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Ubiquitin: Structure and Function

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Julius T. Dongdem, Simon P. Dawson and Robert Layfield

Submitted: 15 December 2022 Reviewed: 05 June 2023 Published: 28 February 2024

DOI: 10.5772/intechopen.112091

Modifications in Biomacromolecules IntechOpen
Modifications in Biomacromolecules Edited by Xianquan Zhan

From the Edited Volume

Modifications in Biomacromolecules

Edited by Xianquan Zhan and Atena Jabbari

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Abstract

Ubiquitin is a small (8.6 kDa) protein that is found ‘ubiquitously’ in eukaryotic organisms and functions as a regulator of numerous cellular processes. It is a multifaceted post-translational modifier of other proteins involved in almost all eukaryotic biology. Once bound to a substrate, ubiquitin initiates a plethora of distinct signals with unique cellular outcomes known as the ‘ubiquitin code’. More recently, much progress has been made in characterising the roles of distinct ubiquitin modifications though it is anticipated that more is yet to be unravelled as several questions remain elusive. The major aim of this chapter is to comprehensively review in detail using published data, the current understanding of the physico-chemical properties and structure (primary, secondary and tertiary) of ubiquitin, outlining current understanding of ubiquitin signal regulatory functions (Ubiquitin Proteasome System) and ubiquitin combinations, with emphasis on the structural relation to its function. Synthesis of ubiquitin (genes) will be illustrated. Additionally, ubiquitin-mediated processes and various possible covalent modifications of ubiquitin and their known functions will be illustrated. Deubiquitinase-dependent deubiquitylation of the ubiquitin code will also be described. Finally, ubiquitin-binding proteins and their ubiquitin-binding domains, the consequences of post-translational modification of ubiquitin by phosphorylation and future prospects will be discussed.

Keywords

  • gene
  • functions
  • ubiquitin
  • structure
  • modifications
  • ubiquitin-mediated processes
  • ubiquitin-binding domain
  • phosphorylated ubiquitin-binding protein

1. Introduction

Ubiquitin is a small (8.6 kDa) globular regulatory protein, which is found ‘ubiquitously’ in the cell-surface membrane, cytoplasm and nucleus of eukaryotic cells [1, 2]. Originally known as ‘ubiquitous immunopoietic polypeptide’ (UBIP), ubiquitin was first identified in calf thymus by Gideon Goldstein in 1975 in the search for thymopoietin and was further characterised through the 1980s [2, 3, 4, 5]. Aaron Ciechanover, Avram Hershko and Irwin Rose first expounded the functions and the components of the ubiquitylation pathway, which earned the group the Nobel Prize for Chemistry, in 2004 [5, 6, 7, 8, 9]. The main function of ubiquitin is labelling of improperly folded, unwanted or damaged proteins for proteasomal degradation [10]. However, ubiquitin may also cause a change in the cellular location, structural conformation or biological function of other target protein substrates.

Understanding of ubiquitin signalling has broadened in recent years. Many novel proteins with non-covalent mono- and/or polyubiquitin binding activity have been discovered. These proteins, which are collectively referred to as ubiquitin-binding proteins (UBPs), contain ubiquitin-binding domains (UBDs, Section 7.0), which interact with ubiquitylated targets and regulate diverse biological processes such as endocytosis and DNA repair. This notwithstanding, the molecular mechanisms governing ubiquitin recognition in most cases have remained elusive [11, 12]. Dysfunction of ubiquitin signalling has been implicated in a wide range of diseases, including cancer, immune disorders, neurodegeneration, cardiovascular and metabolic disorders [13, 14, 15]. More recently, the demonstration that ubiquitin itself can be modified through phosphorylation by PINK1 provided a major breakthrough linking two very important signalling pathways in cells; phosphorylation and ubiquitylation [16, 17, 18]. The scope of ubiquitin signalling functions is probably much broader than first envisioned. The major objective of this chapter is to comprehensively review the current understanding of the structure of the ubiquitin protein, outlining ubiquitin signal regulatory functions, ubiquitin combinations and ubiquitin-mediated processes with emphasis on the structural relation to its function.

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2. Physico-chemical properties of ubiquitin

Ubiquitin is a highly stable molecule over a wide range of pH and temperature values, exhibiting a melting point of ~100°C. Indeed, nuclear magnetic resonance (NMR) studies have shown that there is no significant denaturation of the ubiquitin fold over a temperature range of 23–80°C and a pH range of 1.18–8.48 [19]. The major contributor to ubiquitin stability is the large amount of intra-hydrogen bonding established within its entire structure, however, there are no disulphide bonds, coordinated metal ions or binding cofactors. Interestingly, if the ubiquitin protein is chemically denatured, the molecule can actually refold reversibly in vitro [20, 21, 22]. Ubiquitin is very resistant to tryptic (protease) digestion [2]. Only the two terminals -Gly75-Gly76 are lost during proteolysis. This phenomenon is utilised in liquid chromatography with tandem mass spectrometry (LC-MS/MS) identification of covalent target conjugates of ubiquitin. The ubiquitin protein is composed of all common amino acids in different proportions except Trp. Its molar extinction coefficient [ελ = 280 nm] is 1280 [L/(mmol·cm)]. It has an Eetinction coefficient A280 of 1 = 6.69 mg/mL and an isoelectric point (pI) of 6.79. Net electric charge of ubiquitin at pH = 7 is −0.14 [3, 23].

