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A diagram of ubiquitin. The seven lysine sidechains are shown in orange.
A diagram of ubiquitin. The seven lysine sidechains are shown in orange.
Ubiquitin is a small (8.5 kDa) regulatory protein that has been found in almost all tissues (ubiquitously) of eukaryotic organisms. It was discovered in 1975 by Goldstein and further characterized throughout the 1970s and 80s. There are four genes in the human genome that produce ubiquitin; UBB, UBC, UBA52 and RPS27A.
Ubiquitination is a post translational modification (an addition to a protein after it has been made) where ubiquitin is attached to a substrate protein. The addition of ubiquitin can affect proteins in many ways: it can signal for their degradation via the proteasome, alter their cellular location, affect their activity and promote or prevent protein interactions. Ubiquitination is carried out in three main steps; activation, conjugation and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). The result of this sequential cascade binds ubiquitin to lysine residues on the protein substrate via an isopeptide bond or to the amino group of the protein's N-terminus via a peptide bond.
The protein modifications can either be a single ubiquitin protein or chains of ubiquitin. There are different forms of chains, named by which of the seven lysine amino acids are used to link the chain together. Lysine 48-linked chains, linked by the 48th amino acid (a lysine) have been well studied. They are the forms of chains that signal proteins to the proteasome which destroys and recycles proteins. This discovery won the Nobel Prize for chemistry in 2004.Lysine 63-linked chains, linked by the 63rd amino acid of ubiquitin (a lysine), regulate processes such as endocytic trafficking, inflammation, translation and DNA repair.
Ubiquitin (originally, ubiquitous immunopoietic polypeptide) was first identified in 1975 as an 8.5 kDa protein of unknown function expressed in all eukaryotic cells. The basic functions of ubiquitin and the components of the ubiquitination pathway were elucidated in the early 1980s at the Technion by Aaron Ciechanover, Avram Hershko, and Irwin Rose for which the Nobel Prize in Chemistry was awarded in 2004.
The ubiquitination system was initially characterised as an ATP-dependent proteolytic system present in cellular extracts. A heat-stable polypeptide present in these extracts, ATP-dependent proteolysis factor 1 (APF-1), was found to become covalently attached to the model protein substrate lysozyme in an ATP- and Mg2+-dependent process. Multiple APF-1 molecules were linked to a single substrate molecule by an isopeptide linkage, and conjugates were found to be rapidly degraded with the release of free APF-1. Soon after APF-1-protein conjugation was characterised, APF-1 was identified as ubiquitin. The carboxyl group of the C-terminal glycine residue of ubiquitin (Gly76) was identified as the moiety conjugated to substrate lysine residues.
|Number of residues||76|
|Molecular mass||8564.8448 Da|
|Isoelectric point (pI)||6.79|
|Gene names||RPS27A (UBA80, UBCEP1), UBA52 (UBCEP2), UBB, UBC|
|Sequence in amino acid abbreviations||MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPD |
Ubiquitin is a small protein that exists in all eukaryotic cells. It performs its myriad functions through conjugation to a large range of target proteins. A variety of different modifications can occur. The ubiquitin protein itself consists of 76 amino acids and has a molecular mass of about 8.5 kDa. Key features include its C-terminal tail and the 7 lysine residues. It is highly conserved among eukaryotic species: Human and yeast ubiquitin share 96% sequence identity.
Ubiquitin is encoded in mammals by 4 different genes. UBA52 and RPS27A genes code for a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively. The UBB and UBC genes code for polyubiquitin precursor proteins.
No ubiquitin and ubiquitination machinery are known to exist in prokaryotes. However, ubiquitin is believed to have descended from prokaryotic proteins similar to ThiS or MoaD. These prokaryotic proteins, despite having little sequence identity (ThiS has 14% identity to ubiquitin), share the same protein fold. These proteins also share sulfur chemistry with ubiquitin. MoaD, which is involved in molybdenum cofactor biosynthesis, interacts with MoeB, which acts like an E1 ubiquitin-activating enzyme for MoaD, strengthening the link between these prokaryotic proteins and the ubiquitin system. A similar system exists for ThiS, with its E1-like enzyme ThiF. It is also believed that the Saccharomyces cerevisiae protein Urm-1, a ubiquitin-related modifier, is a "molecular fossil" that connects the evolutionary relation with the prokaryotic ubiquitin-like molecules and ubiquitin.
