Breakthroughs and Views
U-box proteins as a new family of ubiquitin ligases

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Abstract

Ubiquitin-protein ligases (E3s) determine the substrate specificity of ubiquitylation and, until recently, had been classified into two families, the HECT and RING-finger families. The U-box is a domain of ∼70 amino acids that is present in proteins from yeast to humans. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. We recently showed that mammalian U-box proteins, in conjunction with an E1 and an E2, mediate polyubiquitylation in the absence of a HECT type or RING-finger type E3. U-box proteins have thus been defined as a third family of E3s. We here review recent progress in the characterization of U-box proteins and of their role in the quality control system that underlies the cellular stress response to the intracellular accumulation of abnormal proteins.

Section snippets

Ubiquitin-conjugation system for regulation of intracellular protein abundance

The synthesis and functional regulation of proteins have long been key issues in biology. During the past 2 decades, however, it has gradually become recognized that protein degradation is also important for many cellular activities. The discovery of the ubiquitin–proteasome system for the degradation of intracellular proteins represented a major step forward in our understanding of the regulation of protein turnover in eukaryotes [1]. This system is thought to mediate selectively the

HECT type E3 enzymes

Interaction between the human papilloma virus gene product E6 and the tumor suppressor p53 results in the degradation of p53 by the ubiquitin–proteasome pathway in transformed cells [13]. The E6-dependent ubiquitylation of p53 is catalyzed by the human E3 enzyme E6-AP [14]. E6-AP forms a thiol intermediate with a ubiquitin moiety transferred from an E2 (UbcH5A, UbcH5B, UbcH5C, or UbcH7) to a catalytic cysteine near its carboxyl terminus. Ubiquitin is then transferred from E6-AP to, and forms an

RING-finger type E3 enzymes

The really interesting new gene (RING) finger was first described in the early 1990s as a functional module thought to mediate protein–protein or protein–nucleotide interactions [17]. It contains an octet of cysteine and histidine residues that constitute a zinc binding domain and is structurally classified as either RING-H2 (which contains histidines at positions 4 and 5) or RING-HC (which contains only one histidine at position 4). It has become clear that many, but probably not all, proteins

Discovery of E4

Johnson et al. [34] showed that a fusion protein bearing an amino-terminal ubiquitin moiety that was resistant to ubiquitin-specific hydrolase activity was highly unstable and rapidly degraded in vivo (Fig. 3A). The proteolytic system responsible for the degradation of this fusion protein, designated the UFD (ubiquitin fusion degradation) pathway, was characterized in detail by analysis of the stability of a ubiquitin–β-galactosidase fusion construct. Genetic screening in yeast led to the

U-box proteins as a new E3 family

The predicted three-dimensional structure of the U-box is similar to that of the RING-finger, despite the lack in the former of the hallmark metal-chelating residues of the latter [36]. This observation prompted us to investigate the possibility that U-box proteins in general are able to function as E3s in E2-dependent ubiquitylation. We isolated six mammalian U-box proteins and showed that all of them mediate ubiquitylation in conjunction with E1 and E2 and in the absence of other E3

Unusual linkage of ubiquitin mediated by U-box type E3s

The formation of a polyubiquitin chain by linkage of the carboxyl terminus of a new ubiquitin molecule to lysine-48 of the last ubiquitin moiety of the chain is thought to mark a protein for proteolysis by the 26S proteasome. However, recent observations indicate that polyubiquitin chains are also assembled through conjugation to lysine residues of ubiquitin other than lysine-48, and the resulting chains appear to function in distinct biological processes. A short chain of lysine-29-linked

Biological aspects of UFD2 function

Loss of heterozygosity of the distal portion of chromosome 1, which is thought to harbor tumor suppressor genes, is frequently observed in many human cancers, including neuroblastoma. The human UFD2a gene is located in this region of chromosome 1. A 500-kb deletion at chromosome 1p36.2-p36.3 in a human neuroblastoma cell line has been shown to encompass at least six genes, including those for DFF45 (ICAD), PGD, CORT, KIF1B-β, PEX14, and UFD2a (HDNB1) [44]. Although it is not clear which of

U-box proteins as partners of molecular chaperones

Through its interaction with the AAA type ATPase Cdc48, which possesses chaperone activity, Ufd2 is thought to contribute to cell survival under stressful conditions in yeast. Similarly, in mammals, UFD2a binds to VCP, a mammalian ortholog of Cdc48 [35], [48]. The U-box protein CHIP, which contains two tetratricopeptide repeats, also binds to the molecular chaperones Hsp70 and Hsp90, and contributes to the stress response to the accumulation of unfolded or misfolded proteins [49], [50] (Fig. 5

Other U-box proteins

The U-box protein UIP5 (UbcM4/UbcH7-interacting protein 5), also known as KIAA0860, was isolated by yeast two-hybrid screening with UbcM4 as a bait. In addition to the U-box domain, UIP5 contains a RING-finger domain at its carboxyl terminus [54] (Fig. 2B). The E3 activity of UIP5 depends on its U-box domain, however, not on its RING-finger domain. Mutational analysis has also indicated that the U-box domain mediates the physical interaction of UIP5 with E2 enzymes. UIP5 is localized

The ubiquitin system and protein quality control

The inhibition of protein aggregation by molecular chaperones and the degradation of misfolded or unfolded proteins by the ubiquitin–proteasome system are two important aspects of the cellular response to stressful conditions (Fig. 5). It has been unclear, however, how unfolded or misfolded polypeptides are recognized by the ubiquitin–proteasome system. Although molecular chaperones are thought to contribute to this process, direct evidence of a link between molecular chaperones and the

Conclusion

A quality control system for intracellular proteins is pivotal to maintenance of the intracellular environment. The accumulation of abnormal proteins, such as those that are unfolded or misfolded, is likely to impair cellular functions and eventually to result in cell death. Cells have thus developed molecular chaperones and the ubiquitin–proteasome pathway to deal with this problem. Certain U-box type proteins with E3 activity associate with molecular chaperones that mediate the recognition of

Acknowledgements

The authors’ own publication are in part supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sport, Science and Technology of Japan, and by YASUDA Medical Research Foundation.

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