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3. Ubiquitin genes

The human genome consists of several copies of the ubiquitin sequence as a multigene family. The human ubiquitin is encoded by four independent genes: UBB, UBC, UBA52 and RPS27A [24]. UBA52 (located on chromosome band 19p13.1-p12) and RPS27a (chromosome band 2p16) encode a single copy of ubiquitin molecule, which is respectively fused to the ribosomal protein (RP) L40 and S27a (Figure 1). UBB and UBC are polyubiquitin precursor genes, with UBB encoding three ubiquitin moieties and UBC encoding nine ubiquitin monomers linked head-to-tail in tandem repeats by isopeptide linkages and without any spacer (intron) sequences. The last ubiquitin in the sequence is often extended with an additional Val residue in humans, and Tyr in chicken at the C-terminal end [25, 26, 27, 28]. The number of tandem repeats of ubiquitin moieties varies depending on species and can also differ within one species. Species of kinetoplast eukaryotes, for example, Trypanosoma cruzi, contain polyubiquitin coding sequences with 52 tandem repeats of the ubiquitin gene. Generation of free ubiquitin molecules is by post-translational cleavage from the precursor ubiquitin moieties and is achieved by the action of deubiquitinases (Dubs), which hydrolyse the isopeptide bonds linking ubiquitin to the L40, S27a ribosomal proteins or amino acid linking the UBB or UBC translates (Figure 1) [29]. UBB gene and UBB pseudogenes are located on chromosome band 17p12-p11.1 while UBC is located on chromosome band 12q24.3 [25, 27, 30].

Figure 1.

Human ubiquitin genes and the ubiquitin-proteasome system (UPS). Ubiquitin is encoded by four independent genes in the human genome, including UBB, UBC, UBA52 and RPS27A. UBB encodes three ubiquitin moieties, while UBC encodes nine ubiquitin monomers linked head-to-tail in tandem repeats by isopeptide linkages, extended by an amino acid, for example, Val. UBA52 and RPS27a encode a single copy of ubiquitin molecule respectively fused to a RPL40 and RPS27a. This phenomenon is illustrated by the figure. It also illustrates how free ubiquitin molecules are generated by post-translational cleavage of precursor ubiquitin by dubs in order to maintain the ubiquitin pool. The 26S proteasome is responsible for degradation and recycling of unwanted proteins. Proteasomal proteolysis enables the cell to rid itself of these misfolded or damaged proteins and re-adjusts the concentration of essential proteins so that cellular homeostasis is maintained.

The physiological role of ubiquitin-coding genes as a source of ubiquitin remains largely elusive. UBA52 is apparently essential in the development of embryo. It has been demonstrated that UBA52 gene is not only a contributor to the ubiquitin pool, but also a regulator of the ribosomal protein complex (RPL40), downstream protein synthesis and cell-cycle arrest [31]. RPL40 is essential for translation of specific cellular transcripts. RPS27a is a regulator of microglia activation and is biologically relevant in triggering neurodegenerative diseases [32]. Other studies have underscored RPS27a as a signal transmitter between DNA damage response and cell cycle progression/ribosome biogenesis. RPS27a is implicated in the inhibition of apoptosis and promotion of cell proliferation [33, 34]. Both RPS27a and UBA52 are preferentially over-expressed during hepatoma cell apoptosis [35]. An aberrant form of UBB translation has been detected in patients with Alzheimer’s disease as well as patients with Down syndrome. Other diseases associated with UBB include cleft hard palate and submucosal cleft palate. UBC has been reported to be the most responsive gene to UV irradiation, heat shock, oxidative stress, proteotoxic stress and translational impairment, and is upregulated under these conditions [27, 35, 36, 37, 38].

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4. Structure of the ubiquitin protein

4.1 Primary structure

Ubiquitin is a 76-residue protein, which is evolutionarily one of the most highly conserved eukaryotic proteins known to date [39]. The amino acid sequence of the human ubiquitin for instance is 100% identical to that of sea slug aplysia. Similarly, primary sequences of ubiquitin isolated from bovine, fish, insects and humans are identical in the first 74 amino acids [40, 41, 42, 43]. Yeast, barley, soya-bean, arabidopsis and oat ubiquitin primary sequences differ in only 3 out of 76 residues in comparison with that of higher eukaryotes [39]. Comparison of ubiquitin genes across eukaryotes have been extensively documented [44]. The amino acid sequence of a molecule of ubiquitin is shown in Figure 2. Ubiquitin possesses seven Lys residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63), including an N-terminal Met(1) residue, which serves as linkage points for ubiquitylation in the formation of ubiquitin polymers. Each ubiquitin molecule also contains a C-terminal Gly76 whose free carboxylate group is used to bond with an ε-amino group of a substrate’s Lys or that of preceding ubiquitins to generate various polyubiquitin chains (see later) [45].

