*1.1. Monoubiquitination in Protein Function*

Even though K48-linked polyubiquitination is the "canonical" signal for proteasomal degradation, a handful of proteins are degraded following monoubiquitination. Degradative monoubiquitination targets proteins smaller than 150 amino acids with low structural disorder,

whereas polyubiquitination recognizes proteins with highly disorganized structures independently of their size. Monoubiquitination-dependent substrates of proteasomal degradation are enriched for genes associated with carbohydrate transport and oxidative stress response pathways [4]. Because carbohydrate transporters are plasma membrane proteins, this finding is consistent with previous studies linking down-regulation of membrane receptors to monoubiquitination-mediated endocytosis [5]. The stability of ribosomal and proteasomal subunits is also regulated by monoubiquitination [6]. Multi-monoubiquitination could also act as a degradation signal [7]. For instance, multi-monoubiquitination of Cyclin B1 mediated by the Anaphase-promoting complex (APC/C) is su fficient to promote Cyclin B1 proteasomal degradation and mitotic exit [7].

The attachment of a single ubiquitin can serve as a signal for specific subcellular localization. It is well established that monoubiquitination triggers endocytosis of receptor tyrosine kinases (RTKs) and the NOTCH receptor 1 (NOTCH) from the plasma membrane [8,9]. Monoubiquitination targets the TNF receptor associated factor 4 (TRAF4) to cell–cell junctions where it is required to promote cell migration [10]. Dissociation of lipidated proteins from the plasma membrane could also be induced by ubiquitin conjugation [11]. Specifically, ubiquitin conjugated to K170 of the small GTPase Harvey rat sarcoma viral oncogene homolog (HRAS) sequesters its farnesyl and palmitoyl groups, impairing association of HRAS with the membrane [12]. Monoubiquitination is also involved in the control of nuclear/cytoplasmic shuttling. Vascular Endothelial Growth Factor (VEGF)-induced monoubiquitination of the actin-binding protein filamin B (FLNB) was shown to inhibit the nuclear translocation of Histone Deacetylase 7 (HDAC7). The monoubiquitinated form of filamin B binds to the nuclear localization signal of HDAC7, thereby transiently preventing its re-entry into the nucleus and mitigating the transcriptional repressor activity of HDAC7 on the genes required for VEGF-mediated responses [13]. In contrast, ubiquitin conjugation induces the nuclear export of the neddylation regulator, Defective In Cullin Neddylation (DCNL1). It was suggested that ubiquitin conjugation might act as a nuclear export signal that promotes the interaction with nuclear export machinery [14]. Furthermore, the monoubiquitination of the cancer stem cells marker CD133 (prominin-1) promotes its secretion into extracellular vesicles by facilitating the interaction of CD133 with the vesicular sorting protein tumor susceptibility gene 101 (TSG101) [15].

Multiple studies highlight a key function of monoubiquitination in controlling protein–protein interactions, both in positive and negative ways. Ubiquitin conjugation facilitates the interaction either by creating additional interaction surface or by attracting proteins containing ubiquitin-binding domains. Ubiquitin-binding domains have been classified into nearly 25 subfamilies. These domains are typically independently folded modular domains of up to 150 amino acids showing remarkable structural heterogeneity, which utilize diverse surfaces to contact ubiquitin [16,17]. Ubiquitin-binding activities are hard to detect and predict, both structurally and bioinformatically; thus, many of such ubiquitin-binding proteins are ye<sup>t</sup> to be identified. Especially taking into account that a subset of ubiquitin interactors has no obvious structurally defined ubiquitin-binding domains, such as the disordered Proteasome Complex Subunit SEM1/DSS1, in which ubiquitin-binding sites are characterized by acidic and hydrophobic residues [18]. The low-a ffinity interactions between ubiquitin-binding domains and ubiquitin are required for rapid, timely, and reversible cellular responses to a particular stimulus. To illustrate this, insulin-like growth factor 1(IGF-I)-induced monoubiquitination of the insulin receptor substrate (IRS-2) favors its interaction with the ubiquitin-binding protein EPSIN1, thereby enhancing IRS-2-mediated signaling and cell proliferation induced by IGF-I [19]. T-cell stimulation leads to monoubiquitination of the paracaspase MALT1 (mucosa-associated lymphoid tissue protein-1) that promotes the formation and stabilization of the catalytically active MALT1 dimer [20]. On top of this, auto-monoubiquitination of several E3 ligases, such as neural precursor cell-expressed developmentally down-regulated 4 (NEDD4), E3 ubiquitin-protein ligase Itchy (ITCH), and X-linked inhibitor of apoptosis (XIAP), is implicated in the recruitment of substrates and regulation of the ligase activity. For instance, the auto-monoubiquitination of NEDD4 promotes the recruitment of its substrate epidermal growth factor receptor substrate 15 (EPS15) [21].

