In this study, we identified CCDC22 as a novel interacting protein that binds avidly to COMMD family members. Our data indicate that each COMMD protein, from COMMD1 to COMMD10, interacts with CCDC22. However, it is unlikely that this occurs in a single complex containing CCDC22 and all 10 COMMD family members at the same time. Rather, we postulate that different and distinct CCDC22-COMMD complexes exist in vivo. This is based on previously published studies indicating that COMMD proteins form dimers (1
) and on our mass spectrometry data demonstrating that only certain COMMD combinations were present in vivo (Figure A). Thus, based on the available data, we conclude that CCDC22 plays a critical role in controlling the cellular distribution of these complexes (Figure ), and in so doing, it seems to be necessary for their normal function. We postulate that the specific composition of COMMD proteins present in any given CCDC22-COMMD complex, as well as the specific CRLs to which they bind, ultimately determine their unique functions. In addition, we speculate that CCDC22 and COMMD proteins work in concert to regulate these ligases, probably acting as a complex to displace the CRL inhibitor CAND1, as recently reported in the case of COMMD1 (16
Since CRLs are involved in a vast array of cellular processes, we anticipate that CCDC22 will have pleiotropic effects in multiple pathways besides the effects on NF-κB transcription observed here. Indeed, the complex phenotype noted in individuals carrying the hypomorphic T17A mutation indicates that CCDC22
plays an important developmental role in the nervous system and beyond. In this regard, it is noteworthy that Cul4b has been similarly linked to XLID, and CCDC22 may therefore also be involved in the regulation of CRL4B targets that are important for neuronal biology (40
). Other rare variants in this gene were also noted in families with XLID; in the case of 2 of these variants, R128Q and R321W, we demonstrated a clear functional alteration of the mutant proteins, which were mislocalized in the cell. The mechanism for this abnormal localization was not mediated by altered COMMD binding, but may involve other protein-protein interactions that remain to be elucidated. With regard to the T30A and E239K variants, no functional effects were identified by our studies; with respect to E239K, we speculate that this may be a rare but functionally normal protein, since it can be found in the NHLBI exome database. Ultimately, we anticipate that additional mutations in this gene will be uncovered in the context of XLID and other X-linked developmental disorders.
Our data suggested that in the NF-κB pathway, a CCDC22-COMMD8 complex plays an important role in IκB turnover and NF-κB activation through its interaction with Cul1, the ligase that targets IκB (21
). However, we conversely found that CCDC22-COMMD1 complexes bound preferentially to Cul2, a ligase involved in NF-κB/RelA ubiquitination and removal of chromatin-bound NF-κB (12
). With these dual functions, CCDC22 deficiency or mutation would impair the activity of both COMMD1 and COMMD8 complexes, but the lack of IκB degradation, an upstream and initial step in this pathway, had a dominant role and was responsible for the impaired NF-κB activation described herein (Figure ). The role of CCDC22 in IκB degradation is supported by ample analysis of cells derived from individuals with the hypomorphic T17A mutation. Moreover, our concordant findings after RNAi-induced silencing of CCDC22 confirmed the role of this factor in NF-κB activation. In agreement with these observations, some of the individuals with XLID affected by the T17A mutation displayed ectodermal dysplasia, a congenital change that can result from blunted NF-κB activation downstream of EDAR, a member of the TNF receptor superfamily (37
). Moreover, NF-κB plays an important role in neuronal function and learning processes (41
) and is similarly important for myelination and Schwann cell function (43
). Therefore, the alterations in the NF-κB pathway seen in these patients may contribute to their neurologic phenotype.
Role of CCDC22-COMMD complexes in NF-κB pathway regulation.
With respect to the role of CCDC22 in immune function, our observations were mainly restricted to 6 individuals with the T17A mutation, with only 2 of them being of adult age at this point. Nevertheless, increased infections, autoimmunity, or unusual malignancies have not been noted thus far. This may indicate that chronic CCDC22 deficiency is better compensated in vivo than in isolated culture systems or that this hypomorphic mutation is not severe enough to result in an obvious immune phenotype in children. Alternatively, the immune defects in vivo may be restricted to selective microorganisms; this has been observed with important immune regulators, such as TRIF
, for which inactivating mutations lead to a narrow and specific susceptibility to herpes encephalitis (44
). If this were the case here, a larger patient cohort and longer-term follow-up may be needed to fully comprehend the immune phenotypes of CCDC22
mutations. Nevertheless, it is interesting to note that a genetic study has indicated that single nucleotide polymorphisms in the CCDC22
gene affect the risk for allergic rhinitis (45
). However, because the CCDC22
gene is located in the complementary strand and in close proximity to FOXP3
, a major regulator of immune function, it is possible that such polymorphisms may be more relevant to the function of FOXP3
. In any event, it is important to note that the functional analysis of CCDC22
presented here, including various RNAi experiments, did not reflect FOXP3
function, and that the T17A mutation did not affect FOXP3
expression (Supplemental Figure 3B). Finally, the high level of CCDC22
expression observed in the immune system, particularly in myeloid and T cells (Supplemental Figure 1A), suggests an important immunological role, in keeping with the NF-κB regulatory function reported here.
The involvement of CCDC22 and COMMD8 in the ubiquitination of IκB represents a novel aspect in the regulation of this critical pathway that might be amenable to therapeutic manipulation. Disrupting CCDC22-COMMD interactions in a manner akin to the effect of the T17A mutation should result in impaired IκB degradation and NF-κB blockade, an effect that would be desirable in certain contexts, such as chronic inflammatory disorders or specific cancers.