) [19]. Nrx shows sequence similarity to the oxidoreductase thioredoxin (Trx) and was shown to reduce disulfide bonds in vitro [20]. The second of two Trx-like domains of Nrx contains a catalytic WCPPC motif similar to the WCGPC motif of Trx. The influence of Nrx on NF-κB activity depends on its direct binding to the adapter protein MyD88 [19]. This interaction between the oxidoreductase Nrx and MyD88 suggested to us that MyD88 may be redox-sensitive and redox-regulated. MyD88 is a signaling adapter involved in NF-κB activation downstream of IL-1R, IL-18R and most TLRs [21–24]. MyD88 comprises a N-terminal death domain (DD), a short intermediate domain (ID) and a C-terminal Toll/ IL-1R homology (TIR) domain [25]. The MyD88 TIR domain mediates recruitment of MyD88 to activated membrane receptor complexes, while the MyD88 DD homotypically interacts with downstream kinases of the IL-1R-associated kinase (IRAK) family [23]. Upon receptor activation, oligomerization of MyD88 and IRAK proteins leads to formation of a large signal initiation complex termed the Myddosome

Abbreviations: DTT, dithiothreitol; EV, empty vector; IB, immunoblotting; IL-1R, interleukin-1 receptor; MW, molecular weight; MyD88, myeloid differentiation primary response gene 88; NEM, N-ethylmaleimide; NF-κB, nuclear factor kappa B; Nrx, nucleoredoxin; TLR, Toll-like receptor; Trx, thioredoxin; WCL, whole cell lysate ⁎ Corresponding author. E-mail address: [email protected] (T.P. Dick). http://dx.doi.org/10.1016/j.freeradbiomed.2016.10.004 Received 24 May 2016; Received in revised form 19 September 2016; Accepted 3 October 2016 Available online 05 October 2016 0891-5849/ © 2016 Elsevier Inc. All rights reserved.

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κB sites was kindly provided by the laboratory of P.H. Krammer. All reagents were purchased from Sigma-Aldrich unless stated otherwise.

[26]. In this study, we report on observations suggesting that MyD88 may be a mediator of oxidation-dependent regulation of the NF-κB signaling pathway. We demonstrate that MyD88 contains multiple cysteines which are highly sensitive to oxidation by H2O2, forming intermolecular disulfide-linked conjugates and influencing NF-κB activation. We observe that MyD88 is oxidized by low amounts of H2O2 and forms distinct disulfide-linked conjugates with other proteins. By trapping disulfide exchange intermediates, we show that oxidized MyD88 is reduced by Nrx. Moreover, we show a functional impact of MyD88 cysteines on NF-κB signaling pathway activation, potentially linking MyD88 thiol oxidation status to signaling functions.

2.3. Transfection HEK293T cells were transfected using polyethylenimine (Polysciences) and HEK293 cells were transfected using Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. 2.4. Titration of MyD88 oxidation HEK293T cells were transfected with MyD88-Flag in 6-well plates. After 48 h, cells were pulsed with different concentrations of H2O2 for 3 min and then free thiols were blocked by incubation with an ice-cold solution of 100 mM N-ethylmaleimide (NEM) for 5 min. Cells were lysed in 1% NP-40/HEPES buffer (40 mM HEPES, 50 mM NaCl, 1 mM EDTA) supplemented with EDTA-free Complete Protease Inhibitor Cocktail Tablets (Roche) and 20 mM NEM. Cleared lysates were subjected to SDS-PAGE and Western blotting.

2. Materials and methods 2.1. Cell culture HEK293 and HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies) and HAP1 cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM; Life Technologies). Media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Life Technologies) and 100 units/ml of penicillin/streptomycin (Life Technologies). Cells were incubated at 37 °C under humidified atmosphere with 5% CO2.

2.5. Analysis of MyD88 mixed disulfides HEK293 cells were transfected with MyD88-Myc cysteine mutants in 12-well plates. After 48 h, cells were pulsed with 100 μM H2O2 for 3 min and then free thiols were blocked with NEM. Cells were lysed and cleared lysates were subjected to SDS-PAGE and Western blotting.

2.2. DNA constructs and reagents ORFs for Nrx (UniProt identifier: Q6DKJ4-1) and MyD88 (UniProt identifier: Q99836-6) and Gateway destination vectors encoding a Cterminal Flag or Myc tag or a N-terminal Myc tag were provided by the German Cancer Research Center Genomics & Proteomics Core Facility. ORFs were cloned into the respective destination vectors using the Gateway recombination system (Life Technologies). Mutations were introduced using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) or by insertion of synthetic, mutated ORF fragments (GenScript) using available restriction sites. An empty vector control was generated by ORF deletion from an untagged pEXP-Nrx construct using restriction digest and the Q5 Site-Directed Mutagenesis Kit. A pGL-based NF-κB firefly luciferase reporter construct with eight

2.6. Nrx mechanism-based kinetic trapping experiments HEK293T cells were transfected with cysteine mutants of Nrx-Flag as well as MyD88-Myc or Myc-MyD88 in 6-well plates. After 48 h, cells were or were not pulsed with 100 μM H2O2 for 3 min and then free thiols were blocked with NEM. Cells were lysed and cleared lysates were incubated with 12 µl anti-Flag beads (ANTI-FLAG M2 Magnetic Beads) for 4 h. Beads were washed three times with TBS (10 mM Tris, 150 mM NaCl, pH 7.4) and protein was eluted by incubation of beads with 150 µg/ml Flag peptide (3x FLAG Peptide) in TBS for 30 min. Eluates were subjected to SDS-PAGE and Western blotting.

