Xicheng Mao

COMMD1 promotes the ubiquitination of NF‐κB subunits through a cullin‐containing ubiquitin ligase

NF‐κB is a pleiotropic transcription factor involved in multiple processes, including inflammation and oncogenesis. We have previously reported that COMMD1 represses κB‐dependent transcription by negatively regulating NF‐κB–chromatin interactions. Recently, ubiquitination of NF‐κB subunits has been similarly implicated in the control of NF‐κB recruitment to chromatin. We report here that COMMD1 accelerates the ubiquitination and degradation of NF‐κB subunits through its interaction with a multimeric ubiquitin ligase containing Elongins B and C, Cul2 and SOCS1 (ECSSOCS1). COMMD1‐deficient cells demonstrate stabilization of RelA, greater nuclear accumulation of RelA after TNF stimulation, de‐repression of several κB‐responsive genes, and enhanced NF‐κB‐mediated cellular responses. COMMD1 binds to Cul2 in a stimulus‐dependent manner and serves to facilitate substrate binding to the ligase by stabilizing the interaction between SOCS1 and RelA. Our data uncover that ubiquitination and degradation of NF‐κB subunits by this COMMD1‐containing ubiquitin ligase is a novel and critical mechanism of regulation of NF‐κB‐mediated transcription.

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

COMMD1 disrupts HIF-1α/β dimerization and inhibits human tumor cell invasion

The gene encoding COMM domain-containing 1 (COMMD1) is a prototypical member of the COMMD gene family that has been shown to inhibit both NF-kappaB- and HIF-mediated gene expression. NF-kappaB and HIF are transcription factors that have been shown to play a role in promoting tumor growth, survival, and invasion. In this study, we demonstrate that COMMD1 expression is frequently suppressed in human cancer and that decreased COMMD1 expression correlates with a more invasive tumor phenotype. We found that direct repression of COMMD1 in human cell lines led to increased tumor invasion in a chick xenograft model, while increased COMMD1 expression in mouse melanoma cells led to decreased lung metastasis in a mouse model. Decreased COMMD1 expression also correlated with increased expression of genes known to promote cancer cell invasiveness, including direct targets of HIF. Mechanistically, our studies show that COMMD1 inhibits HIF-mediated gene expression by binding directly to the amino terminus of HIF-1alpha, preventing its dimerization with HIF-1beta and subsequent DNA binding and transcriptional activation. Altogether, our findings demonstrate a role for COMMD1 in tumor invasion and provide a detailed mechanism of how this factor regulates the HIF pathway in cancer cells.

Deubiquitination of NF-κB by Ubiquitin-Specific Protease-7 promotes transcription.

NF-κB is the master regulator of the immune response and is responsible for the transcription of hundreds of genes controlling inflammation and immunity. Activation of NF-κB occurs in the cytoplasm through the kinase activity of the IκB kinase complex, which leads to translocation of NF-κB to the nucleus. Once in the nucleus, NF-κB transcriptional activity is regulated by DNA binding-dependent ubiquitin-mediated proteasomal degradation. We have identified the deubiquitinase Ubiquitin Specific Protease-7 (USP7) as a regulator of NF-κB transcriptional activity. USP7 deubiquitination of NF-κB leads to increased transcription. Loss of USP7 activity results in increased ubiquitination of NF-κB, leading to reduced promoter occupancy and reduced expression of target genes in response to Toll-like– and TNF-receptor activation. These findings reveal a unique mechanism controlling NF-κB activity and demonstrate that the deubiquitination of NF-κB by USP7 is critical for target gene transcription.

COMMD1 expression is controlled by critical residues that determine XIAP binding.

COMMD {COMM [copper metabolism Murr1 (mouse U2af1-rs1 region 1)] domain-containing} proteins participate in several cellular processes, ranging from NF-kappaB (nuclear factor kappaB) regulation, copper homoeostasis, sodium transport and adaptation to hypoxia. The best-studied member of this family is COMMD1, but relatively little is known about its regulation, except that XIAP [X-linked IAP (inhibitor of apoptosis)] functions as its ubiquitin ligase. In the present study, we identified that the COMM domain of COMMD1 is required for its interaction with XIAP, and other COMMD proteins can similarly interact with IAPs. Two conserved leucine repeats within the COMM domain were found to be critically required for XIAP binding. A COMMD1 mutant which was unable to bind to XIAP demonstrated a complete loss of basal ubiquitination and great stabilization of the protein. Underscoring the importance of IAP-mediated ubiquitination, we found that long-term expression of wild-type COMMD1 results in nearly physiological protein levels as a result of increased ubiquitination, but this regulatory event is circumvented when a mutant form that cannot bind XIAP is expressed. In summary, our findings indicate that COMMD1 expression is controlled primarily by protein ubiquitination, and its interaction with IAP proteins plays an essential role.

