Mechanism of poly-SUMO chain recognition by the ubiquitin ligase RNF4

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

Post-translational modification is a chemical change to a protein after it has already been synthesised within a cell. This step enables the cell to 'mark' certain proteins so that their behaviour can be later controlled. Ubiquitin (Ub) and Small ubiquitin-like modifiers (SUMOs) form a family of proteins that are chemically attached to and removed from proteins in order to modify their function. SUMOs are involved in three important processes within the cell: they play crucial roles in regulating cell multiplication; are involved in how cells respond to damage to their genetic material (DNA); and control which genes are "switched on" or "switched off" in response to the environment. Diseases such as cancer arise when a series of unwelcome changes occur in some of these cellular processes, causing the cell to multiply out of control. Before we can identify what goes wrong in cancers it is necessary to understand exactly how our cells normally work.

In this proposal two teams of researchers from the groups of Prof Steve Matthews (Imperial College) and Ron Hay (Dundee) have linked up to decipher how poly-SUMO protein chains are recognised and the downstream effects are elicited. The focus is on characterising the mechanism of substrate recognition by RNF4. RNF4 is a small protein that specifically recognises poly-SUMO chains on proteins and then attaches Ub to these substrates, which subsequently targets them to the proteasome for degradation. Regions of RNF4 contain SUMO interaction motif (SIMs) that 'fish-out' poly-SUMO chains. By using nuclear magnetic resonance (NMR), the shape and flexibility of protein complexes will be imaged in solution. Insight from these structural studies will be used to understand the features that control poly-SUMO recognition. This will shed light on the mechanisms that underlie fundamental aspects of cellular regulation, and the aberrant pathways that can lead to disease and cancer. In turn, this could lead to new ways of treating many types of cancer, for example, by developing drugs that block the activity of certain molecules that regulate SUMO activity.

Technical Summary

Conjugation and deconjugation with the Small Ubiquitin-like Modifier (SUMO) protein is an important regulatory process in controlling gene expression. Poly-SUMO chains have been implicated in cellular processes such as meiosis, genome maintenance and stress response; and more than 300 poly-SUMO conjugates have been identified in cultured eukaryotic cells. Lys11-linked poly-SUMO chains are recognized by a wide range of effector proteins that contain SUMO interaction motifs (SIMs). As has been observed for poly-ubiquitin chains it is predicted that specific recognition of SUMO chains is based on conformational preferences. Apart from studies on individual SUMO domains interacting with a single SIM, there is no information regarding the conformational properties of poly-SUMO bound to SIM-containing proteins in solution. Deciphering the details of how Lys11-linked poly-SUMO chains are recognized by effector molecules such as RNF4 is vital for a better understanding of this important regulatory mechanism.

This proposal aims to characterize the solution structure, dynamics, and interactions of poly-SUMO chains. We will use NMR and SAXS approaches to determine the three-dimensional conformation of Lys11-linked tetra-SUMO chain, free in solution, and in complex with the SUMO-interaction-motif (SIM) protein, RNF4, which contains 4 canonical SIM motifs. Using a combination of long-range, orientational constraints derived from residual dipolar coupling measurements, complemented with distance information from paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels, we will derive models for interdomain or intermolecular interfaces. The overall structure and dynamics of the polySUMO-RNF4 complex will be probed by site-directed mutagenesis and functional readouts.