Fig. 1. H2O2 induces formation of MyD88 disulfide conjugates. (A) HEK293T cells were transfected with MyD88-Flag for 48 h and then pulsed with increasing concentrations of H2O2 for 3 min, after which free thiols were blocked with 100 mM NEM. Cells were lysed, samples were separated by SDS-PAGE under non-reducing or reducing conditions and immunoblotted (IB) as indicated. Immunoblots are representative of n =2 independent experiments. MW: molecular weight. (B) Quantification of total H2O2-induced MyD88 disulfide conjugates (MyD88-S-S-X) of n=2 independent experiments. All values were normalized to the untreated control.

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5 min, subjected to SDS-PAGE and transferred to polyvinyldifluoride (PVDF) membranes (Immobilon-P; Millipore). Antibodies used for immunoblotting were mouse anti-Flag (Sigma-Aldrich, F3165), goat anti-Prx2 (R & D Systems, AF3489), mouse anti-ß-actin (SigmaAldrich, A5441), rabbit anti-Myc (Cell Signaling Technology, #2278) and rabbit anti-MyD88 (Cell Signaling Technology, #4283). Signals were visualized with a Peqlab Fusion-SL system using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific).

2.7. Time course of endogenous MyD88 oxidation HAP1 cells in 6-well plates were pulsed with 100 µM H2O2 for the indicated time spans and then free thiols were blocked with NEM. Cells were lysed, and cleared lysates were subjected to SDS-PAGE and Western blotting. 2.8. TrxR inhibition using auranofin HAP1 cells in 6-well plates were treated with different concentrations of auranofin for 1 h and then free thiols were blocked with NEM. Cells were lysed, and cleared lysates were subjected to SDS-PAGE and Western blotting.

2.10. NF-κB luciferase reporter assay HEK293 cells were transfected with MyD88-Flag cysteine mutants, a NF-κB firefly luciferase reporter and a Renilla luciferase reporter in 96-well plates. To analyze LPS-induced signaling events, TLR4 and MD-2 were co-transfected and after 24 h cells were stimulated with 2.2 ng/ml LPS. After 48 h, luciferase reporter assays were performed using the Dual-Glo Luciferase Assay System (Promega) according to manufacturer's instructions and luminescence was read in a BMG Labtech PHERAstar FS micro plate reader. The firefly luciferase signal

2.9. SDS-PAGE and Western blotting Samples were mixed with 4x non-reducing LDS sample buffer (Roth) and half of each sample was reduced by incubation with 20 mM DTT (AppliChem) for 4 min. All samples were heated to 95 °C for

Fig. 2. Multiple MyD88 cysteines control the formation of at least seven distinct disulfide-linked conjugates. (A,C-F) HEK293 cells were transfected with MyD88-Myc wild type (wt) or with the indicated cysteine mutants for 48 h and then pulsed with 100 μM H2O2 for 3 min, after which free thiols were blocked with 100 mM NEM. Cells were lysed, samples were separated by SDS-PAGE under non-reducing conditions and immunoblotted (IB) as indicated. Immunoblots are representative of n ≥2 independent experiments. Reducing gels are shown in Supplementary Fig. S3. EV: empty vector. (B) Positions of the nine MyD88 cysteine residues in relation to the MyD88 domain structure. DD: Death domain. ID: Intermediate domain. TIR: Toll/IL-1 receptor homology domain.

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conjugate 4 depends on the prior formation of conjugate 5 but additionally requires another cysteine contributed by the C-terminal half of the TIR domain. The combined mutation of the four cysteines in the C-terminal half of the TIR domain (C224, C241, C282 and C288) (Fig. 2F and Supplementary Fig. S3E) led to the complete loss of conjugates 1, 2, 3 and 4. The triple mutant C241A/C282A/C288A yielded the same result, identifying these three residues as key determinants for the formation of conjugates 1–4. Formation of conjugate 5 was not impaired, confirming its exclusive dependence on the cysteines in the N-terminal half of the TIR domain. Formation of conjugate 4 appears to require one of the four cysteines in the N-terminal half and one out of three cysteines (C241, C282, C288) in the C-terminal half of the TIR domain. Only the combined mutation of C241, C282 and C288 completely prevented formation of complex 3, in line with the previous findings that C241 was sufficient to form complex 3 (Fig. 2D) although its single mutation did not impair complex 3 formation (Fig. 2C). Formation of conjugate 2 strictly required the concomitant presence of C224 and C241, as already suggested by the single mutants. Also consistent with previous results, formation of conjugate 1 was strongly diminished in all variants lacking C288, although C224 and C282 could partially compensate for the absence of C288, as seen before. Taken together, we found MyD88 cysteines to be partially interchangeable in forming seven distinct mixed disulfides. Nevertheless, we were able to assign subgroups of MyD88 cysteines to the formation of individual conjugates (Table 1). In summary, the cysteines of the Nterminal half of the TIR domain are essential for the generation of conjugates 4 and 5 and the cysteines of the C-terminal half of the TIR domain are essential for the formation of conjugates 1–4.

