What is ubiquitin

Ubiquitin is a small 76 amino acid protein modifier of approximately 8.5 kDa that covalently attaches to the lysine residues of target proteins via its carboxy-terminal glycine residue, forming an iso-peptide linkage, in an ATP-dependent fashion ¹. Four genes in the human genome code for ubiquitin: UBB, UBC, UBA52 and RPS27A ¹. Ubiquitin through its chemical conjugation onto proteins (ubiquitination) plays major roles in regulating almost all cellular processes from tagging them for proteasomal degradation (protein degradation), protein trafficking, cell-cycle regulation, DNA repair, apoptosis and signal transduction ² and protein-protein interaction ³. The ubiquitination process is catalyzed by three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase enzyme (E3). In the first step, ubiquitin is activated and binds to C-terminus of an E1 enzyme with the use of ATP, followed by the conjugation of the activated ubiquitin to an E2 enzyme. Next, E2 interacts with an E3 ubiquitin ligase that binds to a substrate protein, and transfers the ubiquitin to the lysine residues in the substrates. All the enzymes are then dissociated form the substrate protein to complete the ubiquitination process. These steps can be repeated to form polyubiquitinated chains ⁴.

Since ubiquitin itself contains seven lysines, it can attach repeatedly to other ubiquitins, allowing the formation of polyubiquitin chains. Owing to diverse lysine residues in proteins, proteins can be either ubiquitinated on a single lysine residue or on multiple lysine residues, resulting in monoubiquitination and multiubiquitination, respectively. Moreover, proteins can also be polyubiquitinated by homotypically attaching a number of ubiquitin molecules on the same lysine residue sequentially ⁵. In addition, when the attachments of additional ubiquitin to the lysine residues of the previous ubiquitin molecule at different positions, it leads to the formation of branched polyubiquitination chains ⁶. Ubiquitination can also occur on cysteine, threonine and serine residues in some cases, which make protein  ubiquitination more diverse ⁷. Therefore, ubiquitin exists intracellularly either as a monomer, a substrate-conjugated polyubiquitin or monoubiquitin, or free (or unanchored) ubiquitin chains, and there is a dynamic equilibrium among the three forms in the cell. The ubiquitination process can be reversed by deubiquitinating enzymes (DUBs), which are ubiquitin-specific proteases. Up to date, it is estimated that there are more than 30 ubiquitin-conjugating enzymes (E2s), 1000 ubiquitin ligase enzymes (E3s) and 100 deubiquitinases (DUBs) discovered in mammalian cells ⁸. Nevertheless, the mechanism of the distinct selection of E2s and E3s for different types of ubiquitination remains largely elusive ⁹.

Ubiquitin itself can also itself become ubiquitinated, resulting in a poly-ubiquitin chain defined by the specific modified lysine residue or in the case of M1/linear chains, the methionine residue of ubiquitin ¹⁰. The type of chain created has a distinct effect on the fate of its substrate protein; for instance,K48 chains are known for their involvement in targeting proteins for proteasomal degradation ¹¹. Ubiquitiniquitination is a reversible process; deubiquitinases (DUBs) remove ubiquitin from a protein or edit the length and type of a ubiquitinchain ³.

Unanchored ubiquitin chains – that is, poly-ubiquitin that is not tethered onto a substrate protein – also exist in the cell. Unanchored ubiquitin chains can arise when a deubiquitinase removes an intact chain from a protein, or they can be generated anew through E1/E2/E3 cycles. Although unanchored poly-ubiquitin is not well understood, it has been implicated as a participant in several cellular processes, including NF-κB signaling and autophagy ¹². The prevailing view is that unanchored ubiquitin chains are quickly disassembled by deubiquitinases and recycled as mono-ubiquitin ¹³. Studies in yeast and in cultured mammalian cells have suggested that the buildup of free poly-ubiquitin might become toxic by perturbing ubiquitin-dependent proteasomal degradation ¹⁴.

