**1. Introduction**

Cellular functions are highly dependent on the precise control of a single protein abundance within cells that is regulated by the equilibration of protein translation, folding, and degradation. The ubiquitination of proteins is one of the most important post-translational modifications, in which an ubiquitin, a small (8.6 kDa) regulatory protein, is attached to a substrate protein. This mechanism maintains protein homeostasis by regulating the degradation of cellular proteins, such as short-lived or long-lived regulatory proteins and damaged or misfolded proteins, mainly via the ubiquitin–proteasome or the autophagosome–lysosomal pathway (autophagy). Moreover, ubiquitination is involved in cellular processes by coordinating the cellular localization of proteins, activating or inactivating them, and modulating protein–protein interactions [1–3]. These effects are mediated by the addition of either a single ubiquitin (monoubiquitination) or a chain of ubiquitin proteins (polyubiquitination) [4].

In recent years, a considerable amount of research has been focused on the understanding of molecular action of ubiquitination in signaling pathways and how alterations in this mechanism ultimately lead to the development of human diseases. In this review, we summarize current knowledge of ubiquitination types and the sequential enzymatic cascade of ubiquitination as well as the involvement of this cascade with proteasomal degradation, which is known as the ubiquitin–proteasome system (UPS). Although protein homeostasis is regulated by the extensive crosstalk between the UPS and

other degradation pathways (e.g., autophagy), the only focus of this review is providing insight into the failure of accurately regulating cellular enzymatic and proteolytic processes in the most common diseases, such as cancer, neurodegenerative, and immune-related diseases.

### **2. Ubiquitin and Ubiquitin Proteasome System (UPS)**

Ubiquitin is a small, evolutionarily conserved 76 amino acid polypeptide encoded by four genes (*UBA52*, *RPS27A*, *UBB*, and *UBC*) in mammals. This protein was first identified in the 1970s as a protein of unknown function expressed in all eukaryotic cells; later on, the key features of this protein, including its C-terminal tail and the seven Lysine (Lys-K) residues, were revealed in the early 1980s [5–8]. The identification of ubiquitin–protein conjugates followed the discovery of the ubiquitination pathway, which was initially characterized as an ATP-dependent proteolytic system. ATP-dependent proteolysis factor 1 (APF-1) was found as a polypeptide that was capable of covalently binding to protein substrates in an ATP- and Mg<sup>2</sup>+-dependent manner [9]. Afterwards, the APF-1 protein was named as ubiquitin.

Ubiquitination is initiated by the attachment of the last amino acid (Glycine-76) of a single ubiquitin molecule to the Lys residue of one substrate protein that is called monoubiquitination. In case of the addition of one ubiquitin molecule to multiple substrate residues, the mechanism is called multi-monoubiquitination (Figure 1A). Indeed, monoubiquitination is required for the formation of a ubiquitin chain on a single Lys residue of the substrate protein, which is known as polyubiquitination. Moreover, polyubiquitin chains can be modified and turned into more complex structures by the addition of ubiquitin-like proteins (e.g., SUMO "Small Ubiquitin-like Modifier ", NEDD "Neural Precursor Cell Expressed Developmentally Down-Regulated Protein") or some chemical modifications (e.g., acetylation or phosphorylation) [9] (Figure 1B). In polyubiquitination, the ubiquitin protein can be ubiquitinated on its seven Lys residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) or on its N-terminus, which leads to the formation of different ubiquitin chain topologies. In fact, the ubiquitinated Lys residues as well as the ubiquitination of either the same (homotypic) or the different Lys residues (heterotypic) determine the fate of the substrate protein (Figure 1B). In other words, Lys63-linked polyubiquitin chains are associated with non-proteolytic cellular functions such as inflammation, protein trafficking, or DNA repair, while Lys48-linked polyubiquitin chains target substrates that are mostly related to the proteasomal degradation, such that the Lys48-linked ubiquitin chain can be recognized by a specific subunit of the proteasome [10].

