Structural Biochemistry/Enzyme Regulation/Multitasking With Ubiquitin Through Multivalent Interactions
Ubiquitylation is the post-translational modification of proteins with ubiquitin, which is a powerful regulatory enzyme in eukaryotes. It can label proteins for destruction or activate gene transcription. Despite being versatile with its functions, ubiquitin is highly specific at sending signals for cellular events. Ubiquitin signaling pathway use multivalency, namely the coordinated use of multiple interaction surfaces. Multivalent interactions regulate each stage of ubiquitin signaling pathways, and appear within the ubiquitin signal, the ubiquitylated substrate, ubiquitin processing enzymes and ubiquitin recognition proteins.
Importance of multivalency in ubiquitin signaling 
Ubiquitin itself is a small 76 amino acid protein that use covalent bonds to attach to other proteins to exercise its regulatory functions. It is now known to communicate with more than 150 proteins, via discrete interacting surfaces. These proteins are labeled ubiquitin receptors, and together with ubiquitin through ubiquitylation, they regulate a vast array of cellular events including protein degration, protein trafficking, transcription, DNA repair, cell-cycle progression and apoptosis. (Figure 1). The ubiquitin signal itself is diverse and often multivalent, as are ubiquitin receptors and substrates.
The ubiquitin signal is diverse and multivalent 
Ubiquitin has a C-terminal glycine that is activated with ATP to form an isopeptide bond with the primary amino group of its substrate, which is usually the ɛ-amino group of a lysine, and also its amino terminus. Serine hydroxyl and cystein thiol groups can also be modified by ubiquitin. Substrate can be attached with a single or multiple ubiquitin (Figure 2). Polyubiquitylation takes place when ubiquitin is sequentially added to substrates to form ubiquitin chains. The chains can be of one linkage type, of mixed or forked with more than one ubiquitin attached. The multivalency provided by ubiquitin chains can greatly enchance their affinity for binding partners.
Ubiquitylation is a type of modification that is highy variable in length and linkage type. Different linkage result in different ubiquitin chain conformation and in unique binding epitopes, which can define downstream signaling events. When binding to ubiquitin chains of closed conformation, ubiquitin receptors must compete with the intra-chain ubiquitin packing interactions for access to binding surfaces.
Diversity and multivalency of ubiquitin processing enzymes 
The use of ubiquitin as a diverse signaling mechanism is supported by three enzymes classes, E1 activating enzyme, E2 conjugating enzyme and E3 ligase (Figure 2), which catalyze substrate ubiquitylation and define the type of ubiquitin chain linkage. Their actions are often regulated by multivalent interactions with each other and other signaling molecules and pathways. An E1 ubiquitin activating enzyme charges ubiquitin in an ATP-dependent manner to form a thioester bond with its catalytic cysteine. This modification induces structural changes in E1 that promote its binding to an E2 conjugating enzyme, to which ubiquitin is passed. E2 conjugating enzymes typically require E3 ubiquitin ligases to pass activated ubiquitin to a protein substrate; however, they can play defining roles in the ubiquitin chain linkage type.
The timing of substrate ubiquitylation is often relayed through multivalency effects of E3s (Figure 3). E3s can respond to their own phosphorylation status, to that of their substrate and to interactions with proteins that activate or suppress their activity. The use of multiple interactions to activate or suppress substrate ubiquitylation is exemplified through MDM2, the major E3 ligase for tumor suppressor p53, which promotes cell-cycle arrest and apoptosis. In response to DNA damage, the protein kinase ATM (ataxia telangiectasia mutated) phosphorylates the E3 MDM2 at several redundant sites near its RING domain to prevent its oligomerization. Oligomerization of the RING domain ofMDM2 is required for its polyubiquitylation of p53, a signaling event that leads to p53 proteolysis; thus ATMmediated phosphorylation of MDM2 in response to damaged DNA stabilizes p53 protein levels.
Other MDM2 interactions stimulate p53 degradation, including its phosphorylation at Ser260 by polo-like kinase-1 and its interaction with death-domain-associated protein DAXX. DAXX enhances the intrinsic activity of MDM2 towards p53 and functions as a scaffolding protein to recruit the deubiquitylating enzyme HAUSP, which protects MDM2 from degradation by removing ubiquitin chains that were formed by MDM2 autoubiquitylation. In the nucleus, these actions are counteracted by the tumor suppressor RASSF1A (Ras association domain family protein 1A), which binds MDM2 and DAXX, but displaces HAUSP, thereby destabilizing MDM2. The MDM2 example highlights the use of multilayered interactions to modulate E3 activity towards specific substrates in response to distinct cellular events. It is worth noting that phosphorylation of a substrate protein can also promote E3 recruitment or displacement.