The diversity of protein ubiquitination is of great significance for protein functions. Different ubiquitination chains display variant structures that can be recognized by different ubiquitin binding domains in a specific manner, leading to different fates of proteins through different pathways. Monoubiquitination was reported to be important for regulation of histone functions, modulation of DNA repair, receptor endocytosis and gene expression ¹⁵. The polyubiquitination chains through K63 is thought to be involved in the regulation of kinase activity, signal transduction and DNA damage response ¹⁶. The K48-and K11-linked polyubiquitination, on the other hand, mainly guides proteins for proteasome-mediated degradation ¹⁷. The monoubiquitination of histone H2A is mediated by the branched ubiquitination chains on RING1B E3 ubiquitin ligase through ubiquitinating lysine residues at position 6, 27 and 48 ¹⁸. In a word, the structural diversity of ubiquitination chains serves as important signal for distinct functions of proteins. The generation of various types of ubiquitination chains arises from the distinct combination of E2 and E3 enzymes used in the ubiquitination processes.

Ubiquitin is an abundant protein in eukaryotic cells constituting ∼0.1–5% of total proteins, therefore it is assumed that it is redundantly expressed ¹⁹. However, due to its pervasive use and large number of substrates to be ubiquitinated in a cell, ubiquitin does not seem to be produced in excess, rather the free pool of ubiquitin is maintained at an adequate level depending on the cell conditions.

In yeast as well as in most higher eukaryotes, ubiquitin is initially expressed in the form of different precursors: polyubiquitin, a linear fusion protein consisting of four or more ubiquitin copies in a head-to-tail configuration, and fusion proteins between ubiquitin and usually ubiquitinL40 and ubiquitinS27, that are large and small essential ribosomal polypeptides, L40 and S27, respectively ²⁰. These ubiquitin precursors are cleaved by deubiquitinases to release identical functional monomeric ubiquitin units. Mammals have four ubiquitin genes, two of which encode polyubiquitin and the other two encode fusions with ribosomal proteins ²⁰. The two polyubiquitin-encoding genes, Ubb and Ubc, express usually four and nine tandem repeats of ubiquitin, respectively. Thus, the polyubiquitin genes seem redundant; however, the importance of the polyubiquitin-encoding genes was highlighted in the knockout mouse of either Ubb or Ubc ²¹. In the case of Ubc, disruption of Ubc in mice is embryonically lethal, possibly due to the lack of fetal liver proliferation at midgestation ¹⁹. Analysis of Ubc−/− mouse embryo fibroblasts showed 40% reduction in ubiquitin level compared with the control. On the other hand, mice lacking Ubb are born normally at the expected Mendelian frequency ²¹. However, they are infertile due to the failure of progression of meiosis in germ cells ²¹. Consistently, significantly low ubiquitin levels were found in the testis and germinal vesicle oocytes in 5-month-old mice whereas other organs were not significantly affected. Furthermore, the ubiquitinb null mice develop adult-onset obesity due to the degeneration of hypothalamic neurons involved in the control of energy balance and feeding. Although the ubiquitin level was not reduced in the whole brain, it was reduced in the hypothalamus by 30%. Therefore, a modest reduction in ubiquitin level seems to cause infertility and neurodegeneration in mice.

Other than the mutations in ubiquitin-encoding genes, mutations in several Dubs cause reduction of ubiquitin and various defects. In yeast, deletion of Dub encoding genes, including DOA4 and UBP6, can reduce the amount of monomeric ubiquitin ²². These mutants are sensitive to canavanine, and the defects are compensated by expression of excess ubiquitin. In mice, UCH-L1 is an abundant brain-specific deubiquitinase, constituting ∼1–5% of the total proteins in the brain ²³. The mutation in UCH-Ll is responsible for gracile axonal dystrophy (gad) in mice ²⁴. The mice develop synaptic dysfunction and degeneration of neurons. In the brain of gad mice, monomeric ubiquitin level is reduced by 20–30% compared with control mice, suggesting that a low level of ubiquitin is a possible cause of the disease (16). Since UCH-L1 binds ubiquitin, it is suggested that UCH-L1 stabilizes ubiquitin or prevent ubiquitin from degradation ²³. Similarly, ataxia (axJ) mutation, a spontaneous recessive mutation, is caused by reduced expression of Usp14, a homolog of yeast Ubp6 ²⁵. The axJ mice develop neurological dysfunctions including progressive motor system abnormalities, ataxia, loss of movement and premature death. In the axJ mice, monomeric ubiquitin level is reduced by 30–40%.

