Two series of probes that are derived from ubiquitin and UBLs to serve as research tools to identify and detect biological functions of ubiquitination/de-ubiquitination enzymes.
- Can monitor enzymatic activities of multiple deubiquitinating enzymes (DUBs) simultaneously.
- Able to identify novel ubiquitination/de-ubiquitination enzymes as potential targets for drug discovery from crude cell lysate.
- Can be easily equipped with radioactive 125I label or epitope tags for direct visualization, purification or identification purposes.
Ubiquitination is one of the most important post-translational modifications of proteins, and it plays central roles in cell division, differentiation, signal transduction, trafficking, and protein quality control. Aberrations in ubiquitination could result in numerous diseases including cancers and neurodegenerative disorders. Therefore, selective probes targeting at the active sites of ubiquitination enzymes are of critical importance in elucidating the mechanism of ubiquitination and studying pathogenesis of related diseases. They also have the potential to identify novel targets for drug development.
This technology provides two series of activity-based probes derived from ubiquitin and UBLs, both of which can covalently bind to the active-sites of different enzymes from the ubiquitin proteasome system. The first series of probes, UbVS, is based on ubiquitin with C-terminal modified to vinyl sulfone. UbVS has been used to monitor catalytic activities of multiple DUBs at the same time by specifically binding to the targets via a thioether bond. This kind of linkage, unlike other ubiquitin-based probes, is resistant to the buffers used in SDS-PAGE allowing for purification by electrophoresis. Furthermore UbVS can be labeled with radioactive 125I, and it is therefore possible to visualize targeted enzymes directly from crude cell extracts. The other series of probes is comprised of an epitope tag and a UBL linked to various reactive groups. The variety of these reactive groups enables fine-tuned specificity of the probes towards different enzyme subsets in the ubiquitination process, and the epitope tag assures antibody-specific identification as well as purification of the probe-enzyme conjugates. These UBL-based probes successfully facilitated the identification of a novel family of ovarian tumor proteins from mouse cell lysate. Both series of probes have been shown to be powerful research tools for studying and monitoring enzymatic activities of ubiquitination with the potential to discover novel targets for drug development as well.
Affinity and specificity tests of ubiquitin and UBL-derived probes have been conducted on yeast and mouse cell lysate.
Publication: Hemelaar et al. Mol Cell Biol 24:84-95. (2004)