Ubiquitin

Ubiquitin is a small (8.6

The discovery that ubiquitin chains target proteins to the proteasome, which degrades and recycles proteins, was honored with the Nobel Prize in Chemistry in 2004.^[8][11][12]

Identification

Ubiquitin (originally, ubiquitous immunopoietic polypeptide) was first identified in 1975

Ubiquitylation (also known as ubiquitination or ubiquitinylation) is an enzymatic

Ubiquitination requires three types of enzyme: ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, known as E1s, E2s, and E3s, respectively. The process consists of three main steps:

1. Activation: Ubiquitin is activated in a two-step reaction by an E1 ubiquitin-activating enzyme, which is dependent on ATP. The initial step involves production of a ubiquitin-adenylate intermediate. The E1 binds both ATP and ubiquitin and catalyses the acyl-adenylation of the C-terminus of the ubiquitin molecule. The second step transfers ubiquitin to an active site cysteine residue, with release of AMP. This step results in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group.^[15][53] The human genome contains two genes that produce enzymes capable of activating ubiquitin: UBA1 and UBA6.^[54]
2. Conjugation: E2
   eukaryotic organisms have between 16 and 35. They are characterised by their highly conserved structure, known as the ubiquitin-conjugating catalytic (UBC) fold.^[55]

   

3. Ligation: E3
   substrate recognition modules of the system and are capable of interaction with both E2 and substrate. Some E3 enzymes also activate the E2 enzymes. E3 enzymes possess one of two domains: the homologous to the E6-AP carboxyl terminus (HECT) domain and the really interesting new gene (RING) domain (or the closely related U-box domain). HECT domain E3s transiently bind ubiquitin in this process (an obligate thioester intermediate is formed with the active-site cysteine of the E3), whereas RING domain E3s catalyse the direct transfer from the E2 enzyme to the substrate.^[56] The anaphase-promoting complex (APC) and the SCF complex (for Skp1-Cullin-F-box protein complex) are two examples of multi-subunit E3s involved in recognition and ubiquitylation of specific target proteins for degradation by the proteasome.^[57]

In the ubiquitylation cascade, E1 can bind with many E2s, which can bind with hundreds of E3s in a hierarchical way. Having levels within the cascade allows tight regulation of the ubiquitylation machinery.[7] Other ubiquitin-like proteins (UBLs) are also modified via the E1–E2–E3 cascade, although variations in these systems do exist.^[58]

E4 enzymes, or ubiquitin-chain elongation factors, are capable of adding pre-formed polyubiquitin chains to substrate proteins.^[59] For example, multiple monoubiquitylation of the tumor suppressor p53 by Mdm2^[60] can be followed by addition of a polyubiquitin chain using p300 and CBP.^[61][62]

Types

Ubiquitylation affects cellular process by regulating the degradation of proteins (via the proteasome and lysosome), coordinating the cellular localization of proteins, activating and inactivating proteins, and modulating protein–protein interactions.^[4][5][6] These effects are mediated by different types of substrate ubiquitylation, for example the addition of a single ubiquitin molecule (monoubiquitylation) or different types of ubiquitin chains (polyubiquitylation).^[63]

Monoubiquitylation

Monoubiquitylation is the addition of one ubiquitin molecule to one substrate protein residue. Multi-monoubiquitylation is the addition of one ubiquitin molecule to multiple substrate residues. The monoubiquitylation of a protein can have different effects to the polyubiquitylation of the same protein. The addition of a single ubiquitin molecule is thought to be required prior to the formation of polyubiquitin chains.

