Commentary 539Introduction Ubiquitylation [also known as ubiquitination, as coined by the discoverers of this modification with regard to its connection to proteolysis (Wilkinson, 2005)] is a three-step enzymatic reaction that is carried out by several enzymes: the ubiquitin-activating enzyme (E1), a ubiquitin carrier protein (E2; also known as ubiquitin-conjugating enzyme, UBC) and a ubiquitin-protein ligase (E3). An additional component of the ubiquitylation machinery has been described. This E4 enzyme is involved in elongation of short ubiquitin chains (Koegl et al., 1999). However, the requirement for an E4 activity appears to be limited to a small subset of substrates. Ubiquitylation-dependent proteasomal degradation is involved in the regulation of numerous cellular processes, including cell cycle progression, apoptosis, DNA repair, the maintenance of cellular quality control, autophagy, the regulation of transcription and receptor-mediated endocytosis (Mayer et al., 2005;Mayer et al., 2006;Mayer et al., 2008). In general, modification by ubiquitin serves as a recognition element in trans, whereby different downstream effectors bind to the ubiquitin-modified protein to affect its fate and/or function. In the case of proteasomal degradation, the ubiquitylated protein is recognized by the 26S proteasome and subsequently degraded (Dikic et al., 2009;Su and Lau, 2009).The widely accepted canonical signal for proteasomal degradation is a polyubiquitin chain that is anchored to the -NH 2 group of a lysine residue(s) in the substrate by an isopeptide bond and is assembled through the formation of isopeptide bonds between the C-terminal residue of one ubiquitin moiety (glycine 76) and lysine 48 of the previously conjugated ubiquitin moiety (Chau et al., 1989). Recent studies have reported, however, that other types of ubiquitin chains can also be recognized by the proteasome (Figs 1, 2). These include an ester-based linkage that connects ubiquitin to a threonine or serine residue in the substrate, and a thiolester-based linkage whereby ubiquitin is bound to a cysteine residue in the substrate (McDowell et al., 2010;Tait et al., 2007;Vosper et al., 2009). Ubiquitin can also be conjugated to the -NH 2 group of the N-terminal residue of the substrate. Instead of using lysine 48 for the linkage, polyubiquitin chains can also be assembled through one of the six additional lysine residues in the molecule. Such homogenous chains based on, for example, lysine 63 (Saeki et al., 2009), or heterogeneous chains in which different ubiquitinubiquitin linkages are found, have also been reported to target proteins for proteasomal degradation. Linear chains, in which the ubiquitin links are attached to one another 'head-to-tail', and heterologous chains, in which the links are made of ubiquitin and a ubiquitin-like protein, such as small ubiquitin-like modifier (SUMO), have additionally been shown to target proteins for proteasomal degradation. Surprisingly, it has been demonstrated that the proteasome does not necessarily have ...

NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.

Activation of NF-kappaB is regulated via numerous ubiquitin- and proteasome-mediated steps; an important one is processing of the precursor p105 to the p50 active subunit. The mechanisms involved are largely unknown, because this is an exceptional case where the ubiquitin system does not destroy its substrate completely. Here, we demonstrate that proteasomal processing of p105 requires ubiquitin but not generation of polyubiquitin chains. In vitro, ubiquitin species that cannot polymerize mediate processing. In yeasts that express nonpolymerizable ubiquitins, processing proceeds normally, whereas degradation of substrates that are dependent on polyubiquitination is inhibited. Similar results were obtained in mammalian cells. Interestingly, processing requires multiple monoubiquitinations, because progressive elimination of lysines in p105 is accompanied by gradual inhibition of p50 generation. Finally, the proteasome recognizes the multiply monoubiquitinated p105. These findings suggest that a proteolytic signal can be composed of a cluster of single ubiquitins, not necessarily a chain.

Purpose Abnormal activation of the NF-κB pathway induces a more aggressive phenotype of cutaneous melanoma. Understanding the mechanisms involved in melanoma NF-κB activation may identify novel targets for this pathway. KPC1, an E3 ubiquitin ligase, is a regulator of NF-κB pathway. The objective of this study was to investigate the mechanisms regulating KPC1 expression and its clinical impact in melanoma. Experimental Design The clinical impact of KPC1 expression and its epigenetic regulation were assessed in large cohorts of clinically well-annotated melanoma tissues (tissue micro-arrays; n=137, JWCI cohort; n=40) and The Cancer Genome Atlas database (TCGA cohort, n=370). Using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-κB, and the epigenetic regulations of KPC1, including DNA methylation and microRNA expression. Results We verified that KPC1 suppresses melanoma proliferation by processing NF-κB1 p105 into p50, thereby modulating NF-κB-target gene expression. Concordantly, KPC1 expression was down-regulated in AJCC stage IV melanoma compared to early stages (stage I/II p=0.013, stage III p=0.004), and low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (n=137, Hazard Ratio 1.810, p=0.006). Furthermore, our data showed that high miR-155-5p expression, which is controlled by DNA methylation at its promoter region (TCGA; Pearson’s r −0.455, p<0.001), is significantly associated with KPC1 down-regulation (JWCI; p=0.028, TCGA; p=0.003). Conclusions This study revealed novel epigenetic regulation of KPC1 associated with NF-κB pathway activation, promoting metastatic melanoma progression. These findings suggest the potential utility of KPC1 and its epigenetic regulation as theranostic targets.

Background: Most proteins must be ubiquitinated prior to proteasomal degradation.Results: Myelin basic protein (MBP) is hydrolyzed by the 26S proteasome without ubiquitination in vitro and in mammalian cells.Conclusion: Proteasome-mediated hydrolysis of the multiple sclerosis autoantigen MBP is uncontrolled by the ubiquitination system.Significance: Results reveal the first example of an autoantigen degraded by the proteasome without ubiquitin.