Figure 2.

Primary structure of the ubiquitin protein. The human ubiquitin amino acid sequence using their three-letter abbreviations is shown in the figure. Indicated are the seven Lys residues, including the four important C-terminal residues, respectively, highlighted in yellow and red. Positions of amino acids in the sequence are specified as superscripts.

4.2 Secondary structure

The most relevant secondary structural features found throughout ubiquitin include three helices; 3.5 turns of α-helix (15.8% of α-helix) and a short 310 helix (7.9%) (Table 1; Figure 3). Each ubiquitin also contains one mixed β-sheet with five β-strands, no barrel and − 1 3X 1 –2X topology (Table 2) [46]. There are six β-reverse turns (Table 3) and two β-harpins whose characteristics are summarised in Table 4 [47, 48]. Ubiquitin also contains a G1 type β-bulge, which is antiparallel (residue X: Tyr7, residue 1: Gly10 and residue 2: Lys11), two reverse Asx turns and a symmetrical hydrogen bonding region between two α-helices and two reverse turns (Figure 3) [46].

Helix №StartEndType№ of residuesLength [Ǻ]Unit riseResidues per turnPitch [Ǻ]Deviation [degrees]Sequence
12334H1217.521.463.685.3810.8IENVKAKIQDKE
23840G3—————PDQ
35659G46.771.693.455.8240.1LSDY

Table 1.

Secondary motives of the three helices in the human ubiquitin (15.8% of α-helix; 7.9% of 310 helix).

Figure 3.

A wiring illustration of the secondary structure of the human ubiquitin. The figure is an illustration of the most relevant secondary structural motives of ubiquitin stretched out from start to end in the form of a string. One-letter abbreviation of each amino acid has been mapped to the secondary structural motif below the structure. Amino acids that make up β-turns (β-t) are highlighted in red colour. The five β-strands of ubiquitin (β-s1 to 5) are shown as blue sheets (arrows) while the three α-helices (H1, H2 and H3) have been illustrated in grey. Positions of β-hairpins (β-h1 and 2) have also been indicated.

№ of standsStartEndEdge№ of residuesSequence
127—6QIFVKT
21216+5TITLE
34145—5QRLIF
44849+2KQ
56671—6TLHLVL

Table 2.

Secondary motives of the five β-strands in the human ubiquitin (31.6%).

β-turnStartEndTurn typeH-bondSequence
1710I+TLTG
21821I+EPSD
34447IV—IFAG
44548I′+FAGK
55154I+EDGR
66265II+QEKS

Table 3.

Secondary motives of the six β-turns in the human ubiquitin.

Strand 1Strand 2Class
StartEndLengthstartEndLength
278121653:5
41455484922:2

Table 4.

Two β-hairpins in the human ubiquitin.

4.3 Tertiary structure

Naturally, ubiquitin adopts a compact ß-grasp globular fold with a globular surface area of 4800 Å2. It has been demonstrated that ubiquitin folds via a two-state process between a native and unfolded state in aqueous solution [49, 50]. Its terminal Gly76-COOH freely protrudes from the globular structure allowing covalent modification of target proteins, including other ubiquitin moieties (Figure 4). Leu8, Val70 and Ile44 residues of ubiquitin form a hydrophobic surface patch centred around Ile44, which in combination with electrostatic potential resulting from positively charged residues, including Lys6, Arg42, Lys48, His68 and Arg72, are relevant for the non-covalent interaction with many UBDs [51, 52]. The hydrophobic patch also facilitates intra-chain interactions between certain polyubiquitin chains. Additional hydrophobic surfaces include a patch centred on Ile36 involving Leu71 and Leu73 as well as the Phe4 patch, which involves Gln2 and Thr12. The ‘TEK-box’ of ubiquitin found in higher eukaryotes is a three-dimensional motif comprising Thr12-Thr14-Glu34-Lys6-Lys11 and is required for mitotic degradation [53, 54]. Another interaction surface includes the C-terminal di-Gly75,76 motif of ubiquitin, which interacts with the ZnF (Zn finger)-UBP, the UBD of HDAC6 during aggresome formation (i.e. intracellular protein aggregation) [55]. Characterisation of a novel acidic interaction (hydrophilic) surface centred on Asp58 has been found to be recognised by the ZnF_A20 UBD of Rabex-5 [56, 57]. Ubiquitin exhibits several strategies that maintain its three-dimensional structure in a very stable state. There is almost no noticeable change in conformation of ubiquitin in water solution and in crystalline form. All three Pro residues19, 37,38 of ubiquitin (at 1.8 Å crystal structure, 1UBQ. pdb) display the Cγc-exo conformation. Pro19 is a component of a flexible loop that connects the N-terminal β-harpin of ubiquitin to the 3.5 α-helix. Pro37 and Pro38 are, however, placed in an extended loop that links the C-terminal end of the 3.5 α-helix to the β-strands [58]. These pro residues are reported to play critical roles in the conformational stability of ubiquitin [59]. Vijay-Kumar et al. [2] determined the three-dimensional structure of ubiquitin at a resolution of 2.8 Å and stated that the His, Tyr and the two Phe residues are located on the surface of the molecule.