CCDC22 deficiency in humans blunts activation of proinflammatory NF-kappa B 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.

COMMD1 (Copper Metabolism MURR1 Domain-containing Protein 1) Regulates Cullin RING Ligases by Preventing CAND1 (Cullin-associated Nedd8-dissociated Protein 1) Binding

Cullin RING ligases (CRLs), the most prolific class of ubiquitin ligase enzymes, are multimeric complexes that regulate a wide range of cellular processes. CRL activity is regulated by CAND1 (Cullin-associated Nedd8-dissociated protein 1), an inhibitor that promotes the dissociation of substrate receptor components from the CRL. We demonstrate here that COMMD1 (copper metabolism MURR1 domain-containing 1), a factor previously found to promote ubiquitination of various substrates, regulates CRL activation by antagonizing CAND1 binding. We show that COMMD1 interacts with multiple Cullins, that the COMMD1-Cul2 complex cannot bind CAND1, and that, conversely, COMMD1 can actively displace CAND1 from CRLs. These findings highlight a novel mechanism of CRL activation and suggest that CRL regulation may underlie the pleiotropic activities of COMMD1.

Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling

Copper metabolism MURR1 domain containing (COM MD) proteins are a group of highly conserved factors defined by the presence of a unique C-terminal homology domain (Burstein et al., 2005). Ten family members can be identified from mammals to unicellular protozoa (Maine and Burstein, 2007), but little is known about their cellular functions and the underlying reason for their conservation and diversification. Most of our understanding is centered on COM MD1, the first identified member of this family that was initially noted to be the site of a recessive mutation that results in copper toxicosis in a particular dog breed, the Bedlington terrier (van de Sluis et al., 2002). The mechanism for the accumulation of copper in these animals was initially unclear; however, an interaction between COM MD1 and the copper transporter ATP7B was reported early on (Tao et al., 2003). Recently, we demonstrated that COM MD1 regulates the endosomal sorting of the copper transporter ATP7A, such that in the absence of COM MD1, ATP7A is trapped in endosomal vesicles and lacks copper-dependent trafficking to the trans-Golgi network and plasma membrane (Phillips-Krawczak et al., 2015). In addition to its control of ATP7A/7B trafficking, COM MD1 has been linked to the regulation of other transporters, including epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, and sodium-potassium-chloride cotransporter 1 (Biasio et al., 2004; Drévillon et al., 2011; Smith et al., 2013). However, whether these other transporters are similarly regulated at the level of endosomal sorting remains to be examined. Furthermore, COM MD1 has also been linked to other pathways that are seemingly not connected to the endolysosomal system, including nuclear factor κB regulation (Maine et al., 2007; Starokadomskyy et al., 2013) and hypoxia adaptation (van de Sluis et al., 2010). The role of COM MD1 in endosomal sorting is linked to its incorporation into a larger complex containing the coiledcoil proteins CCDC22 and CCDC93 (Phillips-Krawczak et al., 2015). This COM MD–CCDC22–CCDC93 (CCC) complex localizes to endosomes through interactions between CCDC22 and CCDC93 with FAM21 (Harbour et al., 2012; Freeman et al., 2014; Phillips-Krawczak et al., 2015), a component of the Wiskott-Aldrich syndrome protein and scar homolog (WASH) complex (Derivery et al., 2009; Gomez and Billadeau, 2009). WASH is a member of the Wiskott-Aldrich syndrome protein Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosomal system is critical in its regulation. In this study we report that Notch recycling to the cell surface is dependent on the COM MD–CCDC22–CCDC93 (CCC) complex, a recently identified regulator of endosomal trafficking. Disruption in this system leads to intracellular accumulation of Notch2 and concomitant reduction in Notch signaling. Interestingly, among the 10 copper metabolism MURR1 domain containing (COM MD) family members that can associate with the CCC complex, only COM MD9 and its binding partner, COM MD5, have substantial effects on Notch. Furthermore, Commd9 deletion in mice leads to embryonic lethality and complex cardiovascular alterations that bear hallmarks of Notch deficiency. Altogether, these studies highlight that the CCC complex controls Notch activation by modulating its intracellular trafficking and demonstrate cargo-specific effects for members of the COM MD protein family. Endosomal sorting of Notch receptors through COM MD9dependent pathways modulates Notch signaling