Ubiquitin chain variability provides receptor selectivity 
The variable length and linkage type of ubiquitin chains provides selectivity to the outcome of ubiquitylation, because some ubiquitin receptors have strong preferences for ubiquitin chains of certain linkage type or size. Receptor specificity for ubiquitin chains of distinct linkage type couples the activities of ubiquitin processing enzymes with downstream signaling events. More than 20 different ubiquitin-binding domain (UBD) families, which exist in more than 150 human receptor proteins, have been identified to date. The mechanisms that they use to achieve selectivity for specific ubiquitin modifications have been comprehensively reviewed.
Ubiquitin chain multivalency enables simultaneous interaction with multiple ubiquitin-binding domains 
The binding of multiple UBDs to ubiquitin chains provides a general mechanism for enhancing the binding affinity of ubiquitin receptors for ubiquitylated substrate (Figure 3). In humans, the proteasome component S5a has two UIMs, which are separated by flexible linker regions causing their relative orientation to be undefined. This flexibility is markedly restricted when S5a binds to Lys48-linked diubiquitin because each UIM binds a ubiquitin moiety of the same molecule simultaneously. The outcome of this coordinated binding is significantly increased affinity between S5a and diubiquitin, suggesting that its two UIMs are not used to recruit multiple substrates to the proteasome simultaneously, but rather to increase affinity for each ubiquitylated substrate. It is worth noting that S5a and hHR23a can bind a common ubiquitin chain, as can S5a and Rpn13, the other intrinsic ubiquitin receptors of the proteasome. The biological significance of these interactions is not yet clear; however, complexes of multiple ubiquitin receptors with a ubiquitylated substrate provide additional levels of multivalency, which most likely leads to a greater binding affinity. Such complexes also seem to operate during endocytic processes to enhance binding affinity. Although monoubiquitylation is sufficient for receptor internalization during endocytosis, quantitative mass spectrometry indicates that more than half of all ubiquitylated epidermal growth factor receptor (EGFR) is conjugated with ubiquitin polymers (largely connected by Lys63). Most likely, modification with a polymeric ubiquitin chain enables more interactions with the UIMs of endocytic adaptors and in turn, interactions of higher affinity compared with that possible with only one ubiquitin subunit.
Multivalent interactions involving ubiquitin chains and multiple ubiquitin receptors can also transduce signals, as exemplified by the kinase activation mechanism in the NF-κB pathway.
Multivalent interactions of ubiquitin receptors influence the fate of ubiquitylated substrates 
Surfaces of ubiquitin receptors that do not bind ubiquitin play key roles in the trafficking and processing of ubiquitylated substrates (Figure 3). Ubiquitin receptors associated with proteasomal degradation have regions that dock them into the proteasome or that enable transient interaction with its components. The UBD of one such receptor, Rpn13, assembles into the proteasome via a surface opposite to its ubiquitin-binding region and this protein contains another domain that binds and activates Uch37, one of the three DUBs of the proteasome. Before their degradation by the proteasome, substrates are deubiquitylated and unfolded for passage through a narrow chamber leading to the catalytic center of the proteasome. Rpn13 might perform a dual functionality in the capture and deubiquitylation of proteasome substrates through its multivalent interactions with ubiquitylated substrates and Uch37.
The integration of multivalent ubiquitin-dependent interactions with ubiquitin-independent interactions is used extensively for endocytic trafficking to direct proteins from the plasma membrane to multi-vascular bodies (MVBs) (Figure 1).
Intramolecular interactions between ubiquitin-binding regions and covalently attached ubiquitin can inhibit ubiquitin receptor activity 
Ubiquitin receptors can be conjugated with ubiquitin, which in turn binds to their UBDs (Figure 3). Such intramolecular interactions can inhibit intermolecular interactions with their ubiquitylated substrates. This mode of regulation exists in the endocytic pathway. HGS, EPS15 and epsin undergo coupled monoubiquitylation, such that their UBD mediates their own ubiquitylation by binding to a ubiquitylated E3 or to an E3 with a ubiquitin-like domain. This modification leads to cis interactions with the attached ubiquitin, which inhibits trans interaction with ubiquitylated targets. The role of this so-called coupled monoubiquitylation in endocytosis remains poorly understood. It is possible that it weakens the interaction with cargo to enable ready passage of the substrate. The DUB UBPY (ubiquitin-specific protease Y) could relieve this autoinhibition by removing the conjugated ubiquitin from receptors, thus activating them towards new substrates.