Planned Impact

This basic "blue-sky" research project will have an impact on a fundamental process in cellular regulation. The following beneficiaries have been identified. Methods of how they will benefit and what will be done to ensure that they have the opportunity to benefit from this research are detailed. The applicants SM and RH will take collective responsibility for maximizing impact.
1. Beneficiary one: SM and RH research groups The proposal will support an established collaboration between the SM and RH groups working on the interaction between poly-SUMO chains and multiple SIMs. We have maintained active communication channels between the groups by organising regular joint meetings, conferences calls and actively promoting the exchange of research staff. In addition to ensuring that both group thrive during this collaboration, our proactive approach to communication will likely fuel future ventures in related areas. The added value of this collaboration is that it will integrate the NMR expertise of the SM group with the molecular biology and biochemistry skills of the RH group. This will benefit the RH group by making them more aware of modern methods of structural analysis that can be applied to ubiquitin and ubiquitin-like proteins.It will also benefit the SH group by giving them access to quantitative biochemical and functional assays for the activity of proteins involved in pathways of ubiquitin and ubiquitin-like protein modification.
2. Beneficiary two: PDRAs and students. The PDRAs and any undergraduate, postgraduate or intern students contribute to the knowledge economy and increase the economic competitiveness of the UK. SM and RH will train PDRAs, and students in key techniques and good laboratory practice and encourage an innovative approach to research. Those involved will be encouraged to present their work as widely as possible and to communicate with both short-term and long-term user groups. Training in communication will be provided if necessary. Both Universities have a highly active staff development programme, which includes courses on presenting science to a lay audience, writing research papers and proposal, career workshops. It is worth noting that this project has already contributed to the training and progression for several undergraduate students, exchange students and Master Students including our BBSRC MRes in Structural Molecular Biology.
3. Beneficiary three: wider academic community As outlined in our statement on data sharing, we will ensure that research is disseminated widely by the Open Access publication in high-impact journals, presentation at international research meetings, and the development of new collaborations where appropriate and deposition of data into freely-accessible databases (e.g. PDB).
4. Beneficiary four: large Pharma and small Biotech companies Although this is a basic research project, its outcomes will benefit the private sector as SUMO is required for repair of damaged DNA in human cells and that SUMO is recruited to sites of DNA damage induced by ionising radiation. By revealing a deeper molecular understanding of SUMO recognition in the regulation of mammalian cells, this work wil benefit large Pharma (e.g. Pfizer, Novartis, GSK), smaller Biotech companies (Ubiquigent) or not-for-profit organisations that are developing new disease therapies. We will ensure that intellectual property opportunities are maintained by liaison with BBSRC together with Dundee's and Imperial College's technology transfer expertise (IC INNOVATIONS Ltd). This local expertise will advise on how to protect inventions and develop an appropriate IP protection strategy. Furthermore, they will foster relationships with industry partners, to determine the right commercialisation strategy. Where appropriate they will co-invest in new ventures to accelerate development and increase value.

Publications

10 25 50
 
Description The SUMO targeted ubiquitin ligase RNF4 is required for the arsenic induced degradation of the PML-RAR fusion protein that is responsible for curing Acute Promyelocytic Leukaemia. What we have discovered in a collaboration with the laboratory of Prof Steve Matthews is the mechanism by which RNF4 selects its targets for ubiquitination.
Exploitation Route The combination of advanced nuclear magnetic resonance approaches and biochemical analysis has served as a model for the analysis of ubiquitin and ubiquitin-like proteins by effector proteins and substrates. Other researchers are now following our approaches.
Sectors Healthcare

 
Description How ubiquitin E3 ligases recognise substrate has become a key issue in the development of drugs to this class of enzyme. Disrupting the interaction between E3 ligase and substrate would provide a means to inhibit ubiquitination of important substrates.
First Year Of Impact 2014
Sector Healthcare
Impact Types Economic

 
Description Investigator Award
Amount £1,922,744 (GBP)
Funding ID 098391/Z/12/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2012 
End 10/2019
 
Description Substrate recognition by RNF4 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution My laboratory generated purified proteins that were labelled by stable isotopes for analysis by NMR. We also carried out biochemical analysis of the purified proteins.
Collaborator Contribution The laboratory of Prof Steve Matthews carried out NMR analysis to determine the structure of RNF4 bound to polySUMO
Impact This is a multidisciplinary research programme where the Matthews lab provide the NMR expertise and the Hay lab provide the biochemical analysis of the ubiquitin system
Start Year 2010
 
Description Presented to Higher level school pupils 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presented the work of my laboratory to school pupils from the Dundee area. This was organised by Dundee University and was aimed at pupils taking their "Higher" exams.

Hopefully there will be an increase in school pupils that decide to do science at University.
Year(s) Of Engagement Activity 2009
 
Description Scottish Book Week at Dundee Contemorary Arts 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Participated in discussion of science and society and gave readings from The Dinosaur Hunters by Deborah Cadbury at "Books Aloud" at Dundee Contemorary Arts. The audience (about 30) were mainly non-scientists from the Dundee area.

We have been asked to run this again.
Year(s) Of Engagement Activity 2013,2014