CCDC22 deficiency in humans blunts activation of proinflammatory NF-κB signaling

Abstract

NF-κB is a master regulator of inflammation and has been implicated in the pathogenesis of immune disorders and cancer. Its regulation involves a variety of steps, including the controlled degradation of inhibitory IκB proteins. In addition, the inactivation of DNA-bound NF-κB is essential for its regulation. This step requires a factor known as copper metabolism Murr1 domain-containing 1 (COMMD1), the prototype member of a conserved gene family. While COMMD proteins have been linked to the ubiquitination pathway, little else is known about other family members. Here we demonstrate that all COMMD proteins bind to CCDC22, a factor recently implicated in X-linked intellectual disability (XLID). We showed that an XLID-associated CCDC22 mutation decreased CCDC22 protein expression and impaired its binding to COMMD proteins. Moreover, some affected individuals displayed ectodermal dysplasia, a congenital condition that can result from developmental NF-κB blockade. Indeed, patient-derived cells demonstrated impaired NF-κB activation due to decreased IκB ubiquitination and degradation. In addition, we found that COMMD8 acted in conjunction with CCDC22 to direct the degradation of IκB proteins. Taken together, our results indicate that CCDC22 participates in NF-κB activation and that its deficiency leads to decreased IκB turnover in humans, highlighting an important regulatory component of this pathway.

Figure 1. Identification of CCDC22 as a COMMD associated factor.

(A) TAP screen identification of CCDC22. CCDC22 peptides identified with high confidence in TAP screens using 3 different COMMD protein baits are indicated by blue shading. The specific COMMD proteins identified with each bait are shown at right. (B and C) Endogenous CCDC22 coimmunoprecipitated with endogenous COMMD proteins. (B) Endogenous CCDC22 was immunoprecipitated (IP) from HEK 293 cell lysates using 2 anti-CCDC22 antisera, and the recovered material was immunoblotted for COMMD1. Preimmune serum (PIS) or beads only were used as negative controls. (C) COMMD1, COMMD6, COMMD9, and COMMD10 were pulled down with polyclonal immune sera, and the precipitated material was immunoblotted for CCDC22. Some input lanes corresponded to different exposures of the same film. (D) CCDC22 associated with all COMMD family members. COMMD proteins fused to GST were expressed in HEK 293 cells and precipitated from Triton X-100 lysates. The recovered material was immunoblotted for endogenous CCDC22. PD, pulldown; NS, nonspecific band. (E) COMMD proteins were the main interaction partners of CCDC22. Volcano plot representation of CCDC22-interacting proteins. LAP-tagged CCDC22 was immunoprecipitated using an antibody directed against the tag. Nontransfected parental HeLa cells served as control. For each protein identified by mass spectrometry, the ratio of the intensities in the CCDC22 IPs over the control was calculated and plotted against the P value (2-tailed t test) calculated from triplicate experiments, both on a logarithmic scale. Dashed curves represent the cutoff, calculated based on a false discovery rate estimation. Specific interactors (top right) are indicated.

Figure 2. CCDC22 is required for proper cellular distribution of COMMD family proteins.

(A) Colocalization of CCDC22 with COMMD1. GFP-tagged CCDC22 and DsRed2-tagged COMMD1 were overexpressed in U2OS cells. Cells were counterstained with Hoechst and imaged by confocal microscopy. Scale bars: 10 μm. The merged view is also shown enlarged (merged and zoom; enlarged ×3-fold). (B) CCDC22 was not required for COMMD-COMMD interaction. HEK 293 cells were transfected with an siRNA targeting CCDC22 (siCCDC22) or a control duplex. After 48 hours, endogenous COMMD6 was immunoprecipitated, and the recovered material was immunoblotted for endogenous COMMD1 and COMMD10. (C and D) CCDC22 deficiency led to COMMD mislocalization. U2OS cells were transfected with YFP-tagged COMMD1 or COMMD10 together with siRNA against CCDC22 or a control duplex (siControl). (C) Nuclear counterstaining with Hoechst was performed just prior to confocal imaging. Scale bars: 10 μm. (D) The proportion of cells with a large perinuclear punctate pattern was determined by examining more than 100 cells per dish in a blinded manner.

Figure 3. CCDC22-COMMD interactions and the effects of XLID-associated variants.

(A) Schematic representation of CCDC22. Conserved regions and the location of nonrecurrent sequence variants identified in XLID patients are displayed. (B and C) The amino terminus of CCDC22 and the COMM domain of COMMD1 were necessary and sufficient for binding. (B) Full-length (F.L.) and indicated domains of CCDC22 were expressed fused to GST, and their binding to endogenous COMMD1 was examined by coprecipitation. (C) Similar experiments were performed to detect coprecipitation of endogenous CCDC22 with full-length COMMD1 or its aminoterminal (N-term; amino acids 1–118) or carboxyterminal (C-term; amino acids 119–190) domains. (D and E) The XLID-associated mutation CCDC22 T17A impaired COMMD1 binding. (D) Coimmunoprecipitation between endogenous CCDC22 and COMMD1 was examined in LCLs derived from the kindred with the T17A mutation and a healthy control subject (WT). (E) Endogenous COMMD1 was similarly immunoprecipitated from HEK 293 cells expressing CCDC22 T17A or the WT. (F and G) Interaction of COMMD1 with other XLID-associated variants of CCDC22. (F) The ability of endogenous COMMD1 and CCDC22 E239K to interact was examined using available LCLs. (G) Interactions were examined by expressing HA-tagged CCDC22 proteins in HEK 293 cells. Immunoprecipitation of endogenous COMMD1 was followed by immunoblotting for HA-tagged CCDC22. (H and I) Abnormal cellular distribution of CCDC22 variants. (H) Distribution of YFP-tagged CCDC22 variants, determined by confocal microscopy. Scale bars: 10 μm. (I) Cellular distribution after examination of more than 100 cells per group in a blinded manner.

