Assessing the physiological relevance and molecular mechanisms of non-canonical ubiquitylation

Ubiquitin is a small, yet influential, protein that is attached to other proteins in a process termed ubiquitylation. Ubiquitylation is fundamental in maintaining cellular homeostasis.

Ageing or other environmental or genetic insults that disrupt the ubiquitylation process often lead to the development of debilitating human diseases, including cancer, neurodegeneration and immunity disorders. The ubiquitylation machinery is tightly regulated by three enzymes that act sequentially in an enzymatic cascade.

In a first step, E1 enzymes activate ubiquitin to render it chemically susceptible. Activated ubiquitin is then transferred to E2 enzymes (conjugating enzymes) which pass it on to the E3 ligase enzymes. The E3 enzymes then attach the ubiquitin molecule to the correct target protein.

In the last decade, the scientific community has been largely convinced that E3 enzymes were only able to transfer ubiquitin to a specific type of amino acid, called lysine. The attachment of ubiquitin to lysine happens through a chemical linkage called "isopeptide bond". This type of bond is chemically strong, very stable and easy to detect.

Attachment of ubiquitin to other amino acids, such as serine, threonine or cysteine, was considered a rare exception.

When ubiquitin is connected to serine or threonine, the linkage between them, which is named "ester bond", is much more fragile than the "isopeptide" bond found on lysine redisues.

These ester bonds can be easily hydrolysed in mild basic conditions and therefore were considered not stable enough to have a functional role in eukaryotic cells.

However, two prominent groups within the MRC-PPU recently discovered that two eukaryotic E3 ligases, MYCBP2 and HOIL1, specifically ubiquitylate serine and threonine (non-canonical ubiquitylation), therefore breaking the "lysine only" ubiquitylation dogma.

These studies strongly support the idea that serine and threonine ubiquitylation is indeed happening in cells and that the evidence for non-canonical ubiquitylation has been widely missed so far because of the use of inappropriate experimental procedures.

In the past years, the development of new, efficient and high-throughput technologies has enabled the study of new aspects of biology and has transformed and even established new research areas.

For example, the development of phosphoproteomics by Mass Spectrometry (MS) has immensely expanded our knowledge on kinases and the role of phosphorylation on cell homeostasis and disease.

MS is a very important analytical technique that allows researchers with such expertise to detect and quantify proteins and their modifications, including ubiquitylation.

An important aim of this project is to develop MS standard procedures able to specifically identify and quantify serine and threonine ubiquitylation.

This will allow us to determine the extent by which non-canonical ubiquitylation happens in cells as well as its physiological role.

By using a particular type of Mass Spectrometer named Matrix Assisted Laser Desorption/Ionization-Time of Flight MS (MALDI-TOF MS), I aim to quantify and characterize the activity of E2 conjugating enzymes and E3 ligases that are involved in non-canonical ubiquitylation.

I will exploit my expertise in MALDI-TOF MS to answer important biological questions but also move into less familiar research areas, as for structural and chemical biology.

In particular, I aim to define the underpinning molecular mechanisms that determine E2 conjugating enzyme specificity toward serine and threonine by employing structural biology tools.

I will also apply chemical biology tools for the identification of E3 ligases that work in tandem with non-canonical E2 conjugating enzymes. The overarching vision of this research project is to shed light on a new and different type of ubiquitylation.

This will expand our understanding of the ubiquitin system and increase our chances to produce drugs and treatments able to restore cellular health/homeostasis.