Deubiquitylating enzymes (DUBs) are promising therapeutical targets, especially in oncology (see my previous post dedicated to this topic). Today, I’d like to introduce you to new innovative substrates to screen selectively DUB inhibitors.
Before going into details about these new DUB substrates, which are used in drug discovery for the screening of inhibitors and kinetic studies, first a short overview regarding the biology of the (De)Ubiquitin pathway.
Isopeptidases / Ub Ligases for a reversible Ubiquitination pathway
Ubiquitin (Ub or UBL) is a 8.5 kDa protein that can be found ubiqitiously in eukaryotic organisms/cells. Ubiquitination (or ubiquitylation) is referred to as the process in which a target protein is covalently coupled to ubiquitin. Either one ubiquitin is transferred to the target protein (mono-ubiquitination) or several which are linked in a chain (poly-ubiquitination). Ub contains 7 Lysines (K6, K11, K27, K29, K33, K48, and K63) which can be considered as the linking points of ubiquitin. The first ubiquitin is linked through its C-terminus to a Lysine residue in the target protein to form a so-called isopeptide bond. If poly-ubiquitination occurs, the C-terminus of a second ubiquitin binds to a specific lysine residue of the first ubibiquitin and so forth. The type of linkage between ubiquitins in poly-ubiquinated proteins is clearly linked to the effect on the target protein’s fate. While K48 and K29 linkages label the proteins to be degraded through the proteasomal pathway, other linkages types (K63, K11, K6) or mono-ubiqitination rather regulate cellular processes such as DNA repair, endocytotic trafficking, translation and silencing events.
The conjugation of ubiquitin to target proteins requires an orchestrated addition of ubiquitin to lysine residues in the target protein by E1 (Ubiquitin Activating Enzyme), E2 (Ubiquitin Conjugating Enzyme), and E3 (Ubiquitin Ligase) in an ATP dependent manner. The enzymes form an isopeptide bond between the carboxy-terminus of the ubiquitin and the ε-amino group of the lysine residue of target proteins (Fig. 1).
Ubiquitination is a reversible process in which deconjugation is performed in cells by de-ubiquitinating enzymes (DUBs aka Isopeptidases) as they cleave isopeptide bonds. Isopeptidases are cysteine proteases that can be divided into five families, depending on sequence homology:
- the Ubiquitin-C terminal Hydrolases (UCH),
- the Ubiquitin specific processing Proteases (UBPs),
- the Machado-Joseph Disease domain proteases,
- the Otubain proteases (Otu), and
- JAMM domain proteases.
The roles of isopeptidases include recycling of used Ub and processing pro-ubiquitin by cleavage to the mature form. Removal of Ub moieties can affect cellular physiology in a number of ways, and several isopeptidases have been linked to pathologies such as cancer and cardiovascular disease.
DUBs recognize and cleave the isopeptide bond between ubiquitin moieties or between Ub and the conjugate protein.
The Measurement of the activity of these enzymes for kinetic studies or inhibitor screenings in research and drug discovery programs has been traditionally carried out using classical substrates (such as ubiquitin conjugated to fluorescent leaving groups (AMC or rhodamine) at the C-terminus). While they are suitable substrates for many deubiquitinating enzymes, the cleavage of the amide bond serves as a proxy for the true activity of isopeptidases: cleavage of an isopeptide bond.
IQF-diubiquitins: new DUB substrates for reliable DUB inhibitor screenings and kinetic studies
Recently, internally-quenched fluorescent diubiquitins have been developed. These so called IQF-diubiquitins (LifeSensors) consist of two ubiquitins singly labeled with a fluorophore and a fluorescent quencher. The cleavage and separation of the isopeptide bond between the labeled ubiquitins results in a fluorescent signal.
IQF-diubiquitins substrates enable continuous measurement of the isopeptidase activity of DUBs linked through K11, K48 and K63.
Diubiquitin IQF Substrates principle
IQF substrates are ideal for the continuous fluorescent measurement of true isopeptidase activity. The C-terminus of wild type ubiquitin is conjugated via an isopeptide bond to a specific lysine (either K11, K48, or K63) of a second ubiquitin molecule with the resultant diubiquitin forming an internally quenched fluorescent FRET pair (IQF). Each ubiquitin is labeled with a single molecule of either a fluorescent reporter (i.e. TAMRA) or a highly efficient quenching dye. Different diubiquitin variants are available which differ in the position of the labels (e.g. DiUb K48-1, DiUb K48-2 etc). Cleavage of the IQF diubiquitin by deubiquitinases leads to separation of the fluorophore from quencher and subsequent increase in observed fluorescence (see Fig 2).
IQF substrates available
IQF diubiquitins, available from catalogue, are linked through Lysine K48 or Lysine K63. Regarding Diubiquitins linked through Lysine K11, IQF substrate can be made upon request. Feel free to contact me to learn more about this.
To optimize your assay in terms of the best label position for your specific DUB, K48, K63 as well as K48/63 panels can be obtained, containing all diubiquitin variants available.
Classical DUB substrates, ubiquitins and ubiquitin chains for your in vitro research
Besides the IQF technology, you can also choose to go for classical DUB substrates, and wildtype and mutant ubiquitins, or ubiquitin chains.
Classical DUB substrates
Ubiquitin-AMC is prepared via the conjugation of 7-amino-4-methylcoumarin (AMC) to the C-terminus of mature ubiquitin leading to quenching of the intrinsic fluorescence of AMC. Upon incubation with a protease recognizing ubiquitin, such as USP2 or UCHL3, AMC is released and the increase in fluorescence can be measured using at 460 nm (Ex380 nm).
Ubiquitin-rhodamine 110 is a quenched, fluorescent substrate for deubiquitinases, especially ubiquitin C-terminal hydrolases. Cleavage of the amide bond between the C-terminal glycine of ubiquitin and rhodamine results in an increase in rhodamine fluorescence at 535 nm (Exc. 485 nm).
Attachment of luciferase substrates to ubiquitin results in highly sensitive substrates and assays for all DUBs, and, in particular, permits the assay of DUBs for which no high throughput screening (HTS) amenable assay was previously available. These DUBs can only be assessed through analysis of polyubiquitin cleavage by western blotting, which is laborious, time intensive, and not amenable to HTS. Depending upon the DUB, ubiquitin-aminoluciferin substrate yielded a 10- to 1000-fold increase in signal-to-background ratios (S/B) over the fluorophore-conjugated substrates (Ub-AMC, Ub-rhodamine110) or the C-terminal Ub peptide-luciferin conjugate substrate (Z-RLRGG-LUC). Optimal assay parameters and reagent requirements must be determined by the end user.
Wildtype and mutant ubiquitins
Furthermore there is a broad range of ubiquitin variants: wildtype ubiqutin as well as mutants especially variants of the diverse lysines which serve as coupling points for Ubiqitin-Ubiqutin binding. To get the broadest possible overview, take a look at this complete selection of ubiquitins.
And last but not least, a broad range of unlabeled ubiquitin chains is available for research purposes: di-ubiquitin, tri-ubiquitin, tetra-ubiquitin, and penta-ubiquitin, in which different linkage types are present together with non-cleavable di-ubiquitins to be used as controls.
What about you?
Interested in testing the most physiological DUB substrates, or classical substrates, ubiquitin variants or ubiquitin chains in your assay?
Ask your questions or get in touch through the form below, or subscribe to tebu-bio’s Drug discovery eNewsletter here to stay up to date!