Monoubiquitination in proteasomal degradation

Conjugation of ubiquitin to target proteins is a finely tuned process involving a reaction, culminating in the conjugation of a single ubiquitin (1). The first step is conjugation of a single ubiquitin molecule to the substrate’s protein amino group (monoubiquitination) or to multiple amino groups (multimonoubiquitination), which can remain as it is or be further extended by additional ubiquitin molecules to form elongated chains (polyubiquitination). The process is reversible because deubiquitinating enzymes can partially trim the polyubiquitin chains or completely strip all ubiquitin molecules from the protein (2, 3). Protein ubiquitination is of great physiological significance and plays a role in every cellular process. Examples include determining protein subcellular localization, regulating signaling (e.g., transcription factors, protein kinases), and controlling the duration and magnitude of a protein’s activity by promoting its clearance (4, 5).

The diverse functions associated with protein ubiquitination require exquisite regulation to ensure that the cellular machinery can distinguish between ubiquitination that marks a protein for translocation from one that targets the protein for elimination. This process is accomplished, in part, through the length, type, and organization of ubiquitin molecules on the substrate. On a simplistic level, monoubiquitination has largely been linked to chromatin regulation, protein sorting, and trafficking, whereas polyubiquitination has been associated with protein signaling and clearance through proteasomal or autophagic degradation (5–7). An additional layer of regulatory complexity results from the different topologies of polyubiquitin chains conjugated through various lysine residues (e.g., K6, K11, K29, K48, and K63). Different polyubiquitin chain topologies have been documented in vitro and in vivo, and they have been modeled structurally (8–11). In particular, high-resolution MS/MS has revealed the highly heterogenous nature of polyubiquitin chains and their topologies.

Cellular homeostasis critically depends on maintaining a balance between the synthesis of proteins and the clearance of proteins that have fulfilled their cellular duty (e.g., transient signaling) or have aberrant structure (e.g., genetic or cellular stress-induced misfolding). Protein degradation, which is primarily carried out by proteasomes, requires structural organization of the proteasome and of ubiquitin-binding subunits within the 19S proteasome subcomplex. Efficient entry and processing of proteins with monoubiquitin, multimonoubiquitin, or heterogenous ubiquitin linkages into the proteasome also depend on deubiquitinating enzymes and other regulatory factors (6, 12–17).

The study by Braten et al. in PNAS (18) demonstrates that mono- and multimonoubiquitinated proteins are commonly processed by proteasomes, more than we have appreciated to date. To identify and characterize monoubiquitinated proteasomal substrates, the authors silenced expression of endogenous wild-type ubiquitin and expressed a nonpolymerizable lysineless ubiquitin. The ectopically expressed ubiquitin can modify each lysine in the target substrate only once and cannot be further conjugated, creating only monoubiquitinated and multimonoubiquitinated proteins. Braten et al. (18) examined both human and yeast cells using this approach, which allowed a comparison of the significance of monoubiquitination in protein clearance in both species. The conditions used to analyze the yeast and human cells differed technically. In yeast cells, the ubiquitin genes were encoded by a dual promoter system, where galactose-dependent expression is used to achieve tight repression of synthetic wild-type ubiquitin and copper to induce lysineless ubiquitin expression. In human U2OS osteosarcoma cells, endogenous ubiquitin was silenced with tetracycline-inducible shRNA and then wild-type or lysineless ubiquitin was expressed by adenoviral infection. A CRISPR-based approach for expression of the lysineless ubiquitin gene may more closely resemble the approach used in the yeast system. However, the subsequent analytical steps were similar in yeast and human cells. In both cases, differentially ubiquitinated proteins were identified by MS-based stable isotope-labeling by amino acids in cell culture and label-free quantification. The classification of proteins as monoubiquitin- and polyubiquitin-dependent proteasome substrates was based on the ratio of signal intensity between wild-type and lysineless ubiquitin. Braten et al. (18) note that a subset of proteins classified as monoubiquitinated is expected to include proteins harboring multiple monoubiquitinated moieties conjugated to multiple different lysines. Multiple ubiquitin molecules spread across a single protein were shown to provide a degradation signal that can at times mimic polyubiquitination (12). Because polyubiquitinated substrates explore multiple configurations on the proteasomes through stochastic binding (17), one wonders whether this can also be the case for multimonoubiquitinated proteins. Along these lines, SIM domains and SUMOylation may be part of the recognition signal for monoubiquitinated substrates.