was normalized to the Renilla luciferase signal. 3. Results 3.1. MyD88 is sensitively oxidized by H2O2 We started out by asking if MyD88 is sensitive to oxidation by exogenously applied H2O2. To this end, we ectopically expressed MyD88 in HEK293T cells. We then exposed the cells to increasing concentrations of exogenous H2O2. A fraction of the total MyD88 pool formed disulfide-linked conjugates of high molecular weight in response to H2O2 bolus concentrations as low as 5 µM (Fig. 1). Of note, MyD88 was nearly as sensitively oxidized as the thiol peroxidase peroxiredoxin-2 (Prx2) which exhibited increased covalent dimerization at about the same H2O2 concentration. We confirmed that H2O2dependent MyD88 oxidation can also be observed in a different cell line expressing detectable levels of endogenous MyD88 (Supplementary Fig. S1). MyD88 oxidation products also formed in response to thioredoxin reductase (TrxR) inhibition (Supplementary Fig. S2), suggesting that endogenous H2O2 production is sufficient to facilitate MyD88 oxidation when thiol reducing systems are compromised. In summary, we observed that MyD88 is a H2O2-sensitive protein, forming high-molecular-weight disulfide-linked conjugates of unknown nature. 3.2. MyD88 forms at least seven distinct disulfide-linked conjugates Upon exposure to H2O2, wild type MyD88 formed a number of conjugates, seven of which were distinguishable by gel mobility (Fig. 2A and Supplementary Fig. S3A). These were labeled with numbers from 1 to 7, in sequence of increasing apparent molecular weight. Conjugates 1-5 are in the weight range between 75 and 150 kDa and complexes 6 and 7 exceed a weight of 250 kDa. As expected, a MyD88 variant lacking all cysteines did not form any conjugates. We then asked which MyD88 cysteines are involved in the formation of the individual disulfide-linked conjugates. To this end, we separately mutated each of the nine MyD88 cysteine residues (Fig. 2B) to alanine, expressed the mutants in HEK293 cells and analyzed the formation of disulfide-linked conjugates in response to H2O2. The C224 and C241 single mutations each resulted in the complete loss of conjugate 2. The C241 and C288 single mutations each diminished formation of conjugate 1. All other single cysteine mutations did not appreciably alter the conjugate banding pattern, suggesting that cysteines can compensate for each other's absence (Fig. 2C and Supplementary Fig. S3B). Thus, in the next step, we analyzed MyD88 mutants retaining just one cysteine (Fig. 2D and Supplementary Fig. S3C). C288 alone was sufficient to allow formation of conjugate 1. C224 and C282 also allowed formation of conjugate 1, albeit with lower efficiency. This explains why the single mutation of C288 did not lead to a complete loss of conjugate 1 (Fig. 2C). The presence of C241 allowed formation of conjugate 3, although the individual C241 mutation did not impair conjugate 3 formation (see Fig. 2C). Cysteines C166, C168, C192 and C203 all enabled formation of conjugate 5, albeit with varying efficiency. Their individual mutation did not alter the conjugate pattern (Fig. 2C), suggesting that these four cysteines compensate for each other in terms of enabling the formation of conjugate 5. Finally, C113 was the only cysteine that did not lead to any conjugate formation. We then investigated the combined mutation of cysteine subsets. The combined mutation of the four cysteines in the N-terminal half of the TIR domain (C166, C168, C192 and C203) (Fig. 2E and Supplementary Fig. S3D) abolished formation of conjugate 5, as would have been predicted from the previous experiment. But the same quadruple mutation also resulted in the complete loss of conjugate 4 which was not formed by any of the MyD88 variants with a single cysteine (see Fig. 2D), suggesting the possibility that formation of

3.3. Nucleoredoxin reduces MyD88 disulfide-linked conjugates Given the oxidation sensitivity of MyD88 and its known interaction with Nrx, we asked if MyD88 engages in disulfide exchange with Nrx. To this end, we co-expressed MyD88 and Nrx cysteine mutants and tested for mechanism-based kinetic trapping of MyD88 by Nrx in the presence of H2O2. Mutation of Nrx C208, the C-terminal cysteine in the catalytic CXXC motif of the second Trx-like domain of Nrx, resulted in the trapping of other proteins (Fig. 3A and Supplementary Fig. S4A). Correspondingly, MyD88 was trapped by the Nrx C208S mutant, with which it formed two distinct covalent complexes (≈200 kDa and ≫250 kDa) of high molecular weight (Fig. 3B and Supplementary Fig. S4B). These complexes exceeded the expected size of a Nrx-MyD88 one-to-one conjugate (≈90 kDa), either suggesting a different stoichiometric relationship or the involvement of other proteins as part of the trapped complex. The same experiment confirmed that MyD88 also interacts non-covalently with Nrx. To analyze the H2O2-dependency of the Nrx-MyD88 mixed disulfide conjugate, we compared MyD88 trapping by Nrx C208S in the presence and absence of an exogenous H2O2 bolus (Fig. 3C and Supplementary Fig. S4C). Trapping of MyD88 by Nrx only occurred in the presence of H2O2, confirming that H2O2 induces disulfide exchange between MyD88 and Nrx. The non-covalent interaction between Nrx and MyD88 was independent of the presence of H2O2. In conclusion, we found that MyD88 and Nrx engage in Table 1 MyD88 cysteines involved in the formation of disulfide conjugates 1–5. Conjugate Number