The enzymatic mechanism of ubiquitination occurs in a proteolytic and non-proteolytic pathway consisting of three main steps: activation (E1), conjugation (E2), and ligation (E3) (Figure 2A). Initially, the binding of an E1-activating enzyme with a thioester bond activates ubiquitin in an ATP-dependent manner. Then, ubiquitin is transferred from an E1-activating enzyme to the E2-conjugation enzyme; thereafter, the E2 enzyme binds to the substrate-bound E3 ligation enzyme, resulting in a covalent attachment between the C-terminal of ubiquitin and the target substrate. As the cycle repeats, a polyubiquitination chain is formed, and the polyubiquitinated substrate is transferred to the proteasome for proteolytic degradation [11]. Considering the essential role of this enzymatic pathway in the protein homeostasis in the cell, three different enzymes (E1, E2, and E3) have been the subject of detailed research in the last decade. According to current knowledge, there are two E1 and approximately 40 E2 genes that exist in the human genome. Besides, more than 600 E3 ligase genes that are critical for the balance between ubiquitination and deubiquitination are known in the human genome [12,13]. E3 ligases are devided in three distinct groups based on their catalytic domains. The first group of E3s contains two domains: Really Interesting New Gene (RING) and UFD2 homology (U-box). The RING E3s domain coordinates Zn2<sup>+</sup> binding and recruits the ubiquitin-charged E2, while the U-box E3s domain acts in a similar manner without Zn2+ coordination. The second group of E3s includes the homologous to E6–AP carboxyl terminus (HETC) domain. This domain consists of C (contains a catalytic cysteine) and N (recruits ubiquitin-charged E2) lobes that can be modulated depending on the cellular pathway. The third and last group of E3s has a RING-between-RING (RBR) motif. RING1 recruits the ubiquitin-charged E2 and transfers ubiquitin to catalytic cysteine of RING2 that conjugates ubiquitin to the substrate [14]. It is worthy of note that E3 ligases are the well-studied enzyme in this enzymatic cascade, which is most probably due to its critical role in protein quality control and homeostasis in the cell.

**Figure 1.** Types of Ubiquitination. (**A**) Monoubiquitination, (**B**) Polyubiquitination.

The proteasomes (26S proteasome) are large and multi-catalytic proteases that are composed of a 20S barrel-shaped core complex and a 19S regulatory complex. Protein degradation occurs in the 20S complex, which consists of four stacked hollow rings, each of which has coordinately functional distinct subunits: outer α rings and inner β rings (Figure 2B). Outer α rings contain seven α subunits, and these subunits are controlled by the binding of cap structures that recognize polyubiquitin tags on the substrate proteins. β subunits contain three to seven protease active sites involved in degradation processes. Notably, the 20S proteasome has also a critical role in the dissociation of ubiquitin proteins from the substrates via deubiquitinating enzymes (DUB) [15]. Once the ubiquitinated proteins reach the 19S regulatory complex, this complex delivers them to the 20S proteasome. The 19S proteasome has 18 different subunits, and its central part consists of six ATPases that bind to substrate proteins for degradation, unfolding, and translocation into the 20S complex [16]. As a final process, proteins are degraded into polypeptides, which are then chopped into small fragments and ultimately into the single amino acids by the activation of peptidases [17]. The overall system of ubiquitination and proteasomal degradation is known as the ubiquitin–proteasome system (UPS).

Ubiquitination is a reversible and dynamic phenomenon. Deubiquitinating enzymes (DUBs) are a large group of proteases that can partially or completely remove ubiquitin and ubiquitin-like proteins NEDD and SUMO from the target substrate. Currently, 100 DUBs are encoded by the human genome, and they are classified into six different groups: ubiquitin-specific proteases (USPs), ubiquitin COOH-terminal hydrolases (UCHs), Machado–Joseph Domain (Josephin domain)-containing proteins (MJD), the JAB1/MPN/MOV34 family (JAMMs), motif interacting with Ub-containing novel DUB family (MINDYs) [18], and ovarian tumor proteases (OTUs) [19]. DUBs are critical for the regulation of cell survival, immune response [20], and cell differentiation [21,22]; therefore, they are considered as potential drug targets in various diseases.

**Figure 2.** Ubiquitin proteasome system (UPS). (**A**) Enzymatic reaction of ubiquitination. For activation of the ubiquitination process, the E1 enzyme makes a thioester bond with the ubiquitin molecule, and the required energy is supplied by the hydrolysis of ATP. In the conjugation step, the ubiquitin on the E1 enzyme is transferred to the E2 enzyme. Finally, the substrate–E3 enzyme bond is involved, and the C-terminal of the ubiquitin molecule makes a covalent bond with the substrate, which is bound to the E3 enzyme. (**B**) Structure of proteasome and degradation of ubiquitinated substrates. The 26S proteasome has a multicomplex structure that is composed of a 19S regulatory units and a 20S catalytic core unit. In the 19S regulatory complex, the ubiquitinated proteins are unfolded, and ubiquitin tags are separated from the protein by deubiquitinase enzymes. Consequently, the unfolded polypeptide chain is delivered to the 20S catalytic core complex where they are degraded into small peptides.

In the following sections, we would like to emphasize the current understanding of the dysfunction or dysregulation of ubiquitin enzymes and proteasomal degradation in several common diseases. Since the subject is too broad, we try to narrow down the topics by focusing specifically on E1, E2, and E3 enzymes, DUBs, UPS, and their association regarding the progression of cancer, neurodegenerative diseases, immune regulation, and immune-related diseases.