Surprisingly, only ∼25% of proteins degraded by the proteasome were mono- (or multimono) ubiquitinated in yeast (82), compared with ∼50% in human (220), whereas 303 and 416 proteins are dependent on polyubiquitination for proteasomal recognition and processing in yeast and human, respectively (Fig. 1). This unexpected observation led Braten et al. (18) to suggest that monoubiquitination is much more common in human cells than in yeast. One explanation for this difference might be the greater structural diversity of monoubiquitinated proteins in human cells (see below). The authors identify distinct populations of proteasomally processed ubiquitin-modified proteins, leading them to conclude that proteasomal processing of monoubiquitinated proteins is also more common in human cells than in yeast.

Fig. 1.

Recognition of ubiquitinated proteins by the proteasome. The outline summarizes the findings by Braten et al. (18). H, human; Y, yeast.

Further classification of the monoubiquitinated and polyubiquitinated proteins using bioinformatics tools points to an association between protein size, biological process, and sequence in the ubiquitinated domain. In both humans and yeast, shorter proteins (<150 amino acids) were more likely to undergo monoubiquitination, confirming the earlier observations by the Ciechanover group (15, 19). Braten et al. (18) suggest that monoubiquitination may provide a weaker targeting signal to the proteasomes, which is sufficient for degradation of smaller proteins. Because longer proteins were associated with either monoubiquitination or polyubiquitination, properties such as structural disorder are among factors that may govern their recognition by proteasomes.

Protein structural disorder (i.e., disordered regions that lack a defined tertiary structure) has previously been proposed to be a determinant of protein ubiquitination and proteasomal recognition (20–22); the Braten et al. (18) study provides new data to support this. Proteins of higher organisms often contain large regions that become intrinsically disordered upon posttranslational modification or association with other proteins; such regions can also oscillate between ordered and disordered forms. Bioinformatics modeling has shown that disordered proteins are enriched in eukaryotes compared with prokaryotes and are associated with signaling diversity [achieved by posttranslational modification (20–24), of which ubiquitination is one example]. Of note, the presence of disordered regions in a protein is a required signal for degradation, as evidenced by the proximity of long-disordered regions to ubiquitin ligase recognition motifs and ubiquitin-acceptor lysine residues (20–23). Such rearrangement is thought to enable unfolding of the substrate after engagement by the proteasome. Whether proteins with larger disordered regions are generally more dependent on p97, proteasome ubiquitin binding sites, and ATPases, is among the explanations that may enable proteasome recognition by different conformation/degree of disordered proteins.

Protein structural disorder (i.e., disordered regions that lack a defined tertiary structure) has previously been proposed to be a determinant of protein ubiquitination and proteasomal recognition; the Braten et al. study provides new data to support this.

The analysis by Braten et al. (18) suggests that, although structural disorder is seen in both monoubiquitinated and polyubiquitinated proteins in yeast, it is largely confined to polyubiquitinated proteins in humans. Consistent with the notion that polyubiquitination provides a stronger signal, structurally disordered proteins may benefit from the extended ubiquitination chains. The observed enrichment of polyubiquitinated proteins in humans may be a result of factors that define disordered regions, such as posttranslational modifications or the proximity to ubiquitinated sites. Other considerations that may underlie the differences seen in yeast and human cells include the replicative and physiological state of the cells (e.g., higher replication of the U2OS cells, differing levels of internal stress, and disparate degrees of signaling pathway activation, to name a few). Experimentally, it may be possible to establish a series of different structurally disordered domains within the same protein, enabling assessment of the relationship between structural disorder, the ability to undergo monoubiquitination and polyubiquitination, and recognition by the proteasome.

Several considerations may shed further light on the physiological significance of these observations. For example, is a monoubiquitinated protein subject to polyubiquitination under physiological conditions, where the prevalence of lysineless ubiquitin does not preclude chain formation? Are there physiological conditions that may favor the monoubiquitination state over multimono- or polyubiquitnation? Similarly, it will be of interest to determine whether a monoubiquitinated protein can out-compete the multimono- or polyubiquitinated protein for proteasome recognition?

The tools used in the Braten et al. (18) study allow us to further study the ubiquitination requirements for proteasomal degradation. Spatiotemporal analyses of genetically comparable systems could help clarify how changes in ubiquitination/degradation of regulatory pathways affect their recognition by the proteasomes. Similarly, further studies will be required to determine the contribution of cofactors, such as p97, ATPases, and deubiquitinating enzymes, to the recognition of ubiquitin-modified disordered regions by the proteasomes. In all, the findings of Braten et al. (18) are expected to catalyze new lines of studies aimed at better understanding the mechanisms underlying protein recognition by proteasomes, and the role of monoubiquitination vs. polyubiquitination in the context of disordered regions in proteasome recognition. The possibility to predict the type of ubiquitination that takes place in structurally disordered proteins may allow us to better define novel approaches to alter their stability or activity for therapeutic purposes.