Cysteines

1

288 (major) OR 224 OR 282 MW compatible with homodimer 224 AND 241 241 (major) OR 282 OR 288 [166 OR 168 OR 191 OR 203]* AND [241 OR 282 OR 288] *this conjugate may secondarily form from conjugate 5 166 OR 168 OR 191 OR 203

2 3 4 5

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Fig. 3. Nucleoredoxin engages in disulfide exchange with MyD88 disulfide-linked conjugates. (A-C) HEK293T cells were transfected with the indicated expression constructs for 48 h and then pulsed or not pulsed with 100 μM H2O2 for 3 min, after which free thiols were blocked with 100 mM NEM. Cells were lysed and Nrx was immunoprecipitated (IP) with antiFlag beads. Samples were separated by SDS-PAGE under non-reducing conditions and immunoblotted (IB) as indicated. (A) HEK293T cells were transfected with Nrx-Flag wild type or with the indicated cysteine mutants and pulsed with H2O2. (B) HEK293T cells were transfected with Myc-MyD88 and with either Nrx-Flag wild type or with the indicated Nrx-Flag cysteine mutants and pulsed with H2O2. (C) HEK293T cells were transfected with MyD88-Myc and with Nrx-Flag wild type or with the indicated Nrx-Flag cysteine mutants and pulsed or not pulsed with H2O2. All immunoblots are representative of n≥2 independent experiments. Reducing gels are shown in Supplementary Fig. S4. Unfilled arrows: Nrx-trapped complexes. WCL: whole cell lysate.

C192, C203 or C282 were most prominently trapped by Nrx. C241 and C288 also facilitated trapping, albeit with lower efficiency. In contrast, C113 did not facilitate detectable trapping. This finding is broadly in line with the previous observation that all cysteines except C113 were involved in forming disulfide-containing oxidation products. In all cases only the lighter of the two trapping products (≈200 kDa) was formed, suggesting that formation of the other (≫250 kDa) requires a combination of MyD88 cysteines to be present. Notably, the size of the trapping product (≈200 kDa) was exactly the same for all MyD88 mutants, despite the variety of H2O2 induced disulfide conjugates formed by the different mutants (see Fig. 2D). This finding suggests that Nrx removes other covalent interaction partners when it attacks MyD88, always yielding the same trapping product, which may be a 2:2 complex.

disulfide exchange in response to H2O2, probably indicating the reduction of a disulfide bond by the second Trx-like domain of Nrx, which also forms transient disulfide exchange intermediates with other proteins. Apart from their transient covalent interaction, MyD88 and Nrx maintain a non-covalent interaction which seems to be independent of the MyD88 oxidation state and the presence of H2O2.

3.4. Nucleoredoxin reduces distinct MyD88 cysteines We then asked which MyD88 cysteines are involved in the disulfide exchange between Nrx and MyD88. As expected, absence of all cysteines from MyD88 completely abolished trapping by Nrx (Fig. 4A and Supplementary Fig. S5A). As individual cysteine mutants of MyD88 did not influence trapping by Nrx (data not shown), we tested MyD88 variants lacking all cysteines except one (Fig. 4B and Supplementary Fig. S5B). MyD88 variants containing C166, C168, 97

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Fig. 4. Nucleoredoxin targets distinct MyD88 cysteines. HEK293T cells were transfected with Nrx-Flag and MyD88-Myc wild type or with the indicated cysteine mutants of both proteins for 48 h and then pulsed with 100 μM H2O2 for 3 min, after which free thiols were blocked with 100 mM NEM. Cells were lysed and Nrx was immunoprecipitated (IP) with antiFlag beads. Samples were separated by SDS-PAGE under non-reducing conditions and immunoblotted (IB) as indicated. Immunoblots are representative of n≥2 independent experiments. Reducing gels are shown in Supplementary Fig. S5. WCL: whole cell lysate.

NF-κB reporter assays in HEK293 cells. First, we confirmed dosedependent activation of NF-κB signaling by expressing increasing levels of MyD88 (Fig. 5). Next, we tested the signaling capacity of individual cysteine mutants of MyD88 (Fig. 6A). While mutation of C113 led to a significant loss of NF-κB signaling activity, individual mutation of all the other cysteines led to a marked increase. Notably, all cysteines whose individual mutation led to an increase in signaling activity were located in the MyD88 TIR domain. Simultaneous mutation of all cysteines present in the MyD88 TIR domain also led to an

3.5. MyD88 cysteines modulate NF-κB signaling Finally, we asked if the MyD88 cysteines influence MyD88-dependent NF-κB signaling. To evaluate the signaling capacity of MyD88 cysteine mutants, we established a MyD88 self-aggregation assay. Ectopic expression of MyD88 is known to promote MyD88 selfaggregation and to activate MyD88-dependent signaling pathways in the absence of other stimuli [22,27]. We assessed the signaling capacity of MyD88 cysteine mutants in the self-aggregation assay by performing 98