Curiously, not only a small amount of ubiquitin but also ubiquitin surplus is not beneficial to cells. In yeast, overexpression of ubiquitin renders cells sensitive to certain kinds of stresses such as treatment with cadmium, arsenite and paromycin ²⁶. In addition, overexpression of ubiquitin worsens cell growth when it is introduced in mutants of ubiquitin-proteasome system (UPS)-related genes, such as cdc48 temperature-sensitive mutant ²⁷. Such mutants exhibit accumulation of ubiquitinated proteins, which could consequently lead to further accumulation of cytotoxic ubiquitinated proteins ²⁷.

Ubiquitin proteasome pathway

In the first step of the ubiquitin conjugation cascade, the carboxyl group of Gly-76 of ubiquitin is activated by ubiquitin-activating enzyme (E1). This step involves the hydrolysis of ATP to PPi to generate an ubiquitinyl adenylate intermediate bound to an E1 enzyme. Subsequently, an active site Cys residue of E1 covalently links to ubiquitin via a high-energy thioester linkage, with the concomitant release of AMP. Following activation, activated ubiquitin is then transferred by transacylation reaction to a thiol group of an active site Cys residue of E2 (ubiquitin carrier protein or ubiquitin-conjugating enzyme). Finally, E2 shuttles ubiquitin either directly to a protein substrate by itself or in cooperation with ubiquitin-protein ligase (E3), to form an amide isopeptide bond between the carboxyl group of Gly-76 of ubiquitin and an ε-amino group of the protein substrate’s internal Lys residue. The last step occurs by first transferring ubiquitin from E2 to E3, which accepts ubiquitin in a similar thiol linkage and then to the protein substrate. In some cases, however, covalent linkage between E3 and ubiquitin is not observed and it appears that ubiquitin is directly transferred from E2 to the protein substrate in a ternary E2-E3-substrate complex.

Once the protein substrate is mono-ubiquitinated, a polyubiquitin chain is formed through the same ubiquitination conjugation cascade, as described above, in which the carboxyl group of the carboxy terminal Gly-76 of ubiquitin is covalently linked to an internal Lys residue of ubiquitin that is already conjugated to the protein substrate.

It should be emphasized that the specificity of ubiquitination is largely determined by a series of E3 enzymes and E3 multiprotein complexes, each of which is specific to one or a few corresponding protein substrate(s) and of E2 enzymes, each of which is dedicated to their cognate E3 enzyme(s). As a result, different combinations of E2 and E3 enzymes allow selective tagging and degradation of specific intracellular proteins. In contrast, there is a single family of E1 that is highly conserved.

Figure 1. Ubiquitin proteasome pathway

Abbreviations: Ub = ubiquitin; E1 = ubiquitin-activating enzyme; E2 = ubiquitin-conjugating enzyme; E3 = ubiquitin ligase enzyme; Lys = lysine; Cys = cystine

Enzymes of the ubiquitination cascade system

Ubiquitin-activating enzyme (E1)

Ubiquitin-activating enzymes (E1) initiate the activation and conjugation of ubiquitin and ubiquitin-like modifiers (UBLs) in a cascade of events that modifies substrate proteins resulting in proteasomal or lysosomal degradation, changes in intracellular localization, activity or other signalling events. To date eight ubiquitin-activating enzymes (E1) enzymes have been identified, two characterized from the human genome comprise three domains; an adenylation domain which binds ATP and ubiquitin, a catalytic cysteine domain (CCD) which binds activated ubiquitin, and a C-terminal ubiquitin-fold domain (Ufd) which recruits specific E2 conjugating enzymes. E1 enzymes utilise ATP to activate the terminal glycine residue of ubiquitin/UBL generating a covalent thioester linkage between ‘activated’ ubiquitin/UBL and the E1 enzyme itself. Ubiquitin/UBL is then transferred to the sulphydryl group of the active-site cysteine on an E2 conjugating enzyme in a transthiolation reaction.