Polyubiquitylation is the formation of a ubiquitin chain on a single lysine residue on the substrate protein. Following addition of a single ubiquitin moiety to a protein substrate, further ubiquitin molecules can be added to the first, yielding a polyubiquitin chain.^[63] These chains are made by linking the glycine residue of a ubiquitin molecule to a lysine of ubiquitin bound to a substrate. Ubiquitin has seven lysine residues and an N-terminus that serves as points of ubiquitination; they are K6, K11, K27, K29, K33, K48, K63 and M1, respectively.^[8] Lysine 48-linked chains were the first identified and are the best-characterised type of ubiquitin chain. K63 chains have also been well-characterised, whereas the function of other lysine chains, mixed chains, branched chains, M1-linked linear chains, and heterologous chains (mixtures of ubiquitin and other ubiquitin-like proteins) remains more unclear.^{[17][39][63][64][65]}

Lysine 48-linked polyubiquitin chains target proteins for destruction, by a process known as proteolysis. Multi-ubiquitin chains at least four ubiquitin molecules long must be attached to a lysine residue on the condemned protein in order for it to be recognised by the 26S proteasome.^[66] This is a barrel-shape structure comprising a central proteolytic core made of four ring structures, flanked by two cylinders that selectively allow entry of ubiquitylated proteins. Once inside, the proteins are rapidly degraded into small peptides (usually 3–25 amino acid residues in length). Ubiquitin molecules are cleaved off the protein immediately prior to destruction and are recycled for further use.^[67] Although the majority of protein substrates are ubiquitylated, there are examples of non-ubiquitylated proteins targeted to the proteasome.^[68] The polyubiquitin chains are recognised by a subunit of the proteasome: S5a/Rpn10. This is achieved by a ubiquitin-interacting motif (UIM) found in a hydrophobic patch in the C-terminal region of the S5a/Rpn10 unit.^[4]

Lysine 63-linked chains are not associated with proteasomal degradation of the substrate protein. Instead, they allow the coordination of other processes such as endocytic trafficking, inflammation, translation, and DNA repair.^[10] In cells, lysine 63-linked chains are bound by the ESCRT-0 complex, which prevents their binding to the proteasome. This complex contains two proteins, Hrs and STAM1, that contain a UIM, which allows it to bind to lysine 63-linked chains.^[69][70]

Methionine 1-linked (or linear) polyubiquitin chains are another type of non-degradative ubiquitin chains. In this case, ubiquitin is linked in a head-to-tail manner, meaning that the C-terminus of the last ubiquitin molecule binds directly to the N-terminus of the next one. Although initially believed to target proteins for proteasomal degradation,^[71] linear ubiquitin later proved to be indispensable for NF-kB signaling.^[72] Currently, there is only one known E3 ubiquitin ligase generating M1-linked polyubiquitin chains - linear ubiquitin chain assembly complex (LUBAC).^[39][73]

Less is understood about atypical (non-lysine 48-linked) ubiquitin chains but research is starting to suggest roles for these chains.^[64] There is evidence that atypical chains linked by lysine 6, 11, 27, 29 and methionine 1 can induce proteasomal degradation.^[68][74]

Branched ubiquitin chains containing multiple linkage types can be formed.^[75] The function of these chains is unknown.^[8]

Structure

Differently linked chains have specific effects on the protein to which they are attached, caused by differences in the conformations of the protein chains. K29-, K33-,^[76] K63- and M1-linked chains have a fairly linear conformation; they are known as open-conformation chains. K6-, K11-, and K48-linked chains form closed conformations. The ubiquitin molecules in open-conformation chains do not interact with each other, except for the covalent isopeptide bonds linking them together. In contrast, the closed conformation chains have interfaces with interacting residues. Altering the chain conformations exposes and conceals different parts of the ubiquitin protein, and the different linkages are recognized by proteins that are specific for the unique topologies that are intrinsic to the linkage. Proteins can specifically bind to ubiquitin via ubiquitin-binding domains (UBDs). The distances between individual ubiquitin units in chains differ between lysine 63- and 48-linked chains. The UBDs exploit this by having small spacers between ubiquitin-interacting motifs that bind lysine 48-linked chains (compact ubiquitin chains) and larger spacers for lysine 63-linked chains. The machinery involved in recognising polyubiquitin chains can also differentiate between K63-linked chains and M1-linked chains, demonstrated by the fact that the latter can induce proteasomal degradation of the substrate.^[8][10][74]