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MyD88 variant lacking all cysteines had a signaling activity similar to the wild type protein, indicating that the positive influence of C113 and the negative effect of the TIR domain cysteines balance each other out. Stimulation with LPS of cells co-expressing TLR4 yielded similar results (Supplementary Fig. S6). We then analyzed the MyD88 variants containing only a single cysteine (Fig. 6B). In line with our previous results, the presence of only one cysteine in the MyD88 TIR domain led to signaling activity similar to cysteine-less MyD88. This confirmed that the presence of single cysteines in the TIR domain is not sufficient to negatively influence signaling activity, and that the combined presence of several TIR domain cysteines is necessary to do so. We therefore tested the MyD88 variants with combined mutation of cysteine subsets. We first tested combined mutations of the four cysteines in the N-terminal half of the TIR domain (Fig. 6C). All tested combinations led to a signaling activity which was by trend higher (though not significant by statistics) than the mutation of the individual cysteines. We then tested combined mutations of the four cysteines in the C-terminal half of the TIR domain (Fig. 6D). The combined mutation of C224 and C288, with or without C241, increased signaling activity to levels exceeding those exhibited by MyD88 variants lacking all TIR domain cysteines. Other triple and quadruple mutations again led to a signaling activity comparable to the absence of all TIR domain cysteines. In conclusion, MyD88 cysteines influence MyD88 signaling

Fig. 5. Dose-dependent activation of NF-κB signaling by MyD88. HEK293 cells were transfected with increasing concentrations of MyD88-Flag, a NF-κB firefly luciferase reporter and a Renilla luciferase reporter for 48 h before reporter assay analysis. Plot is representative of n=3 independent experiments. Each value represents the mean ± SD. RLU: relative light units.

increase in signaling activity, which was more pronounced than the increase caused by individual mutations, suggesting a cumulative negative effect of TIR domain cysteines on signaling activity. The

Fig. 6. MyD88 cysteines regulate MyD88-dependent NF-κB activation. (A-D) HEK293 cells were transfected with MyD88-Flag wild type or with the indicated cysteine mutants, a NF-κB firefly luciferase reporter and a Renilla luciferase reporter for 48 h before reporter assay analysis. Signals were normalized to an empty vector control. Plots represent quantification of n≥3 independent experiments. Each value represents the mean ± SD. * p < 0.01, ** p < 0.001, *** p < 0.0001. (A,C,D) Statistical significance in comparison to MyD88 wild type is p < 0.0001 unless stated otherwise. (B) No statistical significance in comparison to MyD88 wild type unless stated otherwise. RLU: relative light units. n.s.: not significant.

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for all MyD88 mutants, including those harboring just one cysteine, it may be speculated that the disulfide connectivity within the (Nrx)2: (MyD88)2 complex is MyD88-S-S-Nrx-S-S-Nrx-S-S-MyD88. Thus, it would be predicted that Nrx uses one of its other cysteines (outside the CXXC motif) to form a covalent Nrx dimer as part of the overall complex. Alternatively, an unknown additional protein may be covalently attached to Nrx within a 1:1 Nrx-S-S-MyD88 disulfide conjugate, raising the overall molecular weight from ≈90 to ≈180 kDa. An argument in favor of the (Nrx)2:(MyD88)2 stoichiometry is that Nrx normally exists as a non-covalent dimer, as previously shown for the plant homologue of Nrx [31] and confirmed in our laboratory for human Nrx using a bimolecular fluorescence complementation assay (data not shown). MyD88 also forms homodimers in vivo [27]. Noncovalent Nrx dimers may be oxidized to covalent Nrx dimers, prior to or upon interaction with dimeric MyD88 conjugates. How Nrx and MyD88 dimers interact in detail remains to be studied. The elucidation of this process may shed light on the general function and mechanism of Nrx, which remains largely obscure. Notably, MyD88 cysteine 224 does not take part in a disulfide exchange with Nrx, although it is involved in the formation of disulfidelinked conjugates. This suggests that some conjugates are reduced or rearranged by oxidoreductases other than Nrx. Indeed, we observed MyD88 to form mixed disulfide intermediates with Trx (data not shown). Thus, Nrx and Trx may complement each other by reducing different subsets of MyD88 oxidation products. We observed MyD88 cysteines to influence NF-κB signaling, compatible with the hypothesis that MyD88 thiol oxidation modulates downstream signaling events. Thus far, there has been a single report connecting thiol redox state of MyD88 to signaling, namely S-nitrosylation of the cysteine corresponding to C224 in our study, suggested to influence the MyD88 interaction with TIRAP [32]. We observed that all MyD88 cysteines either promoted or inhibited NF-κB activation, dependent on their localization (Fig. 6). While all TIR domain cysteines inhibited MyD88 function, the presence of C113 enhanced MyD88dependent NF-κB activation. C113 is close to the DD, but appears to be located outside the protein-protein interaction interface that is involved in Myddosome assembly [26]. However, the region surrounding C113 seems to be important for signal initiation, as MyD88 short (MyD88s), a splice variant missing the ID, including C113, is incapable of interacting with IRAK4 or of activating NF-κB [33,34]. Thus, C113, and potentially its oxidation, may be crucial for efficient assembly of DDs into the Myddosome.

