*1.2. Enzymes Controlling Monoubiquitination*

The ubiquitination reaction involves three enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligase (E3) enzymes [32]. Although there are only 2 E1s and 30–50 E2s, approximately 600–700 E3 ubiquitin ligases are encoded in the human genome, conferring target specificity to the ubiquitination reaction. The process is reversible as ubiquitin hydrolases, also called deubiquitinating enzymes (DUBs), can hydrolyze the covalently bound ubiquitin peptides.

E2 conjugating enzymes attach ubiquitin molecules to lysine residues on protein substrate or ubiquitin molecules, prompting mono- or poly-ubiquitination. E2s share a highly conserved catalytic core domain called the ubiquitin conjugation domain (UBC). The UBC contains a specific catalytic cysteine that forms a thioester bond with ubiquitin. Most of the studies have been focused on ubiquitin-conjugating enzymes 2S and 3A (UBE2S, UBE3A) and ubiquitin-conjugating enzyme E2 variant 1 (UEV1A) which are responsible for pro-degradative K48-linked polyubiquitination and UBE2N, which is important for pro-trafficking K63-linked polyubiquitination. These E2-conjugating enzymes also have intrinsic ability to form free polyubiquitin chains. On the other hand, several E2 ubiquitin ligases, such as ubiquitin-conjugating enzymes 2A, 2K, 2T, and 2W (UBE2A, UBE2K, UBE2T, and UBE2W) are only able to generate monoubiquitin chains. When E2s are charged with ubiquitin molecules, some of them exhibit enhanced affinity for co-factors containing ubiquitin-binding domains that confer specificity of ubiquitin linkages to specific substrates [33].

The substrate specificity is given by the E3 ubiquitin ligases that control the transfer of ubiquitin to a lysine residue on the substrate. The E3 ligases identify their protein substrate within the cellular pool of proteins, by recognizing specific protein sequences or chemical motifs in the substrate. The E3 ligases can be classified into RING (really interesting new gene), HECT (homology to E6AP C-terminus), and RING-related types [34]. The activity of the RING E3s is specified by a RING domain, which promotes ubiquitin transfer from the E2 to the substrate, whereas the substrate-recruiting module is responsible for the substrate recognition. The RING domain and the substrate-recruiting module of the RING E3 ligase can be found in a single polypeptide, as in the case of CBL (Casitas B-lineage lymphoma), or in separate subunits of a multi-complex E3s, as for the heterodimer of BRCA1 (breast cancer 1) and BARD1 (BRCA1-associated RING domain 1), Cullin–RING ligases, and APC [34]. The HECT E3s have a catalytic cysteine residue that can form a thioester bond directly with ubiquitin [34]. While the HECT domain represents the catalytic domain, the substrate specificity of HECT E3s is determined by their respective N-terminal extensions. The RING-between-RING (RBR) E3s define a third class of ubiquitin ligases distinct from the RING and HECT types. The RBR E3s are characterized by RING1 and RING2 domains and a central in-between-RINGs (IBR) zinc-binding domain. Substrate ubiquitination by RBR ligases is a multistep process. It starts with the recognition of the ubiquitinated E2 by RING1, followed by the transfer of ubiquitin onto the catalytic cysteine in RING2 to form the thioester intermediate, and finally, the transfer onto the substrate [35].

Most E3 ligases are able to generate different types of ubiquitin chains, depending on the E2 conjugating enzyme with which they preferentially interact. It was shown that the monoubiquitinating E2 enzymes have stronger affinity to RING E3 ligases. For example, UBE2A binds the RING E3 ligase RAD18, whereas UBE2K and UBE2T have high affinity to the RING E3 ligase BRCA1 [36,37]. Furthermore, a process termed coupled monoubiquitination is responsible for the monoubiquitination of ubiquitin-binding domain-containing proteins. The proposed model suggests that monoubiquitinated substrates cannot be further polyubiquitinated because the ubiquitin-binding domain interacts intramolecularly with the attached ubiquitin, thus disrupting the association of the substrate with the monoubiquitinated E3 enzyme [21]. Monoubiquitination can also be achieved by engaging a DUB that trims the assembled poly-ubiquitin chain to produce monoubiquitinated proteins.

Given that around 5% of the human genome encodes ubiquitin system components [38], it is not surprising that alterations of the ubiquitination machinery have been observed in multiple disease conditions [39]. Here, we will discuss how dysregulation of monoubiquitin signaling due to germline mutations of E3 ligases or DUBs could contribute to the development of genetic disorders (Table 1).

**Table 1.** Genetic diseases associated with genes regulating monoubiquitination. Short list of substrates modified by the indicated E2 conjugating enzymes, E3 ligases, and ubiquitin hydrolases (DUBs) are shown, together with the indication of the modulated cellular functions and the type of mutations detected in patients.


### **2. Genetic Diseases Associated with Dysregulated Monoubiquitination**