Function

The ubiquitylation system functions in a wide variety of cellular processes, including:^[77]

• 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
• Maintenance of
  pluripotency^[78]
• 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

Membrane proteins

Multi-monoubiquitylation can mark transmembrane proteins (for example, receptors) for removal from membranes (internalisation) and fulfil several signalling roles within the cell. When cell-surface transmembrane molecules are tagged with ubiquitin, the subcellular localization of the protein is altered, often targeting the protein for destruction in lysosomes. This serves as a negative feedback mechanism, because often the stimulation of receptors by ligands increases their rate of ubiquitylation and internalisation. Like monoubiquitylation, lysine 63-linked polyubiquitin chains also has a role in the trafficking some membrane proteins.^{[10][63][66][79]}

Genomic maintenance

Ubiquitylation of histone H2AX is involved in DNA damage recognition of DNA double-strand breaks. Lysine 63-linked polyubiquitin chains are formed on H2AX histone by the E2/E3 ligase pair, Ubc13-Mms2/RNF168.^[82][83] This K63 chain appears to recruit RAP80, which contains a UIM, and RAP80 then helps localize BRCA1. This pathway will eventually recruit the necessary proteins for homologous recombination repair.^[84]

Transcriptional regulation

Ubiquitin-binding domains (UBDs) are modular protein domains that non-covalently bind to ubiquitin, these motifs control various cellular events. Detailed molecular structures are known for a number of UBDs, binding specificity determines their mechanism of action and regulation, and how it regulates cellular proteins and processes.^[89][90]

Disease associations

Pathogenesis

The ubiquitin pathway has been implicated in the pathogenesis of a wide range of diseases and disorders, including:^[91]

• Neurodegeneration
• Infection and immunity
• Genetic disorders
• Cancer

Neurodegeneration

Ubiquitin is implicated in neurodegenerative diseases associated with proteostasis dysfunction, including

Ubiquitin and ubiquitin-like molecules extensively regulate immune signal transduction pathways at virtually all stages, including steady-state repression, activation during infection, and attenuation upon clearance. Without this regulation, immune activation against pathogens may be defective, resulting in chronic disease or death. Alternatively, the immune system may become hyperactivated and organs and tissues may be subjected to autoimmune damage.

On the other hand, viruses must block or redirect host cell processes including immunity to effectively replicate, yet many viruses relevant to disease have informationally limited genomes. Because of its very large number of roles in the cell, manipulating the ubiquitin system represents an efficient way for such viruses to block, subvert or redirect critical host cell processes to support their own replication.^[95]

The retinoic acid-inducible gene I (RIG-I) protein is a primary immune system sensor for viral and other invasive RNA in human cells.^[96] The RIG-I-like receptor (RLR) immune signaling pathway is one of the most extensively studied in terms of the role of ubiquitin in immune regulation.^[97]

Genetic disorders

• Angelman syndrome is caused by a disruption of UBE3A, which encodes a ubiquitin ligase (E3) enzyme termed E6-AP.
• Von Hippel–Lindau syndrome involves disruption of a ubiquitin E3 ligase termed the VHL tumor suppressor, or VHL
  gene.
• Fanconi anemia: Eight of the thirteen identified genes whose disruption can cause this disease encode proteins that form a large ubiquitin ligase (E3) complex.
• 3-M syndrome is an autosomal-recessive growth retardation disorder associated with mutations of the Cullin7 E3 ubiquitin ligase.^[98]

Diagnostic use

• Neurofibrillary tangles in Alzheimer's disease
• Lewy body in Parkinson's disease
• Pick's disease
• Inclusions in
  motor neuron disease and Huntington's disease
• Mallory bodies in alcoholic liver disease
• Rosenthal fibers in astrocytes