capacity and thus impact on NF-κB signaling activity. NF-κB signaling pathway activity is positively influenced by the presence of MyD88 C113. In contrast, the cysteines localized in the MyD88 TIR domain appear to have a negative influence on signaling. We identified cysteines C224 and C288 to be mainly responsible for the negative impact on NF-κB signaling activity. These findings suggest a link between the formation of mixed disulfides by MyD88 and MyD88's signaling function. 4. Discussion Activation of the NF-κB transcription factor has long been known to be influenced by H2O2. Here, we propose that the signaling adapter MyD88 is a candidate target of redox regulation within the NF-κB signaling pathway. We observed that MyD88 is sensitive to H2O2dependent thiol oxidation (Fig. 1). We found MyD88 to be almost as sensitively oxidized as the thiol peroxidase Prx2, suggesting that MyD88 can be oxidized by signaling concentrations of endogenous H2O2 under physiological conditions. MyD88 oxidation resulted in the formation of distinct mixed disulfides which involved eight of its nine cysteine residues. Notably, all nine MyD88 cysteines are conserved amongst the vertebrates. MyD88 crystal structures [26,28,29] reveal that all thiols are either fully exposed or localized very close to the protein surface, suggesting their accessibility to oxidants or mediators of oxidation. The known structures do not contain intramolecular disulfide bonds and do not suggest that MyD88 cysteines are structurally relevant. Indeed, this is in line with our observations that MyD88 cysteine mutations to not affect protein expression levels (Fig. 2) and, remarkably, that a MyD88 mutant lacking all nine cysteines had wild type activity in the NF-κB reporter assay (Fig. 6). We observed that H2O2-dependent MyD88 oxidation results in the formation of various disulfide-linked conjugates, which involve the eight cysteines of the TIR domain. At least some of the mixed disulfide conjugates seem to contain unknown proteins, as most complexes did not match the expected molecular weight of MyD88 multimers or were formed by MyD88 mutants containing only one cysteine (Fig. 2D). The only clear exception is conjugate 1 (75 kDa), most likely representing a covalent MyD88 dimer (Fig. 2), whose formation is mostly mediated by cysteine 288, but may also be formed through C224 or C282. Indeed, MyD88 was previously shown to exist as a non-covalent homodimer in the cytoplasm, mediated by homotypic DD-DD and TIR-TIR interactions [27]. It may be speculated that the non-covalent MyD88 dimer brings the two C288 residues from the two subunits into close proximity, thus favoring disulfide bond formation at this site. C224 and C282 are close to C288 on the protein surface, potentially explaining why they can provide alternative routes to covalent dimer formation (conjugate 1). The identity of unknown proteins likely to be part of the heavier disulfide-linked complexes remains to be elucidated. Most disulfide-linked conjugates could be formed redundantly by alternative cysteines, thus requiring the mutation of several cysteines to completely abolish their formation (Fig. 2). Similar observations have been made with other redox-sensitive signaling proteins such as Ask1, FoxO or STAT3 [9,11,30]. For example, in the case of Ask1, seven of its 23 cysteines had to be mutated to achieve near-complete inhibition of H2O2-induced oligomerization [9]. Overall, the involvement of multiple (alternative) cysteine residues might be a common feature of many redox-sensitive proteins forming mixed disulfides. Nevertheless, we observed that alternative cysteines displayed variable efficiency in the formation of each specific disulfide conjugate, suggesting that certain cysteines are favored over others. We found that MyD88 disulfide conjugates can be reduced by the Trx family member Nrx, known to be able to reduce disulfide bonds [20]. The size of the trapped mixed disulfide intermediate may be explained by a (Nrx)2:(MyD88)2 stoichiometry, with a predicted molecular weight of ≈180 kDa, compatible with the observed mobility (Figs. 3B, 4B). Considering that the size of this conjugate was uniform

5. Conclusions In this study we have collected evidence suggesting that MyD88 might be a key mediator of H2O2-dependent redox regulation of the NF-κB signaling pathway. We demonstrate that MyD88 is highly sensitive to thiol oxidation and forms distinct disulfide-linked conjugates with itself and other proteins. The oxidoreductase Nrx engages in disulfide exchange with oxidized MyD88, presumably acting as a reductase. MyD88-dependent NF-κB signaling pathway activation is influenced by MyD88 cysteines, suggesting a link between MyD88 thiol oxidation and signal transmission. Acknowledgements We thank Alexander Dalpke (University Hospital Heidelberg) for cell lines, plasmids and technical advice, Stephanie Neuhaus for Nrx bimolecular fluorescence complementation constructs, and Britta Statz and Gaby Kuntz for technical assistance. This work has been supported by the Deutsche Forschungsgemeinschaft (SFB938). Appendix A. Supplementary material Supplementary data associated with this article can be found in the 100