Figure 4. CCDC22 is required for NF-κB activation.

(A) Aplasia cutis (left, arrows) and examples of abnormal dentition (right) in patients with the T17A mutation. (B) Fibroblasts from XLID patients displayed blunted activation of NF-κB–dependent genes. Primary dermal fibroblasts from patients demonstrated decreased TNF-induced activation of IL8 and RELB compared with their mother, a heterozygote mutation carrier (HET). (C) Response of NF-κB genes to CD40L activation was decreased in a LCL derived from an XLID patient. LCLs derived from an XLID patient or a healthy control were stimulated with CD40L. (D) CCDC22 deficiency phenocopied the T17A mutation. LCLs derived from the healthy control in C were transfected with the indicated siRNA oligonucleotides and subsequently stimulated with CD40L. (E) CCDC22 was required for activation of NF-κB–responsive genes in HEK 293 cells. 2 siRNA oligonucleotides targeting CCDC22 were used, and cells were subsequently stimulated with TNF. (B–E) Gene induction was evaluated in triplicate experiments by qRT-PCR; data represent mean and SEM.

Figure 5. CCDC22 is required for RelA nuclear transport and IκB degradation.

(A) CCDC22 deficiency resulted in depressed nuclear accumulation of active NF-κB. HEK 293 cells were transfected with the indicated siRNAs and stimulated with TNF. The presence of active NF-κB complexes in nuclear extracts was assessed by a DNA-protein coprecipitation assay (top) or by direct immunoblotting (input, bottom). RNA polymerase II (Pol II) served as a loading control. (B) TNF-induced degradation of classical IκB proteins was impaired in primary fibroblasts bearing the T17A mutation. Primary dermal fibroblasts from 2 patients demonstrate decreased TNF-induced degradation of IκB-α, IκB-β, and IκB-ε compared with fibroblasts from their mother, a heterozygote mutation carrier. (C) CCDC22 deficiency impaired TNF-induced IκB degradation. HEK 293 cells were transfected with the indicated siRNA oligonucleotides and treated with TNF. IκB degradation was determined by Western blot analysis. (D) CCDC22 deficiency affected IκB-β stability. Cells transfected with the indicated siRNA were subsequently treated with cycloheximide (CHX) to inhibit new protein synthesis. IκB-β stability after TNF stimulation was examined by immunoblotting. (E) IκB-α degradation was impaired in XLID-derived LCLs. LCLs derived from a healthy control subject and an XLID patient with the T17A mutation were stimulated with PMA, and IκB-α degradation was examined by immunoblotting. (F) IκB-α phosphorylation was not affected in XLID-derived LCLs. LCLs derived from a healthy control and an XLID patient were stimulated with PMA for indicated times. Phosphorylation of IκB-α at serines 32 and 36 was determined by immunoblotting using a phosphospecific antibody.

Figure 6. CCDC22 is required for IκB ubiquitination.

(A) IκB-α ubiquitination is reduced in lymphoid cells bearing the T17A mutation. LCLs derived from a healthy control subject and an XLID patient with the T17A mutation were stimulated with PMA. The protease inhibitor MG-132 was concurrently administered. Ubiquitinated phospho–IκB-α levels were determined by immunoprecipitating NF-κB complexes with a RelA antibody, followed by phospho–IκB-α immunoblotting. (B) CCDC22 interacted with various CRL1-βTrCP components. FLAG-tagged Cul1, βTrCP1, or SKP1 were expressed along with CCDC22 in HEK 293 cells. CRL components were immunoprecipitated using a FLAG antibody, and CCDC22 was detected in the recovered material by immunoblotting. (C) Endogenous CCDC22 interacted with Cul1, Cul2, and Cul3. CCDC22 was immunoprecipitated, and the recovered material was immunoblotted for endogenous Cul1, Cul2, and Cul3. Some input lanes corresponded to different exposures of the same film. (D) Cul1 and Cul2 were not required for CCDC22-COMMD interaction. HEK 293 cells were treated with siRNA against Cul1, Cul2, or an irrelevant control. Endogenous CCDC22 was subsequently immunoprecipitated, and the recovered material was immunoblotted for endogenous COMMD1 and COMMD10. (E) CCDC22 was not required for Cullin-COMMD interaction. HEK 293 cells were transfected with GST, GST-COMMD1, or GST-COMMD8 along with control or anti-CCDC22 siRNA. After 48 hours, cell lysates were purified on GST-agarose, and the recovered material was immunoblotted for endogenous Cul1, Cul2, Cul3, and COMMD1.

Figure 7. COMMD8, a partner of CCDC22, is also required for IκB degradation.

(A and B) COMMD8 deficiency impaired TNF-induced IκB-α degradation. (A) HEK 293 cells were transfected with the indicated siRNA oligonucleotides, and the effectiveness of the silencing was determined by qRT-PCR. (B) In parallel, cells were stimulated with TNF, and IκB-α degradation was examined by immunoblotting. (C and D) COMMD8 was required for NF-κB–responsive gene expression. (C) HEK 293 cells were transfected with the indicated siRNA oligonucleotides and subsequently treated with TNF. Induction of several NF-κB–responsive genes was examined. (D) The findings in C were recapitulated using 2 distinct siRNA oligonucleotides against COMMD8. (A, C, and D) Gene induction was evaluated in triplicate experiments by qRT-PCR; data represent mean and SEM.

Figure 8. Role of CCDC22-COMMD complexes in NF-κB pathway regulation.