Monoubiquitination can also block protein–protein interactions by either creating steric hindrance or inducing an autoinhibitory conformation for proteins containing ubiquitin-binding domains. As an example for the first mechanism, the attachment of ubiquitin within the e ffector-binding domain of the RAS-like GTPase, RAS-related protein B (RALB) sterically inhibits its binding to the downstream e ffector exocys<sup>t</sup> complex component EXO84 [22]. Another example is the inhibitory e ffect of monoubiquitination on the aggregation of amyloid proteins. Specifically, N-terminal monoubiquitination of presynaptic neuronal proteins, tauK18 and α-synuclein, changes their aggregation properties, resulting in structurally distinct aggregate structures that are cleared through proteasomal degradation instead of accumulating in cells [23]. On the other hand, the monoubiquitination of proteins that contain ubiquitin-binding domains could impose their autoinhibitory conformation, thus providing an intrinsic switch-o ff mechanism. This mechanism is used to regulate the degradative activity of the proteasome. Ubiquitin conjugation to the proteasome regulatory subunit RPN10 blocks the binding between its ubiquitin-interacting motif and interactors containing ubiquitin conjugates [24]. Monoubiquitination of the endocytic proteins, EPS15, hepatocyte growth factor-regulated tyrosine kinase substrate (HGS or HRS), ubiquitin-associated and SH3 domain-containing protein A and B (UBASH3A and UNASH3B, also called STS2 and STS1) leads to intramolecular interactions between ubiquitin and their ubiquitin-binding domains, thereby preventing their binding to ubiquitinated targets and inhibiting their tra fficking [25].

By di fferentially modifying the protein–protein interaction network, monoubiquitination provides precise and timely transmission of biological information [26]. To illustrate this, ubiquitin conjugation of the RAS-like GTPase RALB modifies its a ffinity for downstream e ffectors, inhibiting the interaction with EXO84 and facilitating its binding to exocys<sup>t</sup> complex component 2 (EXOC2 or SEC5). Thus, monoubiquitination within the e ffector-binding domain provides the switch for the dual functions of RALB in autophagy and innate immune responses [22]. Another example is the monoubiquitination of t-SNARE syntaxin 5 (SYN5) in early mitosis that disrupts SNARE complex formation. Subsequently, ubiquitinated SYN5 recruits p97/VCP (valosin-containing protein) to the mitotic Golgi fragments and promotes post-mitotic Golgi reassembly; thus, ubiquitin conjugation regulates Golgi membrane dynamics during the cell cycle [27].

Importantly, the attachment of a single ubiquitin molecule to specific lysine residues can generate diverse substrate–ubiquitin structures, leading to di fferent functional outcomes. This is well-described for the RAS-like small GTPases that undergo monoubiquitination at several lysines. While monoubiquitination of the GTPase Ras-related protein RAB5 at K165 a ffects its GTPase activity, attachment of ubiquitin at K140 alters the ability of RAB5 to bind and activate its downstream effectors. Ubiquitin conjugation to specific lysines also di fferentially a ffects the activity and subcellular localization of the Rat sarcoma (RAS) proto-oncogenes [28]. Specifically, the monoubiquitination of RAS at K117 promotes GTP loading, whereas monoubiquitination at K147 impairs GTP hydrolysis activity, both resulting in RAS activation. On the other hand, monoubiquitination at K170 impairs RAS association to the membrane and downstream signaling [12,29–31]. Altogether, these examples demonstrate a fundamental role of monoubiquitination in controlling protein function.