Figure 4.

Three-dimensional structure of ubiquitin. Three-dimensional structure of human ubiquitin indicating functionally relevant amino acids with exposed secondary structures; the seven Lys residues, including the Leu8-Ile44-Val70 hydrophobic patch. Hydrophobic patches serve as platforms for many UBDs. Side chains of Lys residues are highlighted in cyan. β-strands are shown in grey arrows while the 3.5-turn of α-helix and 310 helix are in black. The C-terminal Gly76-COOH is shown protruding at the top of the structure. N-terminal met is shown at the bottom of the structure (reproduced from Dikic et al. [51]).

Ubiquitin usually exists either covalently attached to other proteins or another ubiquitin or free (unanchored), that is, covalent assemblies of multiple ubiquitins in a substrate-free form [60, 61, 62]. Lys48-linked diubiquitin adopts a closed conformation with the hydrophobic residues (Leu8, Ile44, Val70) forming the inter-domain interface [63]. Due to weak ubiquitin-ubiquitin interactions, the interface is not rigidly locked. This allows the functional hydrophobic residues to be accessible for interactions with various recognition domains (Figure 5a). Lys63-linked diubiquitin, however, adopts an extended conformation and therefore, no hydrophobic interaction exists between its ubiquitin units (Figure 5b).

Figure 5.

Conformation of (a) Lys48-linked diubiquitin and (b) Lys63-linked diubiquitin. Hydrophobic patches are (recognised by UBDs) highlighted in gold and cyan in the ball and stick model. The side chains of Lys48 and Lys63 are highlighted in red (reproduced from Pickart and Fushman, [64]).

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5. Ubiquitin modifications and functions

5.1 Major functions

As aforementioned, the major function of ubiquitin is regulation of the degradation of other proteins [6, 7, 64]. Ubiquitin plays a role in the intracellular ATP-dependent, non-lysosomal proteolysis and elimination of defective proteins, normal proteins with a rapid turnover, as well as certain short-lived regulatory proteins within the cytoplasm. The mechanism of the 26S proteasomal degradation of unwanted proteins involves covalent binding of a polyubiquitin chain of about four or more ubiquitin moieties to the target proteins to be degraded by formation of isopeptide bonds between the free carboxylate group of ubiquitin’s C-terminal Gly76 and an ε-amino group of the substrates’ Lysyl side chains in a process known as ubiquitylation. Protein ubiquitylation is a post-translational modification (PTM) event carried out in a three-step enzymatic process by an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase [13, 28, 45]. Post-translational modifications (PTMs), which are usually covalent and reversible, may alter the properties and therefore, the functions of the modified protein. Proteasomal proteolysis enables the cell to rid itself of these unwanted, short-lived, inactive, oxidised, unfolded, misfolded or damaged proteins and re-adjusts the concentration of essential proteins so that cellular homeostasis is maintained (Figure 1). Protein ubiquitylation plays a plethora of roles aside from proteasomal degradation [45, 65]. The ubiquitin code is able to modulate cell cycle progression, cell differentiation, several signal transduction pathways and membrane protein trafficking. Ubiquitin induces differentiation of both B (bone-marrow-derived) and T (thymus-derived) lymphocytes [1]. Ubiquitin cross-reactive protein, a 15 kDa protein believed to be an example of functionally distinct family of ubiquitin-like proteins, is essential in various cellular responses to biological effects of interferons [66]. Strikingly, ubiquitin acts as an immunophilin, in that it is able to bind immunosuppressive drugs such as tacrolimus (FK506, Kd = 0.8 nM) and sirolimus (rapamycin, Kd = 0.08 nM). Ubiquitin complexes with tacrolimus, which acts as an inhibitor of calcineurin (protein phosphatase 2B) involved in T cell activation [67]. A peptide fragment of ubiquitin; Leu50-Glu51-Asp52-Gly53-Arg54-Thr55-Leu56-Ser57-Asp58-Tyr59 possesses a very high immunosuppressive activity in both cellular and humoral immune responses that is at the level of cyclosporine, with Leu50-Glu51-Asp52-Gly53-Arg54-Thr55-Leu56 fragment been the shortest possible fragment with effective immunosuppressive activity [68]. Ubiquitin is a component of several cytoplasmic inclusions such as Rosenthal fibres, Mallory bodies, Crooke bodies, Lafora bodies and amyloid bodies, and of neurones such as the giant axonal neuropathy (GAN) and paired helical filaments (PHF), which constitute a distinct type of pathological neuronal fibre. These make up the principal constituent of neurofibrillary tangles (NFL) that occur in the brain of patients with Alzheimer’s and Down disease [69, 70]. Apparently, association of ubiquitin with these cell inclusions may signify ATP-dependent proteolysis of these bodies, for example, NFL. Subcellular localisation of proteins where they control other protein functions and cell mechanisms are regulated by the ubiquitin code. For example, ubiquitin is involved in the ATP-dependent insertion of monoamine oxidase A and B into the outer mitochondrial membrane [71]. Autophagy, transcription, inflammatory signalling, modulation of enzymatic activity, DNA repair, stress responses, embryogenesis, cell apoptosis, virus budding, vacuolar protein sorting, inflammatory response and receptor endocytosis are regulated by ubiquitin-mediated signalling [64, 72, 73, 74]. Ubiquitin is responsible for the organisation and maintenance of chromatin structure by binding histone H2A in the nucleus. The first of such conjugations of ubiquitin described was binding histone H2A through an isopeptide bond with ε-NH2- group of Lys119 of histone H2A [4, 75]. Additionally, ubiquitin regulates heat shock responses and is a constituent of certain cell surface receptors [76, 77]. Ubiquitin has also been demonstrated to play a role in the regulation of gene expression [78].