Substrates modulate the effects of their ubiquitylation 
Although the fate of a ubiquitylated substrate is largely determined by its interaction with ubiquitin receptors, some substrate features can modulate the effects of ubiquitylation even after their recognition by a receptor of a designated function. For example, interaction with the proteasome typically culminates in the degradation of ubiquitylated substrates; this mechanistic pathway is an effective means to control protein lifespan. Such degradation, however, seems to require substrates to harbor, or be ‘complexed’ with, a protein containing an unstructured region, and proteins that are not ubiquitylated can be proteolyzed simply by associating with those that are, so long as they contain an unstructured region. By contrast, folded domains within ubiquitylated proteins appear to protect substrates from degradation.
Ubiquitylation can change binding affinities by adding multivalency to already existing interactions 
In the nucleus, ubiquitylation is widely used to change the affinity of already existing interactions by adding multivalency (Figure 3). For example, PCNA encircles DNA to serve as a ‘sliding clamp’ forDNApolymerases duringDNA replication. When a damaged site is encountered, replication is stalled and PCNA is monoubiquitylated at Lys164 (Figure 1). This modification is recognized by UBDs of trans-lesion polymerases to increase their affinity for PCNA and to promote their error-prone trans-lesion synthesis mode of replication [86,87]. After bypassing the lesion, the error-free, processive polymerase takes over. This switch might be due to PCNA deubiquitylation because the exchange back to the processive polymerase is prohibited when PCNA is monoubiquitylated at Lys164.
Protein ubiquitylation is also used to alter the DNAbinding affinity of nucleotide excision repair (NER) factor xeroderma pigmentosum group C (XPC). Ubiquitylation is also used to weaken substrate interactions with binding partners. Histones H3 and H4 are ubiquitylated in response to UV-induced DNA damage, facilitating the recruitment of NER machinery to damaged sites by weakening histone–DNA interactions.
Conclusion: Future potential 
Ubiquitin-mediated signaling is enabled by a large repertoire of enzymes that control the timing of modification, create diversity in the ubiquitin signal itself and enable dynamic alteration of the modification throughout a signaling pathway or in response to new stimuli. These enzymes communicate through ubiquitin to downstream receptors that operate within a larger context to enable signaling specificity. Versatility and specificity become congruent in ubiquitin signaling pathways through multivalency. Ubiquitin-binding regions are typically just one of many functional surfaces present in the receptor, which can contribute to the binding interaction, subcellular localization or link ubiquitin signaling with other post-translational modifications, such as phosphorylation. Ubiquitin belongs to a family of ubiquitin-like proteins that resemble ubiquitin structurally and perform their own distinct signaling, which can cross-talk with ubiquitin signaling. The ubiquitin signaling network is of therapeutic importance because parts of it are hijacked by pathogens or compromised in human diseases. It therefore is likely to have yet uncharted therapeutic potential and the manipulation of ubiquitinmediated protein degradation is actively being pursued for such purposes. Currently, the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma; this inhibitor preferentially induces apoptosis in tumor cells. The underlying mechanisms of its greater cytotoxicity in tumor cells are complex, ranging from the specific accumulation of proapoptotic proteins such as NOXA (NADPH oxidase activator1) to the activation of apoptosis through an endoplasmic reticulum (ER) stress response. Perhaps not surprisingly, drugs targeting the proteasome suffer from unwanted side effects, because many physiologically important processes are regulated by proteasomal proteolysis. Targeting of specific E3s, DUBs or ubiquitin receptors might afford clinical efficacy with fewer side effects. It is foreseeable that the multivalent interactions that regulate E3 and ubiquitin receptor activities could ultimately be used to target ubiquitin signaling for specifically restricting viral budding, stabilizing tumor suppressors or promoting DNA repair.
- Fen Liu1 and Kylie J. Walters1, 1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA. Trends in Biochemical Sciences Volume 35, Issue 6, June 2010, Pages 352-360 Link text, additional text.