Yelena Kravtsova-Ivantsiv

Non-canonical ubiquitin-based signals for proteasomal degradation

Regulated cellular proteolysis is mediated largely by the ubiquitin–proteasome system (UPS). It is a highly specific process that is time- (e.g. cell cycle), compartment- (e.g. nucleus or endoplasmic reticulum) and substrate quality- (e.g. denatured or misfolded proteins) dependent, and allows fast adaptation to changing conditions. Degradation by the UPS is carried out through two successive steps: the substrate is covalently tagged with ubiquitin and subsequently degraded by the 26S proteasome. The accepted ‘canonical’ signal for proteasomal recognition is a polyubiquitin chain that is anchored to a lysine residue in the target substrate, and is assembled through isopeptide bonds involving lysine 48 of ubiquitin. However, several ‘non-canonical’ ubiquitin-based signals for proteasomal targeting have also been identified. These include chains anchored to residues other than internal lysine in the substrates, chains assembled through linking residues other than lysine 48 in ubiquitin, and mixed chains made of both ubiquitin and a ubiquitin-like protein. Furthermore, some proteins can be degraded following modification by a single ubiquitin (monoubiquitylation) or multiple single ubiquitins (multiple monoubiquitylation). Finally, some proteins can be proteasomally degraded without prior ubiquitylation (the process is also often referred to as ubiquitination). In this Commentary, we describe these recent findings and discuss the possible physiological roles of these diverse signals. Furthermore, we discuss the possible impact of this signal diversity on drug development.

Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination Is Sufficient for Proteasomal Processing of the p105 NF-κB Precursor

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.

KPC1-Mediated Ubiquitination and Proteasomal Processing of NF-κB1 p105 to p50 Restricts Tumor Growth

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.

ABIN-1 negatively regulates NF-κB by inhibiting processing of the p105 precursor

p105 plays dual roles in NF-kappaB signaling: in its precursor form it inhibits NF-kappaB activation, but limited processing by the ubiquitin system generates the p50 active subunit of the transcription factor. Here we show that ABIN-1, an A20-binding protein that is also known to attenuate NF-kappaB activation, inhibits p105 processing. p105 and ABIN-1 physically interact with one another, but the binding is not necessary for inhibition of processing. Rather, it appears to stabilize ABIN-1 and to increase its level, which further augments its inhibitory effect. Deletion of the processing inhibitory domain (PID) of p105 abrogates the inhibition which also requires the ABIN homology domain (AHD)-2 of ABIN-1. Together, the effects of ABIN-1 on p105 processing and of p105 on stabilizing ABIN-1 act to potentiate the NF-kappaB inhibitory activity of ABIN-1.

The N-terminal domain of MyoD is necessary and sufficient for its nuclear localization-dependent degradation by the ubiquitin system

A growing number of proteins, including the myogenic transcription factor MyoD, are targeted for proteasomal degradation after N-terminal ubiquitination (NTU) where the first ubiquitin moiety is conjugated to the N-terminal residue rather than to an internal lysine. NTU might be essential in targeting both lysine-containing and naturally occurring lysine-less proteins such as p16INK4a and p14ARF; however, the mechanisms that underlie this process are largely unknown. Specifically, the recognition motif(s) in the target substrates and the ubiquitin ligase(s) that catalyze NTU are still obscure. Here we show that the N-terminal domain of MyoD is critical for its degradation and that its destabilizing effect depends on nuclear localization of the protein. Deletion of the first 15 aa of MyoD blocked completely its lysine-independent degradation. Importantly, transfer of the first 30 N-terminal residues of MyoD to GFP destabilized this otherwise stable protein, and, here too, targeting for degradation depended on localization of the protein to the nucleus. Deletion of the N-terminal domain of lysine-less MyoD did not abolish completely ubiquitination of the protein, suggesting that this domain may be required for targeting the protein also in a postubiquitination step. Interestingly, NTU is evolutionarily conserved: in the yeast Saccharomyces cerevisiae lysine-less (LL) MyoD is degraded in a ubiquitin-, N-terminal domain-, and nuclear localization-dependent manner. Taken together, our data suggest that a short N-terminal segment of MyoD is necessary and sufficient to render MyoD susceptible for ubiquitin- and nuclear-dependent degradation.

Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

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. The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalyzed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.

Ubiquitination and degradation of proteins.

Modification by ubiquitin (Ub) and ubiquitin-like proteins (UbLs) is involved in the regulation of numerous cellular processes and has therefore become an important subject of research in various areas of biomedicine. The large number of components of the system (∼1,500), most of them being ligases (∼800) that recognize their target substrates specifically, along with the complexity of the ubiquitination process, mostly the synthesis of the hallmark polyubiquitin chains, has rendered studies of many of the processes related to the activity of the system resistant to detailed mechanistic analysis. Thus, our knowledge of the modes of recognition of target substrates by ligases and of consensus ubiquitination sites is sparse. We also lack basic tools such as antibodies directed against specific internal polyubiquitin chain linkages and analytical methods to decipher the structure of intact chains and their formation. All these tools are essential in order to understand the mechanisms that underlie the diverse activities of the system, proteolytic as well as non-proteolytic, and the manner in which it exerts its high specificity and selectivity toward its myriad substrates. Here we describe selected basic procedures that allow one to become acquainted with this rapidly evolving field, realizing that one cannot provide a comprehensive coverage of all or even a small part of the methodologies related to this research area. We provide information on how to set up a cell-free system for ubiquitination - a powerful tool that enables researchers to reconstitute the modification from purified components - and how to identify ubiquitin adducts in cells. Additionally, we describe methods to follow stability (degradation) of proteins in cell-free systems and in cells.

Monoubiquitination joins polyubiquitination as an esteemed proteasomal targeting signal

A polyubiquitin chain attached covalently to the target substrate has been recognized for long as the “canonical” proteasomal degradation signal. However, several proteins have been shown to be targeted for degradation following monoubiquitination, indicating that the proteasome can recognize signals other than a ubiquitin polymer. A comprehensive screen aiming at determining the extent of this mode of recognition revealed that ∼40% of mammalian and ∼20% of yeast proteins are degraded following monoubiquitination. Characterization of these proteins showed that on average, the monoubiquitinated proteins are smaller than the polyubiquitinated ones, and in humans, are less disordered. Further, proteins degraded by the two different modes belong to distinct functional groups. These findings along with detailed structural analysis of the proteasome, its ubiquitin receptors and deubiquitinating enzymes, suggest that the ubiquitin signal – its formation, recognition, editing, and removal – is far more complex and diverse than originally assumed. Also see the video abstract here: https://youtu.be/QKpN9c6Rg20

Epigenetic Regulation of KPC1 Ubiquitin Ligase Affects the NF-κB Pathway in Melanoma

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 the 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 microarrays; 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 miRNA 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 downregulated in American Joint Committee on Cancer stage IV melanoma compared with 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; HR 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; Pearsons r −0.455; P < 0.001), is significantly associated with KPC1 downregulation (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. Clin Cancer Res; 23(16); 4831–42. ©2017 AACR.