Link to cancer

The BRCA1 gene is another tumor suppressor gene in humans which encodes the BRCA1 protein that is involved in response to DNA damage. The protein contains a RING motif with E3 Ubiquitin Ligase activity. BRCA1 could form dimer with other molecules, such as BARD1 and BAP1, for its ubiquitylation activity. Mutations that affect the ligase function are often found and associated with various cancers.^[110][114]

Cyclin E

As processes in cell cycle progression are the most fundamental processes for cellular growth and differentiation, and are the most common to be altered in human carcinomas, it is expected for cell cycle-regulatory proteins to be under tight regulation. The level of cyclins, as the name suggests, is high only at certain a time point during the cell cycle. This is achieved by continuous control of cyclins or CDKs levels through ubiquitylation and degradation. When cyclin E is partnered with CDK2 and gets phosphorylated, an SCF-associated F-box protein Fbw7 recognizes the complex and thus targets it for degradation. Mutations in Fbw7 have been found in more than 30% of human tumors, characterizing it as a tumor suppressor protein.^[113]

Increased ubiquitination activity

Cervical cancer

Oncogenic types of the

Gene amplification often occur in various tumor cases, including of MDM2, a gene encodes for a RING E3 Ubiquitin ligase responsible for downregulation of p53 activity. MDM2 targets p53 for ubiquitylation and proteasomal degradation thus keeping its level appropriate for normal cell condition. Overexpression of MDM2 causes loss of p53 activity and therefore allowing cells to have a limitless replicative potential.^[109][114]

p27

Another gene that is a target of gene amplification is SKP2. SKP2 is an F-box protein with a role in substrate recognition for ubiquitylation and degradation. SKP2 targets p27^Kip-1, an inhibitor of cyclin-dependent kinases (CDKs). CDKs2/4 partner with the cyclins E/D, respectively, forming a family of cell cycle regulators which control cell cycle progression through the G1 phase. Low level of p27^Kip-1 protein is often found in various cancers and is due to overactivation of ubiquitin-mediated proteolysis through overexpression of SKP2.^[111][114]

Efp

Efp, or estrogen-inducible RING-finger protein, is an E3 ubiquitin ligase whose overexpression has been shown to be the major cause of estrogen-independent breast cancer.^[105][115] Efp's substrate is 14-3-3 protein which negatively regulates cell cycle.

Evasion of ubiquitination

Colorectal cancer

The gene associated with

As the most aggressive cancer originated in the brain, mutations found in patients with glioblastoma are related to the deletion of a part of the extracellular domain of the epidermal growth factor receptor (EGFR). This deletion causes CBL E3 ligase unable to bind to the receptor for its recycling and degradation via a ubiquitin-lysosomal pathway. Thus, EGFR is constitutively active in the cell membrane and activates its downstream effectors that are involved in cell proliferation and migration.^[112]

Phosphorylation-dependent ubiquitylation

The interplay between ubiquitylation and phosphorylation has been an ongoing research interest since phosphorylation often serves as a marker where ubiquitylation leads to degradation.^[99] Moreover, ubiquitylation can also act to turn on/off the kinase activity of a protein.^[116] The critical role of phosphorylation is largely underscored in the activation and removal of autoinhibition in the Cbl protein.^[117] Cbl is an E3 ubiquitin ligase with a RING finger domain that interacts with its tyrosine kinase binding (TKB) domain, preventing interaction of the RING domain with an E2 ubiquitin-conjugating enzyme. This intramolecular interaction is an autoinhibition regulation that prevents its role as a negative regulator of various growth factors and tyrosine kinase signaling and T-cell activation.^[117] Phosphorylation of Y363 relieves the autoinhibition and enhances binding to E2.^[117] Mutations that render the Cbl protein dysfunctional due to the loss of its ligase/tumor suppressor function and maintenance of its positive signaling/oncogenic function have been shown to cause the development of cancer.^[118][119]

As a drug target

Screening for ubiquitin ligase substrates

Deregulation of E3-substrate interactions is a key cause of many human disorders, therefore identifying E3 ligase substrates is crucial. In 2008, 'Global Protein Stability (GPS) Profiling' was developed to discover E3 ubiquitin ligase substrates.^[120] This high-throughput system made use of reporter proteins fused with thousands of potential substrates independently. By inhibition of the ligase activity (through the making of Cul1 dominant negative thus renders ubiquitination not to occur), increased reporter activity shows that the identified substrates are being accumulated. This approach added a large number of new substrates to the list of E3 ligase substrates.