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[18] K. Asehnoune, D. Strassheim, S. Mitra, J.Y. Kim, E. Abraham, Involvement of reactive oxygen species in toll-like receptor 4-dependent activation of NF-κB, J. Immunol. 172 (2004) 2522–2529. http://dx.doi.org/10.4049/jimmunol.172.4.2522. [19] T. Hayashi, Y. Funato, T. Terabayashi, A. Morinaka, R. Sakamoto, H. Ichise, H. Fukuda, N. Yoshida, H. Miki, Nucleoredoxin negatively regulates toll-like receptor 4 signaling via recruitment of flightless-I to myeloid differentiation primary response gene (88), J. Biol. Chem. 285 (2010) 18586–18593. http:// dx.doi.org/10.1074/jbc.M110.106468. [20] H. Kurooka, K. Kato, S. Minoguchi, Y. Takahashi, J. Ikeda, S. Habu, N. Osawa, A.M. Buchberg, K. Moriwaki, H. Shisa, T. Honjo, Cloning and characterization of the nucleoredoxin gene that encodes a novel nuclear protein related to thioredoxin, Genomics 39 (1997) 331–339. http://dx.doi.org/10.1006/geno.1996.4493. [21] R. Medzhitov, P. Preston-Hurlburt, E. Kopp, A. Stadlen, C. Chen, S. Ghosh, C.A. Janeway Jr., MyD88 Is an adaptor protein in the hToll/IL-1 receptor family signaling pathways, Mol. Cell 2 (1998) 253–258. http://dx.doi.org/10.1016/ S1097-2765(00)80136-7. [22] M. Muzio, J. Ni, P. Feng, V.M. Dixit, IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling, Science 278 (1997) 1612–1615. http://dx.doi.org/10.1126/science.278.5343.1612. [23] H. Wesche, W.J. Henzel, W. Shillinglaw, S. Li, Z. Cao, MyD88: an adapter that recruits IRAK to the IL-1 receptor complex, Immunity 7 (1997) 837–847. http:// dx.doi.org/10.1016/S1074-7613(00)80402-1. [24] O. Adachi, T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami, K. Nakanishi, S. Akira, Targeted disruption of the MyD88 gene results in loss of IL1- and IL-18-mediated function, Immunity 9 (1998) 143–150. http://dx.doi.org/ 10.1016/S1074-7613(00)80596-8. [25] G. Hardiman, F.L. Rock, S. Balasubramanian, R.A. Kastelein, J.F. Bazan, Molecular characterization and modular analysis of human MyD88, Oncogene 13 (1996) 2467–2475. [26] S.-C. Lin, Y.-C. Lo, H. Wu, Helical assembly in the MyD88–IRAK4–IRAK2 complex in TLR/IL-1R signalling, Nature 465 (2010) 885–890. http://dx.doi.org/10.1038/ nature09121. [27] K. Burns, F. Martinon, C. Esslinger, H. Pahl, P. Schneider, J.-L. Bodmer, F.D. Marco, L. French, J. Tschopp, MyD88, an adapter protein involved in interleukin-1 signaling, J. Biol. Chem. 273 (1998) 12203–12209. http:// dx.doi.org/10.1074/jbc.273.20.12203. [28] H. Ohnishi, H. Tochio, Z. Kato, K.E. Orii, A. Li, T. Kimura, H. Hiroaki, N. Kondo, M. Shirakawa, Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling, Proc. Natl. Acad. Sci. 106 (2009) 10260–10265. http:// dx.doi.org/10.1073/pnas.0812956106. [29] G.A. Snyder, C. Cirl, J. Jiang, K. Chen, A. Waldhuber, P. Smith, F. Römmler, N. Snyder, T. Fresquez, S. Dürr, N. Tjandra, T. Miethke, T.S. Xiao, Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli, Proc. Natl. Acad. Sci. 110 (2013) 6985–6990. http://dx.doi.org/10.1073/pnas.1215770110. [30] M. Putker, T. Madl, H.R. Vos, H. de Ruiter, M. Visscher, M.C.W. van den Berg, M. Kaplan, H.C. Korswagen, R. Boelens, M. Vermeulen, B.M.T. Burgering, T.B. Dansen, Redox-dependent control of FOXO/DAF-16 by transportin-1, Mol. Cell. 49 (2013) 730–742. http://dx.doi.org/10.1016/j.molcel.2012.12.014. [31] C. Marchal, V. Delorme-Hinoux, L. Bariat, W. Siala, C. Belin, J. Saez-Vasquez, C. Riondet, J.-P. Reichheld, NTR/NRX define a new thioredoxin system in the nucleus of arabidopsis thaliana cells, Mol. Plant. 7 (2014) 30–44. http:// dx.doi.org/10.1093/mp/sst162. [32] T. Into, M. Inomata, M. Nakashima, K. Shibata, H. Häcker, K. Matsushita, Regulation of MyD88-dependent signaling events by S nitrosylation retards toll-like receptor signal transduction and initiation of acute-phase immune responses, Mol. Cell. Biol. 28 (2008) 1338–1347. http://dx.doi.org/10.1128/MCB.01412-07. [33] K. Burns, S. Janssens, B. Brissoni, N. Olivos, R. Beyaert, J. Tschopp, Inhibition of Interleukin 1 receptor/toll-like receptor signaling through the alternatively spliced, short form of MyD88 is due to its failure to recruit IRAK-4, J. Exp. Med 197 (2003) 263–268. http://dx.doi.org/10.1084/jem.20021790. [34] S. Janssens, K. Burns, E. Vercammen, J. Tschopp, R. Beyaert, MyD88S, a splice variant of MyD88, differentially modulates NF-kappaB- and AP-1-dependent gene expression, FEBS Lett. 548 (2003) 103–107.