Ubiquitination (also known as ubiquitylation) is an enzymatic, post-translational modification (PTM) process in which a ubiquitin protein is attached to a substrate protein. This process most commonly binds the last amino acid of ubiquitin (glycine 76) to a lysine residue on the substrate. An isopeptide bond is formed between the carboxylic acid group of the ubiquitin's glycine and the epsilon amino group of the substrate's lysine. Cases are known in which the amine group of a protein's N-terminus is used for ubiquitination, rather than a lysine residue. In a few rare cases nonlysine residues have been identified as ubiquitination targets, such as cysteine, threonine and serine. The end result of this process is the addition of one ubiquitin molecule (monoubiquitination) or a chain of ubiquitin molecules (polyubiquitination) to the substrate protein.
Ubiquitination requires three types of enzymes; ubiquitin-activating enzymes, ubiquitin-conjugating enzymes and ubiquitin ligases, known as E1s, E2s and E3s, respectively. The process consists of three main steps:
In the ubiquitination 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 ubiquitination machinary. Other ubiquitin-like proteins (UBLs) are also modified via the E1–E2–E3 cascade, although variations in these systems do exist.
Ubiquitination affects cellular process by regulating the degradation of proteins (via the proteasome and lysosome); coordinating the cellular localisation of proteins; activating and inactivating proteins; and modulating protein-protein interactions. These effects are mediated by different types of substrate ubiquitination, for example the addition of a single ubiquitin molecule (monoubiquitination) or different types of ubiqutin chains (polyubiquitination).
Monoubiquitination is the addition of one ubiquitin molecule to one substrate protein residue. Multi-monoubiquitination is the addition of one ubiquitin molecule to multiple substrate residues. The monoubiquitination of a protein can have different effects to the polyubiquitination of the same protein. The addition of a single ubiquitin molecule is thought to be required prior to the formation of polyubiquitin chains. Monoubiquitination affects cellular processes such as membrane trafficking, endocytosis and viral budding.
Polyubiquitination 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. 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 may serve as points of ubiquitination, they are K6, K11, K27, K29, K33, K48 and K63. 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, N-terminal linear chains and heterologous chains (mixtures of ubiquitin and other ubiquitin like proteins) remains more unclear.
Lysine 48-linked polyubiquitin chains target proteins for destruction, by a process known as proteolysis. At least four ubiquitin molecules must be attached to a lysine residue on the condemned protein in order for it to be recognised by the 26S proteasome. This is a barrel-shaped structure comprising a central proteolytic core made of four ring structures, flanked by two cylinders that selectively allow entry of ubiquitinated 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. Although the majority of proteasomal substrates are ubiquitinated, there are examples of non-ubiquitinated proteins targeted to the proteasome. 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.
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. 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.
Less is understood about atypical (non-lysine 48-linked) ubiquitin chains but research is starting to suggest roles for these chains. There is evidence to suggest that atypical chains linked by lysine 6, 11, 27, 29 and N-terminal chains can induce proteasomal degradation.
Differently linked chains have specific effects on the protein to which they are attached, caused by differences in the conformations of the protein chains. Lysine 63-linked and N-terminal chains produce fairly linear chains known as open conformation chains. Lysine 6-, 11- and 48-linked chains form closed conformations. The ubiquitin molecules in linear 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. The proteins that bind ubiquitin have 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 the linear lysine 63-linked chains and linear N-terminal chains, demonstrated by the fact that the latter can induce proteasomal degradation of the substrate.
The ubiquitination system functions in a wide variety of cellular processes, including:
Multi-monoubiquitination 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 ubiquitination and internalisation. Like monoubiquitination, lysine 63-linked polyubiquitin chains also has a role in the trafficking some membrane proteins.
Proliferating cell nuclear antigen (PCNA) is a protein involved in DNA synthesis. Under normal physiological conditions PCNA is sumoylated (a similar post-translational modification to ubiquitination). When DNA is damaged by ultra-violet radiation or chemicals, the SUMO molecule that is attached to a lysine residue is replaced by ubiquitin. Monoubiquitinated PCNA recruits polymerases that can carry out DNA synthesis with damaged DNA; but this is very error-prone, possibly resulting in the synthesis of mutated DNA. Lysine 63-linked polyubiquitination of PCNA allows it to perform a less error prone mutation bypass known by the template switching pathway.
Ubiquitination 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. 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.
Histones can be ubiquitinated and this is usually in the form of monoubiquitination (although polyubiquitinated forms do occur). Histone ubiquitination alters chromatin structure and allows the access of enzymes involved in transcription. Ubiquitin on histones also acts a binding site for proteins that either activate or inhibit transcription and also can induce further post-translational modifications of the protein. These effects can all modulate the transcription of genes.
