Exposure to oxidizing conditions can induce protein disulfides in many cytosolic proteins, which can sometimes affect protein function [18
]. In this report, we present evidence that NEMO forms disulfide-bonded homodimers in vivo
. In addition, we show that Cys54 and Cys347 of NEMO are required for disulfide bond formation by NEMO, and our predicted structural model of the NEMO dimer indicates that Cys54-Cys54 and Cys347-Cys347 disulfide bonds can form. We cannot distinguish whether there are separate populations of NEMO dimers containing either Cys54-Cys54 or Cys347-Cys347 disulfides or whether both disulfide bonds usually occur in a single dimer. Nevertheless, our analysis of C54A and C347A single mutants shows that NEMO dimers containing either single disulfide bond can exist in cells. Marienfeld et al.
] also showed that NEMO migrates as a dimer on SDS-polyacrylamide gels; however, they did not characterize the NEMO dimer as a disulfide-bonded species. Similarly, Drew et al.
] showed that some fragments of NEMO, most of which include either Cys54 or Cys347, form stable high molecular weight species.
Our molecular model of NEMO predicts that each monomer has an elongated α-helical structure and that these monomers are positioned in a head-to-head orientation in the dimer. These structural properties are consistent with gel filtration [6
], chemical crosslinking [7
] and circular dichroism [19
] experiments. Our model positions the leucine zipper to mediate dimerization of NEMO, consistent with mutagenesis studies showing that these leucine residues are required for NEMO oligomerization [5
]. Our model also places NEMO residues involved in interaction with IKKβ [19
] towards the outside of the NEMO dimer.
Fontan et al.
] reported that NEMO is mainly a monomer in unstimulated mouse fibroblasts. Indeed, in NEMO-deficient mouse cells reconstituted for NEMO expression, we find that most NEMO protein appears as a monomer (). In contrast, the monomer-to-dimer ratio of endogenous NEMO differs in various human cell types (). Therefore, we believe that there exists an equilibrium of NEMO monomer-to-dimer, which may vary in different cell types depending on the redox state of the cytosol in a given cell. Furthermore, our results enable us to speculate that formation of the disulfide-bonded NEMO dimer can be modulated by phosphorylation of Ser68 [20
The NEMO mutant C54/347A, which cannot efficiently form disulfide-bonded dimers, has a delayed and blunted ability to activate NF-κB DNA binding in response to acute one-time treatment with TNF. Nevertheless, there is no difference between NEMO-deficient fibroblasts reconstituted with wild-type vs C54/347A mutant NEMO in sustained reporter gene activation in response to TNF or the long-term survival of cells after treatment with TNF (Fig. S1
). Thus, although the C54/347A NEMO mutant is slightly defective in short-term activation, it is not clear if such a mutant would have severe biological consequences. Of note, Marienfeld et al.
] reported that a NEMO mutant missing amino acids 47-56 (which includes Cys54) also has a minor defect in short-term NF-κB activation in response to TNFα and a reduced ability to form dimers on SDS-polyacrylamide gels. Whether the defects in NEMOΔ47-56 and Cys54/347A mutants for activation of NF-κB are due to their reduced abilities to form disulfide-bonded dimers is not clear.
Activation of NF-κB by oxidative stress appears to be cell type-specific and dose-dependent [10
]. Moreover, in several instances, oxidative stress has been reported to impair NF-κB signaling [10
]. Here we show that H2
at a physiological concentration (200 μM) inhibits TNFα-induced IKK activation, and that H2
exerts the same effects in cells expressing either wild-type NEMO or NEMO-C54/347A. This result is consistent with H2
directly inhibiting IKKβ’s kinase activity, as reported previously [21
]. Therefore, although we show that H2
can modulate the NEMO monomer vs dimer ratio, H2
most likely inhibits IKK activation by directly affecting IKKβ in our system.
Previously, it has been demonstrated that p50, p65, c-Rel and IKKβ are targets of Cys-mediated protein modification [10
]. This report is the first demonstration that NEMO also contains redox-sensitive Cys residues. Further characterization of NEMO’s Cys residues may identify novel forms of redox regulation of NF-κB signaling.