5.2 Ubiquitin-mediated processes

The cell must continually maintain its internal homeostatic conditions. The lifespan of each protein is highly regulated. The ubiquitin-proteasome system (UPS) is a major ubiquitin-mediated process recognised as the cellular protein quality control system in that it selectively targets all unwanted or damaged proteins, which would otherwise accumulate and destroy neurones among others for proteasomal degradation. The UPS, which involves a complex combination of several enzymes (over 1000 proteins in human ubiquitylation), is now one of the most important systems required for the regulation of protein function because it is involved in nearly all the important cell biological activities, such as cell metabolism, cell proliferation, glycogen synthesis and cell death, as well as in disease pathogenesis, for example, inflammation, arthritis, heart disease and cancers [79]. The UPS plays essential role in protein homeostasis in that the system regulates the turnover of proteins required for the plethora of regulatory pathways responsible for several cellular processes, for example, DNA damage and repair, cell cycle progression, apoptosis, etc. As such dysfunction of the ubiquitin pathway often results in pathological conditions.

Ubiquitin conjugated on target proteins can, however, be removed by Dubs into free ubiquitin or unanchored ubiquitin chains (Figure 1). Degradation of ubiquitin chains by the UPS machinery also recycles ubiquitin molecules to maintain homeostasis in the cells [80]. Dubs, therefore, further add complexity to the inherent ubiquitin code by acting as proofreading enzymes, which increase the specificity of the UPS (see Section 6.0). The occurrence of different ubiquitin-ubiquitin linkage types further complicates the process and also indicates other potential regulatory functions yet to be discovered.

5.3 Role of ubiquitin modules

Polyubiquitin chains, when attached to a target protein, exhibit different functions depending on the Lys residue of the ubiquitin involved in the formation of the linkage. This arises as a result of changes in shape of the polyubiquitin chain in which a unique and complex ubiquitin code is generated depending on the specific Lys residue on which the preceding ubiquitin molecule is covalently attached to the next ubiquitin molecule on the target protein [81]. The versatility of ubiquitin to potentially exhibit diverse and highly complicated linkage-specific type PTMs of target proteins is by virtue of the occurrence of an N-terminal Met (1) together with the seven Lys residues per ubiquitin moiety. Monoubiquitylation refers to the conjugation of a single ubiquitin molecule to a single Lys of the target protein (Figure 6). Multimonoubiquitylation implies that a target protein is tagged with more than one single molecule of ubiquitin. When the target protein is tagged with a polyubiquitin chain linked through the C-terminal Gly76 of each ubiquitin unit and a specific internal Lys of the previously attached ubiquitin through a series of ubiquitylation, the module is known as polyubiquitylation (Figure 6). A polyubiquitin chain is termed homogenous when the ubiquitin monomers are joined together through a single ubiquitin-ubiquitin linkage-type, whereas a heterogeneous polyubiquitin chain contains more than one single linkage type (Figure 6) [81]. Mixed polyubiquitin chains on the other hand contain one linkage type, which is extended by a second type and so on, forming a non-branched structure. However, a polyubiquitin chain is said to be branched when different linkage types form one or more branches linking multiple Lys residues in the same ubiquitin [82].

Figure 6.

Multifaceted ubiquitin modifications. The figure illustrates different architectural modules of ubiquitin-ubiquitin linkage and some of the known cellular processes they have been found to regulate to date.

