Deconjugating - Inhibitors
Protein modification by ubiquitin (Ub) or ubiquitin-like proteins (UBLs) is a reversible process. These deconjugation reactions are performed by specific cysteine proteases which generate monomeric Ub or UBL proteins from a variety of C-terminal adducts.
These enzymes Ub/UBL process precursor Ub/UBL forms to their mature or active protein forms, they can process linear Ub/UBL fusion proteins, they can disassemble Ub/UBL isopeptide bonds present in poly chains species, and also remove Ub/UBL modifications from various protein substrates.
Deubiquitinating enzymes (DUBs) are one of the largest enzyme families in the Ub system with diverse functions, making them key regulators of ubiquitin-mediated pathways and they often function by direct or indirect association with the proteasome. DUBs regulate and maintain optimal Ub cellular levels. The activity of DUBs has been implicated in several important pathways including cell growth and differentiation, development, oncogenesis, neuronal disease and transcriptional regulation. It is noteworthy too, that several bacteria and viruses encode for DUBs that allow them to exploit UPP pathways to enable viral replication, infection and pathogenesis. Despite their importance, most DUBs are completely uncharacterized, and to date only a few have been purified and crystallized with little specificity ascribed to them. DUB substrate specificity has largely gone uninvestigated, but likely results from recognition of both Ub and target proteins. Specificity determinants likely arise from non-conserved surface regions which make specific contacts necessary for target recognition and orientation of substrate relative to the active site. In contrast to many proteases, DUBs are produced as active enzymes. Structural studies indicate that an active conformation is adopted only when they bind to Ub, thus preventing spurious activities. Alternatively, activity and conformation may also be regulated by interaction or localization with other proteins, co-factors or complexes (eg. E3 ligase- and proteasome-associated DUBs). DUBs catalyze the removal of Ub from native conjugates, Ub C-terminal extension peptides and linear poly-ubiquitin fusion or precursor proteins. DUB activity is also crucial for the pre-processing of substrate proteins destined for degradation by the proteasome. Many DUBs have esterase and amidase activities in vitro, and Ub-esters or Ub-amidocoumarins (Ub-AMC) have been widely used as general substrates for activity assays. These activities could be physiologically relevant for the removal of linkages that might arise in vivo from the nucleophilic attack by small molecules (glutathione, spermidine) on reactive Ub thiolester linkages. In addition, DUB activity can be assessed kinetically in vitro by other fluorescent substrates (Ub-AFC, Ub-R110) and purified Ub chains. Inhibitors that specifically interact with DUB active sites include Ub C-terminal derivatives (Ub-aldehyde, Ub-vinylsulfone) which are useful for the kinetic analysis or identification of DUB activities.
In humans, there is ~100 putative members of the DUB super-family and they are divided into 5 broad categories based on Ub-protease domain structure and mechanism of action. These include the ubiquitin-specific protease (USP), ubiquitin C-terminal hydrolase (UCH), ovarian tumor-related protease or otubain (OTU), Machado-Joseph disease (MJD) and JAB1/MPN/Mov34 metalloprotease (JAMM) enzymes. JAMM enzymes are metalloproteases, while the other four subfamilies are cysteine proteases that hydrolyze their substrates through a three step mechanism compromising substrate binding, acyl-enzyme formation and hydrolysis of this intermediate to liberate Ub and free enzyme. The catalytic domains contain two short and well-conserved Cys and His box motifs forming a catalytic triad with these Cys and His residues together with either an Asp or Asn. DUBS contain ubiquitin-interaction motif (UIM) domains which bind to and recognize Ub/Ub chains helping to recruit poly-ubiquitinated substrates. UCHs have a preference for small substrates and their primary role is Ub recycling from C-terminal fusions such as lysine, glutathione, or peptide remnants. High MW DUBs (such as USPs) primarily remove Ub from larger proteins and disassemble poly-Ub chains, and regulate rates of proteasomal degradation. USPs vary greatly in size (50-300 kDa) usually with N-terminal extensions which may function in substrate recognition, subcellular localization and protein-protein interactions. Several DUBs function freely or in association with the 26S proteasome. DUBs also act on mono-ubiquitinated substrates regulating protein-protein interactions and signaling pathways.
Similar to DUBs, there also exist specific deconjugation enzymes for other UBL proteins. These enzymes also process UBL precursor forms, specifically hydrolyze C-terminal isopeptide bonds between UBL chains, and remove UBL proteins from modified substrates. There are SUMO-specific proteases (SENPs ), NEDD8-specific proteases (NEDP1, USP21, COP9 signalosome), ISG15-specific proteases (UBP43), Apg8-specific proteases or autophagins (Apg4A, Apg4B, Apg4C), and UFM1-specific proteases (UfSP1, UfSP2). There are undoubtedly other as yet identified deconjugating enzymes that might be specific for FAT10, Apg12 and other UBLs. The substrates for these deconjugating enzymes include purified UBL precursor proteins, UBL C-terminal derivatives (AMC versions) and UBL chains (eg. SUMO2 or SUMO3) and specific inhibitors (C-terminal aldehyde or vinylsulfone derivatives).