Deubiquitinating enzyme (DUB) is a large class of proteases that cleave ubiquitin from proteins and other molecules. Ubiquitin attaches to proteins to regulate protein degradation through the proteasome and lysosomes; coordinates cellular localization of proteins; activates and inactivates proteins; and regulates protein-protein interactions. DUB can reverse these effects by cleaving peptide or isopeptide bonds between ubiquitin and its substrate proteins. In humans, there are nearly 100 DUB genes, which can be divided into two main categories: cysteine proteases and metalloproteinases. Cysteine proteases include ubiquitin-specific protease (USP), ubiquitin C-terminal hydrolase (UCH).
Figure 1. Structure of Deubiquitinating enzyme.
Deubiquitinating enzyme (DUB) is a protease that cleaves ubiquitin or ubiquitin-like proteins from a proprotein or target protein. They play multiple roles in the ubiquitin pathway. First, DUBs may activate ubiquitinogen by co-translation. Ubiquitin is always expressed as a mature ubiquitin monomer that is fused to a ribosomal protein or as a linear polyubiquitin protein that must be processed to produce multiple copies of monoubiquitin. The polyubiquitin gene product also contains an additional residue at the C-terminus that must be removed to activate ubiquitin. Second, DUBs recovered ubiquitin, which may have been accidentally captured by the reaction between small cell nucleophiles and the thiol ester intermediates involved in protein ubiquitination. Third, reversed DUB ubiquitination or modification of ubiquitin-like target proteins. In this role, DUB antagonizes the ubiquitination of proteins and plays a similar role as phosphatase in the kinase/phosphatase regulatory pathway. Finally, the deubiquitinating enzyme is also responsible for the regeneration of the monoubiquitin protein from the un-anchored polyubiquitin, ie the free polyubiquitin is synthesized de novo by conjugation or mechanically from the target protein through other deubiquitinating enzymes.
Most DUB activities are mysterious. That is, energy associated with the substrate or scaffold protein is required to achieve a catalytically competent conformation. Therefore, like most other proteases, their activity should be carefully controlled to prevent accidental cleavage of inappropriate substrates. Other DUBs are covalently modified by phosphorylation, ubiquitination or sulfonation, all of which may affect activity, localization or half-life. DUB is modular, encompasses not only catalytic domains, but also other ubiquitin binding domains and various protein-protein interaction domains. These modules facilitate the binding and recognition of different linkages  and guide the assembly of multiprotein complexes that localize DUBs and assist in substrate selection. DUB requires these localization and substrate specific determinants to perform physiological functions. The binding of DUB to the substrate adaptor, the scaffold and the inhibitor is a regulatory specific regulatory interaction. There has also been a recurring theme in which DUB associates with a complex containing E3 ligase to negatively regulate ubiquitin binding. One of the most unique features of DUB is the removal of monoubiquitin and polyubiquitin chains from proteins. These modifications are the addition of a single ubiquitin protein or ubiquitin chain to the lysine of the substrate protein. These ubiquitin modifications are added to the protein by a ubiquitination mechanism. The end result is that ubiquitin binds to lysine residues via isopeptide bonds. Proteins are affected by these modifications in a number of ways: they regulate protein degradation by the proteasome and lysosomes; coordinate the cellular localization of proteins; activate and inactivate proteins and regulate protein-protein interactions.
Figure 2. Schematic representation DUBs function.
1. Catalytic domain
The catalytic domain of DUB is the domain in which DUB is divided into specific groups. USP, OTU, MJD, UCH and MPN+/JAMM. The first four groups are cysteine proteases and the latter one is zinc metalloproteinase. The cysteine protease DUB is papain-like and therefore has a similar mechanism of action. They use catalytic dimers or triplets (two or three amino acids) to catalyze the hydrolysis of the amide bond between ubiquitin and the substrate. The active site residues that contribute to the catalytic activity of the cysteine protease DUB are cysteine (diad/triad), histidine (diad/triad) and aspartic acid or asparagine (triad only). Histidine is polarized by aspartic acid or asparagine in the catalytic triad and otherwise polarized in the doublet. This polarized residue reduces the pKa of the cysteine, allowing it to undergo a nucleophilic attack on the isopeptide bond between the C-terminus of the ubiquitin and the lysine substrate. Metalloproteinases coordinate zinc ions with histidine, aspartic acid and serine residues, thereby activating water molecules and attacking isopeptide bonds.
Figure 3. Catalytic domain of USP7.
2. UBL domains
The ubiquitin-like (UBL) domain has a similar structure (folding) to ubiquitin, except that they lack terminal glycine residues. The function of the UBL domain differs between USPs, but typically they modulate USP catalytic activity. They can coordinate the localization of the proteasome (USP14); negatively regulate USP by competing with the USP catalytic site (USP4) and induce conformational changes to increase catalytic activity (USP7), like other UBL domains, the structure of the USP UBL domain Showing beta-grip folding.
3. DUSP domains
A single or multiple tandem DUSP domains of approximately 120 residues were found in six USPs. The function of the DUSP domain is currently unknown, but may play a role in protein interactions, particularly in DUBs substrate recognition. It can be predicted that this is due to the presence of hydrophobic cracks in the DUSP domain of USP15 and, if these domains are absent, will not interact with certain proteins of the DUSP-containing USP. The DUSP domain shows a novel tripod-like fold comprising three helices and an anti-parallel β-sheet consisting of three chains. This fold is similar to the legs (spiral) and seat (β-fold) of the tripod. In most DUSP domains of USP, there is a conserved amino acid sequence called the PGPI motif. This is a sequence of four amino acids; valine, glycine, valine and isoleucine, arranged in close proximity to the three helical bundles and highly ordered.
Figure 4. Solution structure of the DUSP domain of HUSP15.
Deubiquitination reverse ubiquitination plays an important role in the regulation of various cellular processes, including: 1) maintaining the steady state level of monoubiquitin; 2) regulating the degradation of the substrate of the proteasome; 3) passing the group protein deubiquitination and remodeling of chromatin's subsequent effects on other histone modifications; 4) cell cycle regulation, 5) DNA damage repair process 6) activation of kinases and other enzymes, and 7) endocytosis. DUB is highly specific and is affected by a variety of regulatory patterns, including transcriptional control, substrate or scaffold-induced activation, post-translational modification, and rapid degradation. DUB is usually part of a large multiprotein complex that regulates its localization, substrate availability and activity during cellular processes. As a result, DUB has become an important pharmacological target in the search for the development of more specific agents for the treatment of various diseases. It is becoming more and more obvious that DUB is involved in the regulation of almost every cellular process, and with the development of knowledge systems, DUB may prove to be an important target for therapeutic drugs.
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