Typically, both monoubiquitylation and multimonoubiquitylation on a single protein regulate intra- and inter-molecular interactions [81]. For instance, response to a genotoxic stress is mediated by monoubiquitylation of PCNA’s Lys164 resulting in subsequent recruitment of DNA polymerases lacking proofreading activity to overcome a DNA replication block and bypass DNA lesions [83]. Additionally, monoubiquitylation has been implicated in the endocytic trafficking of certain cargo proteins, for example, small GTPases and receptors such as the EGFR to specific cellular compartments at different stages of the endocytic pathway. Monoubiquitylation is known to regulate histone modification [84]. Monoubiquitylation has also been implicated in the regulation of gene expression as well as DNA repair [85, 86, 87, 88]. Multimonoubiquitylation is important for receptor endocytosis [89]. As noted previously, Lys48-linked polyubiquitylation (i.e. polyubiquitin chains linked through Lys48 of the proximal ubiquitin to the next ubiquitin moiety in the chain) are the primary targeting signals for proteasomal degradation [90, 91, 92]. Lys63-linked polyubiquitin chains function as scaffolds to assemble signalling complexes as in the activation of the transcription factor NF-κB, which is involved in inflammatory and immune response, DNA damage tolerance, the endocytic pathway and ribosomal protein synthesis [93]. Unanchored (substrate-free) polyubiquitin also has distinct signalling roles, including activation of protein kinases (Figure 6). Xia et al. [94] showed that unanchored Lys63-linked polyubiquitin chain assembled through the action of UBE2N/UBE2V1 (an E2 conjugating enzyme) and TRAF6 (the E3 ligating enzyme) also activate the NF-κB pathway by activating the kinase TAK1, which in turn phosphorylates and activates IκB kinase (IKK). While Lys6-linked ubiquitin chains are reported to be involved in DNA repair, Lys11-linked polyubiquitin chains are involved in endoplasmic reticulum-associated degradation (ERAD) as well as the regulation of cell-cycle. Lys11-linked polyubiquitin chains also target proteins for proteasomal degradation with Lys48-linked polyubiquitin being the most efficient trigger of degradation [95]. Whereas Lys29-linked polyubiquitylation is involved in proteotoxic stress response and cell cycle regulation, Lys33-linked ubiquitylation is apparently required for kinase modification. Linear linkage via N-terminal Met is reported to regulate cell signalling such as NF-κB signalling [96, 97, 98].

Though the function of phospho-Ser65-(poly)ubiquitin in parkin activation has been established, the molecular consequences of ubiquitin phosphorylation have not been fully explored. Additionally, little is known about the existence and therefore functions of phosphorylated unanchored (poly)ubiquitin. Also, much less is known about more complicated polyubiquitin architectures, including heterogenous chains having branches or mixed chains [99].

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6. Deubiquitylation

The action of the E1, E2 and E3 enzyme cascade in the process of ubiquitylation is antagonised by deubiquitylation proteases or Dubs. Dubs are proteases involved in the removal of ubiquitin from ubiquitylated proteins, as well as cleaving ubiquitin from its precursors [100]. Dubs cleave the isopeptide bonds between the tagged protein and ubiquitin or ubiquitin molecules. Immediately after a target protein has been delivered to the 26S proteasome, the tagged ubiquitin chain is cleaved off by proteasome-associated Dubs prior to target destruction after which the ubiquitin chain is likewise cleaved to produce free ubiquitin, which is recycled [101]. Generically, Dubs are capable of exo-activity (hydrolysis of ubiquitin-linkage from ends) and endo-activity (hydrolysis from within an ubiquitin polymer), however, in each case, an existing scissile linkage between an ε-NH2 group of a Lys in the distal ubiquitin or substrate protein and the C-terminus of the proximal ubiquitin is cleaved.

Approximately, 100 putative Dubs have been characterised from the human genome and have been grouped into five distinct families based on the architecture of their catalytic domains [100]. They include ubiquitin C-terminal hydrolases (UCH), ubiquitin-specific proteases (USP), the ovarian tumour proteases (OTU) and the five Machado-Josephin domain proteases (MJDs/Josephins). These four subfamilies are Cys proteases, which rely on a conserved Asp/Asn, His and Cys catalytic triad mechanism of action. In contrast, the fifth subfamily, the Jun activating binding protein 1 (JAB1/MPN/MOV34) also known as JAMMs are Zn2+ metalloproteases, which invariably utilise Asp, His and Ser residues to coordinate a catalytic Zn2+ [102]. Dubs recognise (poly)ubiquitin non-covalently through intrinsic catalytic core domains or mediated by UBDs, including the ZnF-UBP domain, ubiquitin-interacting motif (UIM), ubiquitin-associated (UBA) domain and ubiquitin-like (Ubl) domain. Dubs, therefore, represent a specialised class of UBPs [80].

Dubs have been implicated in the regulation of various cellular events and have recently emerged as attractive therapeutic targets. They play several significant roles in the ubiquitin-mediated processes [103]. Dubs carry out processing of ubiquitin precursors, most probably co-translationally (Figure 1). As already mentioned, ubiquitin is expressed as a pro-protein fused to either ribosomal proteins or as linear polyubiquitin plus an additional amino acid and must, therefore, be hydrolysed to yield the free mature (poly)ubiquitin [28, 104, 105]. They also hydrolyse unanchored polyubiquitin released from target proteins or synthesised by de novo conjugating machinery into free ubiquitin molecules, for example, Ubp1 [106, 107, 108]. By so doing, Dubs maintain the homeostatic control of cellular flux of free ubiquitin levels [108, 109]. Dubs are also responsible for rescuing proteins from ubiquitin-mediated proteasomal degradation thereby stabilising the proteins. By antagonising the process of ubiquitylation, Dubs reverse the ubiquitin-mediated signalling cascades in response to cellular environmental changes [100, 103]. Dubs may also edit the form of ubiquitin modification by trimming polyubiquitin chains on substrate proteins [80, 102, 110].