Possible therapeutic applications

Blocking of specific substrate recognition by the E3 ligases, e.g. bortezomib.^[115]

Challenge

Finding a specific molecule that selectively inhibits the activity of a certain E3 ligase and/or the protein–protein interactions implicated in the disease remains as one of the important and expanding research area. Moreover, as ubiquitination is a multi-step process with various players and intermediate forms, consideration of the much complex interactions between components needs to be taken heavily into account while designing the small molecule inhibitors.^[105]

Similar proteins

Ubiquitin is the most-understood post-translation modifier, however, several family of

UBLs are structurally similar to ubiquitin and are processed, activated, conjugated, and released from conjugates by enzymatic steps that are similar to the corresponding mechanisms for ubiquitin. UBLs are also translated with C-terminal extensions that are processed to expose the invariant C-terminal LRGG. These modifiers have their own specific E1 (activating), E2 (conjugating) and E3 (ligating) enzymes that conjugate the UBLs to intracellular targets. These conjugates can be reversed by UBL-specific isopeptidases that have similar mechanisms to that of the deubiquitinating enzymes.[77]

Within some species, the recognition and destruction of sperm mitochondria through a mechanism involving ubiquitin is responsible for sperm mitochondria's disposal after fertilization occurs.^[124]

Prokaryotic origins

Ubiquitin is believed to have descended from bacterial proteins similar to

Prokaryotic ubiquitin-like protein (Pup) is a functional analog of ubiquitin which has been found in the gram-positive bacterial phylum Actinomycetota. It serves the same function (targeting proteins for degradations), although the enzymology of ubiquitylation and pupylation is different, and the two families share no homology. In contrast to the three-step reaction of ubiquitylation, pupylation requires two steps, therefore only two enzymes are involved in pupylation.

In 2017, homologs of Pup were reported in five phyla of

The finding of the Pup/UBact-proteasome system in both gram-positive and gram-negative bacteria suggests that either the Pup/UBact-proteasome system evolved in bacteria prior to the split into gram positive and negative clades over 3000 million years ago or,[131] that these systems were acquired by different bacterial lineages through horizontal gene transfer(s) from a third, yet unknown, organism. In support of the second possibility, two UBact loci were found in the genome of an uncultured anaerobic methanotrophic Archaeon (ANME-1;locus CBH38808.1 and locus CBH39258.1).

Human proteins containing ubiquitin domain

These include ubiquitin-like proteins.

• Ubiquitin-associated protein domain

Prediction of ubiquitination

Currently available prediction programs are:

• UbiPred is a
  SVM-based prediction server using 31 physicochemical properties for predicting ubiquitylation sites.^[132]
• UbPred is a random forest-based predictor of potential ubiquitination sites in proteins. It was trained on a combined set of 266 non-redundant experimentally verified ubiquitination sites available from our experiments and from two large-scale proteomics studies.^[133]
• CKSAAP_UbSite is SVM-based prediction that employs the composition of k-spaced amino acid pairs surrounding a query site (i.e. any lysine in a query sequence) as input, uses the same dataset as UbPred.^[134]

Podcast

Investigating the ubiquitin proteasome system was the focus of a Dementia Researcher Podcast.^[135] The podcast was published on 16 August 2021, hosted by Professor Selina Wray from University College London.

See also

• Autophagy
• Autophagin
• Endoplasmic-reticulum-associated protein degradation
• JUNQ and IPOD
• Prokaryotic ubiquitin-like protein
• SUMO enzymes

References