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Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of B1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed. Cell Death and Differentiation (2011) 18, 1393-1402 doi:10.1038/cdd.2011; published online 4 March 2011One of the major roles of the covalent modification of cellular proteins by ubiquitin is signaling them for proteasomal degradation ( Figure 1). The first step of the modification is catalyzed by the ubiquitin-activating enzyme, E1, which generates a high-energy thiol ester intermediate that is subsequently transferred to the second enzyme, a ubiquitinconjugating enzyme, E2. The third step ascertains substrate specificity, and is catalyzed by one of the numerous (B650) ubiquitin ligases, E3s. Typically, it results in the formation of an isopeptide bond between the C-terminal Gly of ubiquitin and an e-NH 2 group of an internal Lys of the substrate. Less frequently, it can generate a linear peptide bond with the a-NH 2 group, a thiol ester bond with an internal Cys, or an ester bond with a Thr or Ser. The three-step cascade of reactions is repeated, where additional ubiquitin moieties are attached sequentially to one another in an isopeptide bond involving one of the seven internal Lys residues in the ubiquitin moiety, thus generating a polyubiquitin chain. Lys48-based chains serve as a signal for proteasomal degradation, whereas chains based on other internal Lys residues, or modification by single moiety(ies) can serve non-proteolytic functions.Ligases fall into two main families: RING (really interesting new gene) and HECT (homologous to the E6-AP carboxy terminus) domain-containing E3s. RING ligases serve as scaffolds that facilitate direct transfer of ubiquitin from the E2 to the target protein. HECT E3s contain an active Cys residue to which ubiquitin binds prior to its transfer to the substrate (Figure 1). There are B600 RING finger and B30 HECT ligases in humans. Smaller families of ligases (U-box, plant homology domain, and zinc finger) have also been described.An important problem relates to regulation of the ubiquitin system components, and in particular to that of the ligases that are the specific substrate-recognizing elements. 1,2 Phosphorylation of an E3 can activate the protein, such as the ca...

Background/Aims-Wilson disease is characterized by hepatic copper overload and caused by mutations in the gene encoding the copper transporting P-type ATPase ATP7B. ATP7B interacts with COMMD1, a protein that is deleted in Bedlington terriers with hereditary copper toxicosis. Here we characterized the implications of the interaction between COMMD1 and ATP7B in relation to the pathogenesis of Wilson disease.

Wilson disease (WD) is an autosomal recessive copper overload disorder of the liver and basal ganglia. WD is caused by mutations in the gene encoding ATP7B, a protein localized to the trans-Golgi network that primarily facilitates hepatic copper excretion. Current treatment comprises reduction of circulating copper by zinc supplementation or copper chelation. Despite treatment, a significant number of patients have neurological deterioration. The aim of this study was to investigate the possibility that defects arising from some WD mutations are ameliorated by drug treatment aimed at improvement of protein folding and restoration of protein function. This necessitated systematic characterization of the molecular consequences of distinct ATP7B missense mutations associated with WD.

COMMD [copper metabolism gene MURR1 (mouse U2af1-rs1 region 1) domain] proteins constitute a recently identified family of NF-kappaB (nuclear factor kappaB)-inhibiting proteins, characterized by the presence of the COMM domain. In the present paper, we report detailed investigation of the role of this protein family, and specifically the role of the COMM domain, in NF-kappaB signalling through characterization of protein-protein interactions involving COMMD proteins. The small ubiquitously expressed COMMD6 consists primarily of the COMM domain. Therefore COMMD1 and COMMD6 were analysed further as prototype members of the COMMD protein family. Using specific antisera, interaction between endogenous COMMD1 and COMMD6 is described. This interaction was verified by independent techniques, appeared to be direct and could be detected throughout the whole cell, including the nucleus. Both proteins inhibit TNF (tumour necrosis factor)-induced NF-kappaB activation in a non-synergistic manner. Mutation of the amino acid residues Trp24 and Pro41 in the COMM domain of COMMD6 completely abolished the inhibitory effect of COMMD6 on TNF-induced NF-kappaB activation, but this was not accompanied by loss of interaction with COMMD1, COMMD6 or the NF-kappaB subunit RelA. In contrast with COMMD1, COMMD6 does not bind to IkappaBalpha (inhibitory kappaBalpha), indicating that both proteins inhibit NF-kappaB in an overlapping, but not completely similar, manner. Taken together, these data support the significance of COMMD protein-protein interactions and provide new mechanistic insight into the function of this protein family in NF-kappaB signalling.

The polycomb repressive complex (PRC) 1 protein Ring1B is an ubiquitin ligase that modifies nucleosomal histone H2A, a modification which plays a critical role in regulation of gene expression. We have shown that self-ubiquitination of Ring1B generates multiply branched, "noncanonical" polyubiquitin chains that do not target the ligase for degradation, but rather stimulate its activity toward histone H2A. This finding implies that Ring1B is targeted by a heterologous E3. In this study, we identified E6-AP (E6-associated protein) as a ligase that targets Ring1B for "canonical" ubiquitination and subsequent degradation. We further demonstrated that both the self-ubiquitination of Ring1B and its modification by E6-AP target the same lysines, suggesting that the fate of Ring1B is tightly regulated (e.g., activation vs. degradation) by the type of chains and the ligase that catalyzes their formation. As expected, inactivation of E6-AP affects downstream effectors: Ring1B and ubiquitinated H2A levels are increased accompanied by repressed expression of HoxB9, a PRC1 target gene. Consistent with these findings, E6-AP knockout mice display an elevated level of Ring1B and ubiquitinated histone H2A in various tissues, including cerebellar Purkinje neurons, which may have implications to the pathogenesis of Angelman syndrome, a neurodevelopmental disorder caused by deficiency of E6-AP in the brain.polycomb complexes | ubiquitin-proteasome system

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Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1 Δhep) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1 Δhep mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1 Δhep mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1 Δhep mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1 Δhep mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1 Δhep mice could be detected. Despite the absence of hepatocellular toxicity in Commd1 Δhep mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.