Dubs not only frequently associate with E3 ligases but are themselves often modified by ubiquitin. This suggests that Dub activity may also be regulated by E3 ligases and vice versa. While the basic enzymatic function of Dubs is understood, how Dub activity is regulated, the cellular pathways that are regulated by Dub activity, the cellular target substrates of Dub regulation as well as how substrate recognition is regulated have not been fully explored. Notably, ubiquitin may become Dub-resistant once phosphorylated. Significant inhibition of the hydrolytic activity of Dub enzymes by phosphorylation of ubiquitin has been reported [111, 112, 113, 114, 115].

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7. Ubiquitin-binding proteins and their ubiquitin-binding domains

A UBD is a modular structural motif, typically less than 50 residues of a protein that can bind non-covalently to a ubiquitin moiety. UBDs mediate the non-covalent recognition of (poly)ubiquitin modifications and interpret and transmit information conferred by protein ubiquitylation to control various cellular events.

Proteins that contain UBDs are collectively referred to as UBPs and are classified according to the type of UBD they possess. Most UBPs contain one or more recognisable UBD(s) (Figure 7). The majority of UBPs interact through ubiquitin’s canonical Ile44/Val70 hydrophobic patch on its β-sheet surface (Figure 4). A number of UBPs, however, bind to non-canonical sites such as the C-terminal di-Gly, the polar surface centred on Asp58, and the hydrophobic patch centred on Leu8 or Ile36 [55, 116]. Although individual UBDs bind ubiquitin with low affinity, they do so with high specificity, a probable requirement for rapid assembly and disassembly of ubiquitin–UBD complexes. To date, most isolated UBDs bind monoubiquitin, as such do not exhibit linkage specificity, and are involved in the regulation of a variety of cellular processes such as endocytosis, DNA repair, vesicular trafficking or signal transduction (Figure 7, left panel). A number of UBDs bind polyubiquitin and can differentiate the eight ubiquitin linkages, which are essential for linkage-selective functions (Figure 7, middle panel) [117]. Where two or more UBDs simultaneously recognise different ubiquitin moieties within a specific polyubiquitin chain via avidity effects, they typically align in tandem on the ubiquitin chain (Figure 7, right panel) [118]. Currently, more than 20 different families of UBDs have been characterised based on structure. Nevertheless, additional UBDs will undoubtedly be discovered [51, 118, 119].

Figure 7.

Ubiquitin interactions with ubiquitin-binding proteins in the control of cellular dynamics. An illustration of UBPs which contain UBDs typically less than 50 residues in length. UBDs adopt many different folds and are capable of interacting non-covalently with both mono- (left panel) and polyubiquitylated (middle panel) targets. In so doing, UBPs interpret and transmit information conferred by covalent protein ubiquitylation to control various cellular events.

7.1 Families of ubiquitin-binding proteins

Several different families of UBPs have been described based on the type of UBDs they possess. Proteins that contain UBDs include ubiquitylation enzymes and Dubs as well as ubiquitin receptors. Ubiquitin-receptors (non-catalytic effector proteins) carry UBDs, thus, they recognise and bind ubiquitylated proteins and subsequently decode information into a specific cellular response. The activity of some ubiquitin receptors is controlled by self-monoubiquitylation, referred to as coupled monoubiquitylation. Coupled monoubiquitylation has been shown to involve monoubiquitylation of a ubiquitin ligase and its subsequent interaction with a ubiquitin receptor. Coupled monoubiquitylation provides an efficient switch from an active to an inactive conformation via an auto-ubiquitylation mode where the conjugated ubiquitin masks the UBD sequence [120].

Some examples of UBPs include S5a/Rpn10/Pus1 and S6’/Rpt5, which are two proteasomal subunits identified to interact non-covalently with Lys48-linked polyubiquitin chains of four or more moieties. The first UBP to be identified was S5a. The S5a/Rpn10/Pus1 ubiquitin-binding region was mapped to an approximate 20 residue region near the C-terminus. Mammalian S5a/Rpn10/Pus1 contains two of such regions, now recognised as the ubiquitin-interacting motif (UIM), and binds ubiquitin through hydrophobic interactions [121, 122, 123]. The Rpn13 protein also plays a role in proteasome function and is characterised by the Pleckstrin-like receptor for ubiquitin (PRU) domain [124]. Yeast protein Rad23/hHR23A (the human homologue) and Dsk2/Dhp1 are ubiquitin-associated domain (UBA) containing UBPs involved in shuttling ubiquitylated proteins to the proteasome whereas that of neighbour of BRCA1 gene 1 (NBR1) plays an important function in autophagy [125]. Whereas NF-kß essential modulator (NEMO), ABIN1-ABIN3 and OPTN are involved in NF-kß signalling and bind ubiquitin via their ubiquitin binding in ABIN and NEMO domain (UBAN) domain [126, 127], the Prp8 is involved in RNA splicing and employs its JAB1/MPN domain to bind ubiquitin [128]. UBCH5C (UBE2D3) contains the UBC domain. UBCH5C accepts ubiquitin from the E1 complex and catalyses its covalent attachment to other proteins [129]. Several other UBPs (e.g., NDP52, p62), their domain structure and functions have been extensively reviewed by many researchers [118, 130, 131].