Ubiquitin-conjugating enzyme or ubiquitin-carrier protein (E2)

Ubiquitin conjugating enzyme (E2) form a ubiquitin-thioester intermediate in the ubiquitylation pathway. There are predicted to be 38 ubiquitin-conjugating enzyme (E2) conjugating enzymes (of which Ubiquigent offer 32) and four classes (Class I-IV) of the enzyme exist, all of which contain a catalytic (Ubc) domain with an active site cysteine. After transfer of ubiquitin/UBL (ubiquitin-like modifier) from the ubiquitin-activating enzyme (E1) activating enzyme to the ubiquitin-conjugating enzyme (E2) via a transthiolation reaction, the loaded ubiquitin-conjugating enzyme (E2) interacts with E3 ligases to transfer the activated ubiquitin/UBL to the substrate lysine acceptor residue; either directly from the E2 or via a lysine residue on the E3 ligase itself.

All ubiquitin-conjugating enzymes (E2s) contain a conserved ~130 amino acid-long catalytic core sequence i.e., UBC domain. An active-site Cys residue within the UBC domain is essential since it is required to form an ubiquitin-thioester intermediate. As a result, mutation of an active-site Cys residue abolishes UBC activity. Ubiquitin-conjugating enzymes (E2s) are subdivided on the basis of their distinct primary sequences, presumably reflecting their different specificities for their cognate E3s. For example, class I E2s, such as UBC4, UBC5, UBC7, and UBC9–13 consist exclusively of UBC domain and may require their cognate ubiquitin ligase enzymes (E3s) for recognition of substrates, since they cannot alone transfer ubiquitin to substrates. Class II (UBC1, UBC2/RAD6, UBC3/CDC34, UBC6, and UBC8), class III (UBC6), and class IV ubiquitin-conjugating enzymes (E2s) contain unique carboxy or amino terminal extensions or both that may mediate substrate specificity as well as intracellular localization.

Among the 13 known yeast ubiquitin-conjugating enzymes (E2s), only UBC3 and UBC9 are encoded by essential genes and both are involved in regulating cell cycle progression. UBC3/CDC34, for example, is involved in the degradation of SIC1 CDK inhibitor, which is necessary for the G1 to S phase transition, in conjunction with CDC53, SKP1, and CDC4. In addition, UBC2/RAD6 is involved in DNA repair in cooperation with its cognate E3α15 to degrade so-called N-end rule protein substrates. It is generally believed that E2s together with their cognate E3s recognize specific protein substrates. Such examples and specific E3s will be described below in more detail.

Ubiquitin ligase enzymes (E3s)

E3 ligases or ligase complexes recognize specific motifs in their substrate(s) and catalyze the transfer of ubiquitin directly or indirectly from a thioester intermediate from their cognate ubiquitin-conjugating enzyme (E2) to form an amide isopeptide bond between protein substrate and ubiquitin. In general, there are a few possible mechanisms for recognition of protein substrates by the different ubiquitin ligase enzymes (E3s). For example, some substrates carry degradation signals that are constitutively active, whereas other substrates require a covalent modification of the sequence motif, such as phosphorylation. Conversely, ubiquitin ligase enzymes (E3s) themselves can be altered or bound with an ancillary protein to recognize a specific subset of substrates.

E3 ligases confer specificity to the ubiquitylation pathway by recognising target substrates and mediating transfer of ubiquitin or ubiquitin like modifiers (UBLs) from an E2 conjugating enzyme to a substrate. There are estimated to be >700 E3 ligases forming several classes of enzyme; N-end rule ubiquitin E3 ligases, Homology to E6AP C-Terminus (HECT) domain E3s, Really Interesting New Gene (RING) domain E3s, U-Box containing E3s, Inhibitor of Apoptosis (IAP) E3s, and Cullin-RING E3 ligases (CRLs). The classes of E3 ligases are continually expanding. HECT E3 ligases form a thioester intermediate with the C-terminus of activated ubiquitin via a catalytic cysteine residue on the E3. In this case, ubiquitin is transferred from an E2 via the E3 to a substrate protein lysine side chain. RING and U-Box E3 ligases do not possess a catalytic cysteine residue and bring the E2-ubiquitin complex and substrate into close proximity to mediate the transfer of the ubiquitin directly from the E2 to the substrate.