Deubiquitinating enzymes (DUBs) oppose the role of ubiquination by removing ubiquitin from substrate proteins. They are cysteine proteases that cleave the amide bond between the two proteins. They are highly specific, as are the E3 ligases that attach the ubiquitin, with only a few substrates per enzyme. They can cleave both isopeptide (between ubiquitin and lysine) and peptide bonds (between ubiquitin and the N-terminus). In addition to removing ubiquitin from substrate proteins, DUBs have many other roles within the cell. Ubiquitin is expressed either as multiple copies joined in a chain (polyubiquitin) or attached to ribosomal subunits. DUBs cleave these proteins to produce active ubiquitin. They also recycle ubiquitin that has been accidentally bound to small nucleophilic molecules during the ubiquitination process. Monoubiquitin is formed by DUBs that cleave ubiquitin from free polyubiquitin chains that have been previously removed from proteins.
|Domain||Number of Proteins |
|Ubiquitin Binding |
|CUE||S. cerevisiae 7 |
H. sapiens 21
|GATII||S. cerevisiae 2 |
H. sapiens 14
|GLUE||S. cerevisiae ? |
H. sapiens ?
|NZF||S. cerevisiae 1 |
H. sapiens 25
|PAZ||S. cerevisiae 5 |
H. sapiens 16
|UBA||S. cerevisiae 10 |
H. sapiens 98
|UEV||S. cerevisiae 2 |
H. sapiens ?
|UIM||S. cerevisiae 8 |
H. sapiens 71
|VHS||S. cerevisiae 4 |
H. sapiens 28
redirect Ubiquitin Binding Domains
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.
The ubiquitin pathway has been implicated in the pathogenesis of several diseases and genetic disorders:
Immunohistochemistry using antibodies to ubiquitin can identify abnormal accumulations of this protein inside cells, indicating a disease process. These protein accumulations are referred to as inclusion bodies (which is a general term for any microscopically visible collection of abnormal material in a cell). Examples include:
Although ubiquitin is the most well understood post-translation modifier, there is a growing family of ubiquitin-like proteins (UBLs) that modify cellular targets in a pathway that is parallel to, but distinct from, that of ubiquitin. Known UBLs include: small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 ISG15), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), MUB (membrane-anchored UBL), ubiquitin fold-modifier-1 (UFM1) and ubiquitin-like protein-5 (UBL5, which is but known as homologous to ubiquitin-1 [Hub1] in S. pombe). Whilst these proteins share only modest primary sequence identity with ubiquitin, they are closely related three-dimensionally. For example, SUMO shares only 18% sequence identity, but contain the same structural fold. This fold is called "ubiquitin fold" or sometimes called ubiquiton fold. FAT10 and UCRP contain two. This compact globular beta-grasp fold is found in ubiquitin, UBLs, and proteins that comprise a ubiquitin-like domain e.g. the S. cerevisiae spindle pole body duplication protein, Dsk2, and NER protein, Rad23, both contain N-terminal ubiquitin domains.
These related molecules have novel functions and influence diverse biological processes. There is also cross-regulation between the various conjugation pathways since some proteins can become modified by more than one UBL, and sometimes even at the same lysine residue. For instance, SUMO modification often acts antagonistically to that of ubiquitination and serves to stabilize protein substrates. Proteins conjugated to UBLs are typically not targeted for degradation by the proteasome, but rather function in diverse regulatory activities. Attachment of UBLs might alter substrate conformation, affect the affinity for ligands or other interacting molecules, alter substrate localization and influence protein stability.
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.
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.
Recently, a functional analog of ubiquitin has been found in prokaryotes. Prokaryotic ubiquitin-like protein (Pup) serves the same function (targeting proteins for degradations), although the enzymology of ubiquitination and pupylation is different. In contrast to the three-step reaction of ubiquitination, pupylation requires two steps, therefore only two enzymes are involved in pupylation.
ANUBL1; BAG1; BAT3/BAG6; DDI1; DDI2; FAU; HERPUD1; HERPUD2; HOPS; IKBKB; ISG15; LOC391257; MIDN; NEDD8; OASL; PARK2; RAD23A; RAD23B; RPS27A; SACS; 8U SF3A1; SUMO1; SUMO2; SUMO3; SUMO4; TMUB1; TMUB2; UBA52; UBB; UBC; UBD; UBFD1; UBL4; UBL4A; UBL4B; UBL7; UBLCP1; UBQLN1; UBQLN2; UBQLN3; UBQLN4; UBQLNL; UBTD1; UBTD2; UHRF1; UHRF2;
Currently available prediction programs are:
Programs for ubiquitination prediction: