Easily customizable E3-ubiquitin ligase assay kits

A ready-to-use E3 ligase activity enzymatic assay, the E3LITE kit, has been recently designed enabling researchers to screen for E3 enzyme inhibitors and potentially new therapeutical drugs. What’s it for and why is it of interest?

Well, the conjugation of ubiquitin (Ub) to a target protein requires the co-ordinated function of a “ubiquitination machinery” involving 3 distinct ligases. Among these enzymes involved in the proteasome pathway, Ubiquitin E3 ligases act as scaffold proteins, providing docking sites for the conjugation of Ub.

E3 ligases are known to regulate any cellular process linked to ubiquitination (ex. cell cycle, DNA repair…). They are frequently described as being overexpressed in human cancers. They are considered to participate actively in the development of cancers (ex. the components of E3 ligases complexes (FBW7, MDM2, RBX1, RBX2, cullin 4…) but also of neurodegenerative disorders. Mutations in the PARK2 gene (encoding for the Parkin RBR E3 ubiquitin protein ligase) have been described in a familial form of autosomal recessive juvenile Parkinson’s disease (AR-JP).

Such discoveries have led to the development of a novel class of anticancer drugs targeting selectively E3 ubiquitin ligases, with the need to develop reliable in vitro assays to evaluate E3 ligase activities.

E3LITE Customizable Ubiquitin Ligase Kit for measuring E3 activity

E3lite assay results

Premixed enzyme cocktail was added to serial 2-fold dilutions of control E3 ligase CARP2 in coated wells in the presence (triangle) or absence (circle) of N-ethylmaleimide (NEM) which blocks the active site of ubiquitination enzymes. Subsequent autoubiquitination of CARP2 was allowed to progress prior to detection by luminescence.

The E3LITE Customizable Ubiquitin Ligase Kit produced by LifeSensors and available through tebu-bio in 96- and 384-well plate format for HTS application) has been developed for the exploration of the mechanistic basis underlining the activity of E3 ligase enzyme of choice. At the core of the assay, microtiter plate strips, pre-coated with a proprietary reagent, are used for the capture of polyubiquitin chains formed in an E3 ligase-dependent reaction.

For the assay, an E1-E2 enzyme cocktail (the enzymatic partners of E3) is added first, in the presence of ubiquitin, to the coated microtiter plate wells. An E3 ligase under consideration is then added to the wells, and the reaction is initiated with ATP. During the reaction, polyubiquitin chains generated by the E1-E2-E3 machinery are recognized and captured in the wells.

Following the reaction and subsequent wash steps, the isolated polyubiquitinated product is incubated with Detection Reagent and streptavidin-HRP allowing for detection by chemiluminescence. Thus, the signal generated by captured polyubiquitinated product in this “sandwich” ELISA-like assay is a quantitative measure of E3 ligase activity. Furthermore, this detection strategy does not require additional non-native tagging or labeling of ubiquitin, which could lead to experimental artifact.

E3LITE Customizable Ubiquitin Ligase Kit for flexible experimental design when measuring E3 activity

Interestingly, the E3LITE Kit itself can be assembled with any one of 20+ E2 conjugation enzymes and with a selection representing members from each Ub enzyme class.
You now attain a unique level of flexibility when designing your Ubiquination-based assays just by selecting the particular enzymatic partners you want for investigating any E2/E3 enzyme pair, as well as any particular polyubiquitination linkage type like:

Go a step further in your Ubiquitination / proteasome pathway studies

To broaden your panel of E2 enzymes to be tested with your E3 ligase of interest you can assemble a panel of 4 different E2 enzymes through the E2 Selection Panel. The recombinant ubiquitin component of this kit is available as wild-type or variants that have been engineered to form linkages only through one of the seven possible surface exposed lysines (e.g. K6, K11, K27, K29, K33, K48, K63). Of course these ubiquitin variants can be purchased separately as well.
Applications:
  • E3 activity monitoring in solution phase, with the E2 of your choice
  • Quantitative Analysis of Ubiquitin E2 Conjugation or E3 Ligase Activity
  • Identification of novel E2 or E3 Activity
  • High Throughput Screening (HTS) of antagonist or agonists of ligase activity
  • Monitor enzyme activity during purification

Interested in measuring E3 ligase activity or screening for E3 inhibitors?

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Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.