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

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.

A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis.

Ubiquitination can have profound effects on the stability and function of cellular proteins. Mass spectrometry (MS) can be used to map the specific amino acid residues that are conjugated to ubiquitin in a target protein. However, the purification required for proteomic analysis can be challenging. In this paper, we describe a bimolecular affinity purification scheme for the isolation of a specific ubiquitinated protein in which affinity moieties are fused to ubiquitin and to a target protein of interest. After ubiquitin conjugation in vivo, the protein target acquires two affinity tags, allowing the specific purification of its ubiquitin-modified forms. To prevent deubiquitination after lysis or the copurification of interacting cofactors, this procedure is performed after protein denaturation using polyhistidine and biotinylation tags. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified in large amounts of sufficient purity for MS analysis and for mapping of ubiquitin acceptor sites.

GCN5 is a required cofactor for a ubiquitin ligase that targets NF-kappaB/RelA.

The transcription factor NF-kappaB is a critical regulator of inflammatory and cell survival signals. Proteasomal degradation of NF-kappaB subunits plays an important role in the termination of NF-kappaB activity, and at least one of the identified ubiquitin ligases is a multimeric complex containing Copper Metabolism Murr1 Domain 1 (COMMD1) and Cul2. We report here that GCN5, a histone acetyltransferase, associates with COMMD1 and other components of the ligase, promotes RelA ubiquitination, and represses kappaB-dependent transcription. In this role, the acetyltransferase activity of GCN5 is not required. Interestingly, GCN5 binds more avidly to RelA after phosphorylation on Ser 468, an event that is dependent on IKK activity. Consistent with this, we find that both GCN5 and the IkappaB Kinase (IKK) complex promote RelA degradation. Collectively, the data indicate that GCN5 participates in the ubiquitination process as an accessory factor for a ubiquitin ligase, where it provides a novel link between phosphorylation and ubiquitination.

Bimolecular affinity purification (BAP): tandem affinity purification using two protein baits.

The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.