Ubiquitin function

Ubiquitin through its chemical conjugation onto proteins (ubiquitination) plays major roles in regulating almost all cellular processes from tagging them for proteasomal degradation (protein degradation), protein trafficking, cell-cycle regulation, DNA repair, apoptosis and signal transduction ² and protein-protein interaction ³. Among the various functions of ubiquitin, the most characterized function is serving as a tag for selective proteolysis by the 26S proteasome. Multiple ubiquitins are covalently added to a substrate successively by ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes, producing a substrate conjugated with polyubiquitin. The ubiquitinated substrates are recognized and degraded by the 26S proteasome after the polyubiquitin chain is processed off and recovered by deubiquitinases.

The ubiquitination system functions in a wide variety of cellular processes, including:

• Antigen processing
• Apoptosis
• Biogenesis of organelles
• Cell cycle and division
• DNA transcription and repair
• Differentiation and development
• Immune response and inflammation
• Neural and muscular degeneration
• Morphogenesis of neural networks
• Modulation of cell surface receptors, ion channels and the secretory pathway
• Response to stress and extracellular modulators
• Ribosome biogenesis
• Viral infection

In addition, ubiquitination is also critical in the vacuolar sorting process of both endocytic and biosynthetic membrane proteins ². At the plasma membrane, ubiquitin serves as a signal for endocytosis, and at the endosome, ubiquitin serves as a signal to sort cargo proteins into the multivesicular body, which is a critical step to their transport to lysosomes. ubiquitin is removed from the cargo by deubiquitinases before its entry into the multivesicular body.

Human diseases due to defects of the ubiquitin proteasome pathway

Reflecting the fact that the regulation of diverse short-lived proteins and regulatory proteins is mediated by the ubiquitin-proteasome pathway, it is not surprising that defects of various components of this complex biochemical machinery result in a range of human diseases. As a result, these components would provide an attractive novel target for therapeutic intervention. A few examples of inherited and acquired human diseases due to the defects of the ubiquitin-proteasome pathway are described below, emphasizing the molecular mechanism of pathogenesis.

Inherited Disorders

Angelman’s syndrome

Angelman’s syndrome is a complex neurological disorder ²⁸ due to various genetic mechanisms that map to human chromosome 15q11–q13 ²⁹. Clinical characteristics of this disorder include developmental delay, speech impairment, movement or balance disorder, and behavioral uniqueness ³⁰. Further genetic analysis revealed that truncated mutants of the UBE3A gene on chromosome 15q were identified in some patients with Angelman’s syndrome ³¹. UBE3A gene encodes a protein called E6-AP protein ligase (also known as ubiquitin-protein ligase or E3). Although the true substrate(s) of this E6-AP in Angelman’s syndrome patients has not yet been identified, the ubiquitin-proteasome pathway plays an important role during brain development and perturbation of this pathway causes a neurolopathological disorder.

Liddle syndrome

Liddle et al. ³² recognized this rare hereditary disorder in a family who were unable to maintain the proper balance of salt and water in the body, resulting in abnormally high blood pressure. The molecular mechanism of Liddle syndrome was later understood by Yale researchers ³³ who observed mutations in the carboxy terminus of β and γ subunit of the epithelial sodium channel in the kidney. Among many different mutations, deletion or mutation of the proline-rich (PPxY) region leads to constitutive activation of the kidney sodium channel, resulting in retention of excessive amounts of salt and water. Using a yeast two-hybrid screen and in vitro binding studies ³⁴, WW domain of an ubiquitin ligase, Nedd4, is found to bind specifically to the PPxY sequences of the above-mentioned kidney sodium channel. Taken together, these findings suggests a novel mechanism regulating sodium reabsorption, in which failure of proper ubiquitination of the sodium channel results in increased retention of the sodium channel at the cell surface and thus excessive reabsorption of sodium and water leading to hypertension. It should be noted, however, that this short-lived sodium channel, after ubiquitination, is targeted and degraded in the lysosome, but not by the 26S proteasome machinery.

