Novel Technique to Study Ubiquitination

Once human proteins are synthesized, they undergo some chemical changes. These adjustments control their stability, functionality, and structure. The Bhogaraju Group at EMBL Grenoble has devised a novel approach to investigating ubiquitination, a crucial process of protein modification.

Numerous biological processes depend on ubiquitination, and its deregulation is linked to several human illnesses, such as cancer and dementia.

During ubiquitination, a small protein known as ubiquitin is attached to other proteins by a class of enzymes known as E3 ubiquitin ligases. Thus, the targeted protein's destiny is partly determined by this labeling. It is believed that every human protein experiences ubiquitination at least once during its lifespan. Ubiquitination is a relatively common process in humans.

Approximately 3 % of the human genome, or more than 600 E3 ligase genes, are involved in ubiquitination's diverse range of biological functions. Understanding the landscape of human E3 target proteins will help target them for treatments and eventually understand their function.

The incredibly fleeting nature of their interaction is one reason why many E3 ligases and their targets are still not well characterized. Their high resource requirements further limit the utility and scalability of current methods for mapping such interactions.

The Bhogaraju Group, which studies ubiquitination pathways in several physiological circumstances, devised the straightforward, affordable Ub-POD technique to swiftly and simply identify the targets of a particular E3 ligase enzyme directly in human cells to address this issue.

Urbi Mukhopadhyay, an EMBO Postdoctoral Researcher in the Bhogaraju group, led the work published in the journal Science Advances. He discovered a mechanism to efficiently identify the ubiquitinated targets of a particular E3 ligase with biotin inside of cells.

This makes it possible to identify the targets using quantitative mass spectrometry in the future. A little organic substance called biotin can be utilized to biochemically bind to specific proteins and separate them from a mixture of proteins.

The technique is so straightforward and uses common chemicals that it may be applied in any lab with rudimentary molecular biology tools anywhere in the world.

Using this technique and as a proof of concept, the researchers discovered additional targets of E3 ligases, RAD18 and CHIP, which are implicated in cancer and neurological illnesses.

Christian Behrend's group at Ludwig-Maximilians University (LMU), Munich, and the Bhogaraju Group's collaborators have also effectively used this technology to find known and novel substrates for TRAF6, a vital immunological signaling regulator and another E3 ligase.

The group intends to use this technique for all known human E3 enzymes.

We believe this will help fill the disparity in the therapeutic space between the kinase family of proteins and the ubiquitin ligase family. Despite hosting a similar number of enzymes, there are ~80 FDA-approved therapeutic agents that target kinases, while only a handful of drugs target the ubiquitin system. The method we developed would contribute to expanding the scope of E3 ligases or their substrates as drug targets.”

Sagar Bhogaraju, Group Leader, EMBL Grenoble