Researchers from CIC bioGUNE develop a strategy capable of identifying the cellular components on which the E3 ubiquitin ligase enzymes act

Researchers from CIC bioGUNE, member of BRTA, have developed a strategy capable of identifying the specific substrates or compounds on which the E3 ligase enzymes of interest act. The Ubiquitin and Development laboratory, led by Dr. Rosa Barrio at CIC bioGUNE, has implemented the BioE3 strategy, which has identified the substrates of five different ligases. These ligases, which are involved in human diseases, including some rare diseases and cancer, are located in different subcellular compartments and with different regulation.

The importance of the BioE3 strategy is related to the fundamental role that E3 ligase enzymes have for the development of new therapies and drugs through Targeted Protein Degradation (TPD).

The work, which has been developed over the last three years, has been published by the scientific journal Nature Communications. The research opens the door for the development of future drugs that have human proteins involved in diseases as therapeutic targets.

“Despite there being more than six hundred E3 ligases in the human proteome, only a handful of them are currently used for TPD drug development. Most of the substrates of E3 ligases are still unknown and there is an urgent need to characterize new E3s and to identify E3s substrates of interest, as well as new substrates acquired after treatment with TPD drugs. However, the identification of direct E3s substrates of interest remains a major challenge and considerable time has been spent searching for robust and reliable methodologies for that purpose. In recent years, new strategies have been developed, although most of them are indirect and do not allow us to distinguish direct substrates from secondary substrates or from other proteins that interact with E3s or interactors”, says Dr. Rosa Barrio.

Furthermore, depending on the function of each E3, these enzymes may also be involved in various diseases (minor diseases, cancer, etc.), so the characterization of their substrates will help us understand their mechanism of action and search for effective treatments.

The ubiquitin-proteasome system is the basis of a fundamental process inside cells, which regulates the degradation of proteins that have reached the end of their active life, either because their function is no longer desired or because they are damaged. Specifically, the natural degradation process begins with the binding of a small protein, called ubiquitin, to the target proteins that need to be degraded, and this binding is carried out by a series of specific enzymes. Alterations in this process usually give rise to multiple diseases, such as cancer or some neurodegenerative diseases.

““E3 ligase enzymes are important because they confer specificity to the system. On the one hand, they bind to these targets, which are their specific substrates that must be degraded. On the other hand, they facilitate the binding of ubiquitin to these substrates, which are thus marked for degradation. Therefore, ubiquitinated proteins are recognized by the proteasome, which is the degradative molecular machinery. Despite being very important enzymes for the functioning of the organism, most of the specific substrates of most E3 ligases are unknown,” explains Dr. Rosa Barrio in relation to the causes that have motivated the research.

Regarding the obstacles that had to be overcome during this research, the CIC bioGUNE researcher highlights that “the main objective of our group, at the beginning of the project, was to develop a robust strategy that would allow the identification of specific substrates and differentiate them from other proteins that could also interact with the E3 enzymes, but that were not modified by them. To do this we had to adapt the BirA and biotinylating technology. This took us more than a year, as we had to perform multiple control and validation experiments to demonstrate that the identified proteins, labelled by biotin, were indeed the substrates of the E3 ligase enzymes. Finally, we managed to develop the BioE3 technology, and demonstrate that it can identify the direct substrates of E3s with the required specificity and sensitivity.”

In the research of the Ubiquitin and Development laboratory of CIC bioGUNE, led by Dr. Rosa Barrio, Dr. James D. Sutherland has participated, as senior researcher and responsible for the conceptual development of the project; Dr. Orhi Barroso Gomila, who carried out his doctoral thesis in the group; and Laura Merino Cacho, who is completing her doctoral thesis, as well as other members of the group.

In addition, the group has collaborated with Dr. Ugo Mayor, Ikerbasque researcher at the Faculty of Science and Technology of the UPV/EHU; with Professor Alfred C.O. Vertegaal, researcher at Leiden University (Netherlands); and with the group of Professor Simona Polo, researcher at IFOM (The AIRC Institute of Molecular Oncology, Milan, Italy).

Reference: Orhi Barroso-Gomila, Laura Merino-Cacho, Veronica Muratore, Coralia Perez, Vincenzo Taibi, Elena Maspero, Mikel Azkargorta, Ibon Iloro, Fredrik Trulsson, Alfred C. O. Vertegaal, Ugo Mayor, Felix Elortza, Simona Polo, Rosa Barrio & James D. Sutherland. Nature Communications. DOI: 10.1038/s41467-023-43326-8

About CIC bioGUNE

The Centre for Cooperative Research in Biosciences (CIC bioGUNE), member of the Basque Research & Technology Alliance (BRTA), located in the Bizkaia Technology Park, is a biomedical research organisation conducting cutting-edge research at the interface between structural, molecular and cell biology, with a particular focus on generating knowledge on the molecular bases of disease, for use in the development of new diagnostic methods and advanced therapies.

About CIC bioGUNE

About BRTA

BRTA is an alliance of 4 collaborative research centres (CIC bioGUNE, CIC nanoGUNE, CIC biomaGUNE y CIC energiGUNE) and 12 technology centres (Azterlan, Azti, Ceit, Cidetec, Gaiker, Ideko, Ikerlan, Lortek, Neiker, Tecnalia, Tekniker and Vicomtech) with the main objective of developing advanced technological solutions for the Basque corporate fabric.

With the support of the Basque Government, the SPRI Group and the Provincial Councils of the three territories, the alliance seeks to promote collaboration between the research centres, strengthen the conditions to generate and transfer knowledge to companies, contributing to their competitiveness and outspreading the Basque scientific-technological capacity abroad.

BRTA has a workforce of 3,500 professionals, executes 22% of the Basque Country's R&D investment, registers an annual turnover of more than 300 million euros and generates 100 European and international patents per year.