Acquired Disorders

Cervical cancer

The most important risk factor for cervical cancer is human papillomavirus (HPV) infection. In cases of cervical cancer caused by the high-risk strains of HPV, levels of the tumor suppressor protein p53 were found to be unusually low due to the presence of the E6 proteins encoded by the oncogenic HPV ³⁵. This E6-promoted degradation of p53 involves the ubiquitin-proteasome pathway and represents a major mechanism, by which virus transforms the host cells. The E6 oncoprotein was later found to modify the functions of E6-AP ³⁶ whose absence prevents ubiquitination of p53 by E6-AP ³⁷. Specifically, E6 appears to function as an adapter protein to maintain a ternary complex of E6-AP ubiquitin-protein ligase (E3), its cognate UbcH8 ubiquitin-conjugating enzyme (E2), and a target substrate to ensure specific ubiquitination of substrate molecules ³⁸. Interestingly, E6 also promotes the ubiquitination and degradation of E6-AP ³⁹.

Colorectal cancer

Colorectal cancer, which arises in the epithelium of the lumen of the colon and rectum, is the second most common cause of cancer death in the United States. Generally speaking, the transformation of the normal epithelial cells into cancer cells occurs through a multistage process as a result of many different defects. One of most well-characterized genetic defects is found in the APC (adenomatous polyposis coli) gene located on chromosome 5q21. Mutations of the APC gene have been documented in familial adenomatous polyposis and in more than 80% of the sporadic colorectal cancer patients ⁴⁰.

Among its many functions, APC is known to control the cellular level of β-catenin by forming a complex with Axin, which recruits and facilitates ubiquitination of β-catenin for subsequent degradation by the 26S proteasome ⁴¹. This process appears to be dependent on the phosphorylation of both APC and β-catenin by glycogen synthase kinase-3 (GSK-3) ⁴². In cancer cells expressing mutant APC, β-catenin accumulates ⁴³ and interacts with T-cell factor as well as activates transcription of the proto-oncogenes, such as cyclin D1 and c-myc ⁴⁴. Mutations of Ser to amino acid residues that cannot be phosphorylated stabilize β-catenin ⁴⁵. It appears that ubiquitination of β-catenin is catalyzed by the SCF complex and its cognate E2. A receptor component of the SCF E3 ligase complex, i.e., F box- and WD domain-containing β-TrCP binds to the phosphorylated form of β-catenin, and over-production of a defective β-TrCP blocks β-catenin degradation ⁴⁶.

Alzheimer’s disease

Alzheimer’s disease is a chronic brain disorder due to a progressive degeneration of brain tissue, leading to loss of mental functions, such as memory and learning. Alzheimer’s disease is marked by abnormal aggregates (senile plaques) and irregular knots (neurofibrillary tangles) of brain matter. By immunohistochemical analysis of protein contents in intracellular inclusion bodies, ubiquitin is found in both neurofibrillary tangles and senile plaques. Although it is not yet clear whether accumulation of ubiquitin conjugates is primary or secondary, a growing number of evidence indicate the involvement of the ubiquitin-proteasome pathway in Alzheimer’s disease. For example, findings of a simultaneous frameshift mutation of both β amyloid precursor protein (βAPP) and ubiquitin have implicated the ubiquitin-dependent pathway in the pathogenesis of Alzheimer’s disease ⁴⁷. In addition, treatment of cells expressing presenilins 1 and 2 (PS1 and PS2) with proteasome inhibitors was shown to increase the PS1/PS2-mediated production of both APPα and Aβ40, which are βAPP maturation products ⁴⁸. Senile plaques are thought to derive from the abnormal level of Aβ40 production ⁴⁹. PS1 and PS2 are also found to be the proteasome substrates ⁵⁰. Taken altogether, the ubiquitin-proteasome pathway appears to control the level of PS1 and PS2, thereby regulating the βAPP maturation pathway.

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