Ubiquitination is a reversible, post-translational modification in which the 76 amino acid polyubiquitin peptide is added to proteins by the sequential action of three enzymes: E1 ubiquitin-activiating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubuiquitin ligase. Protein ubiquitination is a highly dynamic process and the removal of ubiquitin from proteins is now considered to be equally important for protein regulation.
The large group of enzymes that are responsible for the removal of ubiquitin from proteins are known as deubiquitination enzymes (also known as DUBs, deubiquitylating enzymes or ubiquitin deconjugating enzymes). The human genome encodes nearly 100 deubiquitinating enzymes, making them the largest family of enzymes in the ubiquitin system. DUBs are responsible for ubiquitin precursor processing, ubiquitin recycling, trimming of ubiquitin chains, as well other diverse roles in cell growth and differentiation, devlopment, DNA damage, disease pathways, transcriptional regulation and chromatin remodeling.
Deubiquitination enzymes can be divided into five families: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumour proteases (OTUs), Machado-Joseph disease protein domain proteases (MJDs) and JAB1/MPN/MOV34 metalloproteases (JAMM). The first four families are classified as cysteine proteases and comprise the majority of deubiquitinating enzymes. The last family, JAMM, bind zinc and therefore are classified as metalloproteases. The cysteine hydrolases specifcally cleave ubiquitin substrates with the general structure UB1-72-Leu73-Arg74-Gly75-Gly76-X, where X can be any small thiol, amine, ubiquitin molecule or even another protein.
USPs are the largest family of deubiquitination enzymes and they are also the group with the largest size variation (50-300 kDa). These high molecular weight DUBs can process ubiquitin precursors, remove ubiquitin from protein conjugates and disassemble long ubiquitin chains. In contrast to the USPs, the UCHs are relatively small enzymes (<30 kDa) that catalyze the removal of peptides and small molecules from the C-terminus of ubiquitin. The table below provides a sample of deubiquitination enzymes and their associated biological function.
|USP3, USP7, USP16, USP21, USP22, MYSM1, BRCC36
|USP1, USP3, USP28
|USP7, USP16, USP19, USP28, CYLD
Figure 1: Kinetic analysis of DUB activity in HeLa nuclear extract.
HeLa nuclear extracts were assayed at 0.313, 0.625 and 1.25 µg per reaction in the presence or absence of 1 µM inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate (100 nM) was added to each well and the fluorescent intensity was measured every two minutes. Data shown are the results from duplicates.
Figure 2: Endpoint analysis of DUB activity in HeLa nuclear extract.
The DUB-Detector Assay was used to assay activity of HeLa nuclear extracts at 0.313, 0.625 and 1.25 µg per reaction in the presence or absence of 1 µM inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate (100 nM) was added to each well and the reaction proceeded for 60 minutes before the fluorescent intensity was measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fold change in fluorescence was plotted. Data shown are the results from duplicates.
Figure 3: Recombinant USP16 deubiquitination activity.
The DUB-Detector Assay was used to assay activity of recombinant USP16 at 12.5, 25 and 50 pM per reaction in the presence or absence of 100 nM inhibitor for 20 minutes. Following the incubation, Fluorescent Substrate (100 nM) was added to each well and fluorescent intensity was immediately measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescent intensity was measured every minute with a total reaction time of 60 minutes. Data shown are the results from duplicates.
Why use DUB-Detector™?
- Fluorescent assay can be detected with an excitation wavelength of 485 nm and an emission wavelength of 535 nm
- Complete assay with optimized buffers for enhanced enzymatic acitivity
- Includes both positive control extract and a universal DUB inhibitor
- Fast procedure can be completed in less than 1 hour
- Great for either kinetic or endpoint analysis
- Quickly screen for DUB inhibitors
Despite the differences in their cellular roles and molecular sizes, the deubiquitinating enzymes all appear to hydrolzye their substrates through a common mechanism. Active Motif's DUB-Detector Assay uses a universal ubiquitin substrate to detect enzymatic activity or screen for potential inhibitors. The fluorescent substrate is based on a C-terminal derivative of ubiquitin which is hydrolyzed by the cysteine protease class of enzymes to release a fluorescent signal proportional to the amount of enzyme activity. The fluorescence can be detected using a microplate reader with excitation of 485 nm and emission of 535 nm. For added convenience, the assay also includes HeLa nuclear extract and an inhibitor to all classes of deubiquitinating enzymes as positive and negative controls.