Mechanisms of protein SUMOylation and its functional consequences

Project Information

SUMOwriteNread

Grant agreement ID: 101078837

DOI

10.3030/101078837

EC signature date 5 December 2022

Start date 1 January 2023

End date 31 December 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 493 515,00

EU contribution

€ 1 493 515,00

Coordinated by

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
France

Periodic Reporting for period 1 - SUMOwriteNread (Mechanisms of protein SUMOylation and its functional consequences)

Reporting period: 2023-01-01 to 2025-06-30

Summary of the context and overall objectives of the project

Small ubiquitin-like modifiers (SUMOs) are members of the superfamily of small ubiquitin-like proteins. SUMOs become covalently attached to intracellular target proteins in the process known as SUMOylation. When a substrate protein becomes SUMOylated, it can change its inherent activity, interactions, subcellular location, or stability. SUMOylation plays an essential role in almost all biological functions and is implicated in diseases such as neurodegeneration and cancer.
The SUMOylation process typically depends on an interplay between an enzyme called E2 (its technical name in humans is UBC9), which acts as a “carrier” of SUMO, and a scaffold called a SUMO E3 ligase that can accelerate discharge of SUMO from the E2 onto a specific set of substrates. While SUMO E3 ligases are arguably key players in this modification system, contributing to its substrate specificity, few SUMO E3 ligases are known, and even fewer are well understood in terms of their mechanism of action. Furthermore, although we know that SUMOylation of a protein substrate can change its function in various ways listed above, there are hardly any examples available where this change is precisely understood in molecular, mechanistic terms.
In this project, we aim to illuminate the two main aspects of protein SUMOylation using approaches of mechanistic and structural biology. The first part of the project concerns the process of SUMO ligation onto substrates, catalysed by the interplay between SUMO E2 and E3 enzymes. We will move from characterising some enzymes that have been reported to have SUMO E3 ligase activity but remain poorly understood – to identifying new enzymes with this function, using both bioinformatic tools and chemical biology probes. In the second part of the project, we will explore how SUMO modification of substrates impacts their properties.
Given the biological importance of protein SUMOylation - which is an essential process in humans and other eukaryotic model organisms - the mechanistic insights gained through this project will inform research in various parts of biology.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

A big part of our efforts in the first two years was invested in the study of selected potential SUMO E3 ligases, that is to say proteins for which the SUMO E3 ligase activity has been proposed but not properly characterised in mechanistic terms. To characterise SUMO E3 ligases in this sense, you need to “capture” them (using tools of structural biology) in the process of catalysing the transfer of SUMO from E2 onto a substrate – or at least in the process of stabilising a SUMO-E2 molecule in an active conformation from which SUMO can more easily be dispatched onto a substrate. Thus, complexes between a SUMO E3 ligase candidate, SUMO molecules, the E2 enzyme, and (optionally) a substrate need to be assembled, and stabilised through the introduction of stable covalent bonds (achieved either by mutagenesis or with chemical biology tools). To do this, all these components need to be recombinantly produced and purified, and strategies of stabilising the assembly with covalent bonds need to be envisioned. Building on existing methods and developing them further, we have recently developed such strategies and achieved the reconstitution of required complexes, and began with their structural analysis, obtaining first results.
Work on other objectives, including the identification of novel SUMO E3 ligases and production of chemical probes is ongoing.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The expected potential impact of the project includes improved understanding of the protein SUMOylation process and its molecular consequences. More specifically, we envision that we will be able to elucidate the mechanism of action of some so far poorly understood SUMO E3 ligases and possibly identify new enzymes with this function. We also expect to demonstrate – through specific case studies – what the molecular effect of SUMOylation of a specific substrate can be in terms of new intramolecular interactions and other properties.
In addition to these expected results, we are also open to any unexpected discoveries that may come our way. One such discovery we have already made. Driven by our interest in identifying new potential SUMO E3 ligases, we analysed a published list of SUMOylated human proteins, reasoning that SUMO E3 ligases are likely to undergo extensive autoSUMOylation. Among the top 500 SUMOylated sites in human cells (the top 1% of all reported sites), we identified 14 sites belonging to different members of the poorly characterised ZBTB protein family. Given that ZBTB proteins contain a BTB domain – also found in some subunits of ubiquitin E3 ligase complexes – and zinc-finger domains involved in DNA binding, we hypothesised that they might act as SUMO E3 ligases that localise to specific DNA sites and catalyse SUMOylation through their BTB domains. However, instead of confirming SUMO E3 ligase activity, we found that the BTB domain of some ZBTB proteins forms large homomultimers in the form of filaments. When expressed inside cells, these filaments drive the proteins’ localisation to nuclear condensates and contribute to the repression of genes whose promoters are bound by their zinc-finger domains. While we are still investigating why ZBTB proteins undergo such efficient SUMOylation, our research has unexpectedly uncovered a new aspect of cellular biology.