online version at doi:10.1016/j.freeradbiomed.2016.10.004. References [1] M. Sundaresan, Z.-X. Yu, V.J. Ferrans, K. Irani, T. Finkel, Requirement for generation of H2O2 for platelet-derived growth factor signal transduction, Science 270 (1995) 296–299. http://dx.doi.org/10.1126/science.270.5234.296. [2] Y.S. Bae, S.W. Kang, M.S. Seo, I.C. Baines, E. Tekle, P.B. Chock, S.G. Rhee, Epidermal growth factor (EGF)-induced generation of hydrogen peroxide role in EGF receptor-mediated tyrosine phosphorylation, J. Biol. Chem. 272 (1997) 217–221. http://dx.doi.org/10.1074/jbc.272.1.217. [3] B. Meier, H.H. Radeke, S. Selle, M. Younes, H. Sies, K. Resch, G.G. Habermehl, Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha, Biochem. J. 263 (1989) 539–545. [4] M. Ohba, M. Shibanuma, T. Kuroki, K. Nose, Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells, J. Cell Biol. 126 (1994) 1079–1088. http://dx.doi.org/ 10.1083/jcb.126.4.1079. [5] A.M. Zafari, M. Ushio-Fukai, M. Akers, Q. Yin, A. Shah, D.G. Harrison, W.R. Taylor, K.K. Griendling, Role of NADH/NADPH oxidase–derived H2O2 in angiotensin II– induced vascular hypertrophy, Hypertension 32 (1998) 488–495. http:// dx.doi.org/10.1161/01.HYP.32.3.488. [6] C. Patterson, J. Ruef, N.R. Madamanchi, P. Barry-Lane, Z. Hu, C. Horaist, C.A. Ballinger, A.R. Brasier, C. Bode, M.S. Runge, Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin evidence that p47 phox may participate in forming this oxidase in vitro and in vivo, J. Biol. Chem. 274 (1999) 19814–19822. http://dx.doi.org/10.1074/jbc.274.28.19814. [7] M. Ushio-Fukai, Localizing NADPH oxidase–derived ROS, Sci. STKE 2006 (2006). http://dx.doi.org/10.1126/stke.3492006re8. [8] Y.-A. Suh, R.S. Arnold, B. Lassegue, J. Shi, X. Xu, D. Sorescu, A.B. Chung, K.K. Griendling, J.D. Lambeth, Cell transformation by the superoxide-generating oxidase Mox1, Nature 401 (1999) 79–82. http://dx.doi.org/10.1038/43459. [9] P.J. Nadeau, S.J. Charette, M.B. Toledano, J. Landry, Disulfide bond-mediated multimerization of Ask1 and its reduction by Thioredoxin-1 regulate H2O2induced c-Jun NH2-terminal kinase activation and apoptosis, Mol. Biol. Cell. 18 (2007) 3903–3913. http://dx.doi.org/10.1091/mbc.E07-05-0491. [10] S.-R. Lee, K.-S. Kwon, S.-R. Kim, S.G. Rhee, Reversible inactivation of proteintyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor, J. Biol. Chem. 273 (1998) 15366–15372. http://dx.doi.org/10.1074/ jbc.273.25.15366. [11] M.C. Sobotta, W. Liou, S. Stöcker, D. Talwar, M. Oehler, T. Ruppert, A.N.D. Scharf, T.P. Dick, Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling, Nat. Chem. Biol. 11 (2015) 64–70. http://dx.doi.org/10.1038/nchembio.1695. [12] X.-L. Chen, Q. Zhang, R. Zhao, R.M. Medford, Superoxide, H2O2, and iron are required for TNF-α-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase, Am. J. Physiol. - Heart Circ. Physiol. 286 (2004) H1001–H1007. http://dx.doi.org/10.1152/ajpheart.00716.2003. [13] Q. Li, M.M. Harraz, W. Zhou, L.N. Zhang, W. Ding, Y. Zhang, T. Eggleston, C. Yeaman, B. Banfi, J.F. Engelhardt, Nox2 and Rac1 regulate H2O2-dependent recruitment of TRAF6 to endosomal interleukin-1 receptor complexes, Mol. Cell. Biol. 26 (2006) 140–154. http://dx.doi.org/10.1128/MCB.26.1.140-154.2006. [14] H.S. Park, H.Y. Jung, E.Y. Park, J. Kim, W.J. Lee, Y.S. Bae, Cutting edge: direct interaction of TLR4 with NAD(P)H oxidase 4 isozyme is essential for lipopolysaccharide-induced production of reactive oxygen species and activation of NF-κB, J. Immunol. 173 (2004) 3589–3593. [15] R. Schreck, P. Rieber, P.A. Baeuerle, Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1, EMBO J. 10 (1991) 2247–2258. [16] J.R. Matthews, N. Wakasugi, J.L. Virelizier, J. Yodoi, R.T. Hay, Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62, Nucleic Acids Res 20 (1992) 3821–3830. [17] M. Herscovitch, W. Comb, T. Ennis, K. Coleman, S. Yong, B. Armstead, D. Kalaitzidis, S. Chandani, T.D. Gilmore, Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347, Biochem. Biophys. Res. Commun. 367 (2008) 103–108. http://dx.doi.org/10.1016/j.bbrc.2007.12.123.

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