7.2 Phosphorylated ubiquitin and phosphorylated ubiquitin-binding proteins

Eukaryotes have a large repertoire of PTMs, which include phosphorylation, acetylation, methylation, ubiquitylation, SUMOylation neddylation, glycosylation and glycation. PTMs act as a common mechanism for modulating and regulating protein function by considerably changing surfaces and binding properties of a protein. Phosphorylation is the biochemical process by which a phosphoryl (PO32−) group is added to a protein. Protein phosphorylation often results in the alteration of the tertiary structure, function and activity of the modified protein. Ubiquitin function can in turn be modulated by PTMs such as acetylation (on Lys residues) or phosphorylation. Each of these modifications has potential to dramatically alter the fate or signalling outcome of the modified protein [115, 132, 133, 134]. Phosphorylation of ubiquitin on Thr7, Thr12, Thr14, Ser20, Thr22, Thr55, Ser57, Ser65, Thr66 and Tyr59 has been reported in various studies [133, 135, 136], however, the kinases that instal phosphorylations on these residues are largely unknown with the exception of PINK1 [18] and the molecular consequences of phosphorylations on these sites have not been fully characterised [132, 133, 134, 135, 136, 137, 138, 139, 140].

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8. Conclusion and perspective

Although the ubiquitin structure has been resolved and is well documented, and recent studies have made creditable advances to understand the molecular basis of ubiquitin regulatory roles in higher-order polyubiquitin architecture, many questions have remained elusive. Much less is known about functions of more complicated polyubiquitin architectures such as heterogenous (branched and mixed) chains. The molecular mechanisms governing ubiquitin recognition by UBDs are not also well understood. It is believed that there are many more UBPs and UBDs than are currently known to exist. Additionally, little is known about the linkage type-specific functions of unanchored (poly)ubiquitin and phosphorylated unanchored polyubiquitin. The complex nature of ubiquitin modulation by phosphorylation has raised further questions. There are nine phosphorylatable Ser and Thr residues in ubiquitin besides Ser65. The molecular significance of their phosphorylation has not been fully investigated. The kinases (besides PINK1, i.e., the Ser65-ubiquitin kinase), phosphatases and selective recognition of ubiquitin targets induced by phosphorylations or dephosphorylations at these sites in brain and neurones have not been fully understood. How Dub activity is regulated, cellular pathways regulated by Dubs and how substrate recognition is regulated are yet to be explored. The current limitations preventing more insights into unanswered questions are probably due to lack of the most relevant and versatile technology for assessing these principles. However, it is envisioned that more insights regarding unanswered questions on ubiquitin biology will be unravelled in the near future.

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Acknowledgments

We thank IntechOpen Ltd. UK, for an opportunity to contribute to a book project.

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Conflict of interest

The authors declare no conflict of interest.

Funding

Self-funding and OAPF Waiver from Intech Open Ltd.

Author contribution

RL conceived the idea. JTD drafted the manuscript. All authors contributed to reviewing the manuscript.

Abbreviations

B lymphocytes

Bone-marrow-derived

Dub(s)

Deubiquitinase(s)

ERAD

Endoplasmic reticulum-associated degradation

GAN

Giant axonal neuropathy

H1–3

α-helice (1 to 3)1

JAB

Jun activation domain-binding protein 1

LC–MS/MS

Liquid Chromatography with tandem mass spectrometry

MJDs/Josephins

Five Machado-Josephin domain proteases

MPN/MOV34

Mpr1, Pad1 N-terminal domain

NFL

Neurofibrillary tangles

NMR

Nuclear magnetic resonance

OUT

Ovarian tumour proteases

PHF

Paired helical filaments

PTM

Post-translational modification

RP

Ribosomal protein

T lymphocytes

Thymus derived

UBD(s)

Ubiquitin-binding domain(s)

UBP(s)

Ubiquitin-binding protein(s)

UCH

Ubiquitin C-terminal hydrolases

UPS

Ubiquitin-proteasome system

USP

Ubiquitin-specific proteases

ZnF_A20

Zinc finger domain of A20

ZnF-UBP

Zinc finger ubiquitin-binding protein

β-h1

β-hairpin1

β-h2

β-hairpin2

β-s

β-strand

β-t

β-turn

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Written By

Julius T. Dongdem, Simon P. Dawson and Robert Layfield

Submitted: 15 December 2022 Reviewed: 05 June 2023 Published: 28 February 2024

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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