Determining the role of the SUMO-specific, ubiquitin ligase RNF4

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

Abstract

The work here is directly relevant to finding out how cellular components are destroyed. Understanding how this is done is important as it is involved in the regulation of basic cellular processes. The subject of this proposal, RNF4 is a unique enzyme that adds chains of ubiquitin molecules onto proteins already modified with another ubiquitin-like protein known as SUMO. This targets the modified proteins for proteasomal degradation. It is RNF4 that mediates the therapeutic effect of arsenic in treatment of Acute Promyelocytic Leukaemia, by inducing degradation of the oncogenic PML-RAR fusion protein responsible for the disease. Understanding the detailed mechanism of RNF4 action could therefore have important implications for the development of future therapeutic agents. The remarkable efficacy of the proteasome inhibitor Velcade in Multiple Myeloma suggests that more substrate specific inhibition of proteasomal degradation could allow the development of drugs with increased efficacy and reduced side effects. Identification of suitable targets for the development of new drugs requires a detailed knowledge of the molecules involved in protein degradation and their mechanisms of action. The work described here is an attempt to obtain a detailed mechanism for the function of RNF4.

Technical Summary

Our objective is to determine the role of the SUMO-specific ubiquitin ligase RNF4 in DNA damage and other stress responses. As RNF4 contains multiple SUMO Interaction Motifs (SIMs) and a RING domain our hypothesis is that RNF4 targets proteins bearing poly-SUMO chains for ubiquitination and subsequent degradation via the proteasome. To test this hypothesis and establish the biological pathways that RNF4 participates in, we will ablate RNF4 expression either by siRNA in human cells or by gene knock out in chicken DT40 cells, and determine the phenotypes of the RNF4 depleted cells. As the yeast homologues of RNF4 have a role in DNA damage responses and RNF4 can complement the yeast mutants it is likely that vertebrate RNF4 will have a role in DNA damage responses. We will therefore expose the wild type or RNF4 depleted cells to a variety of genotoxic insults and test a range of DNA damage responses. This should reveal the steps in the DNA damage response that are subject to RNF4 dependent regulation. Quantitative proteomics will be used to identify the cellular substrates targeted for ubiquitination by RNF4. Bioinformatic analysis of the data will reveal the additional biological processes that RNF4 participates in. This will be confirmed by further siRNA analysis. In Acute Promyelocytic Leukaemia the PML gene is fused to the retinoic acid receptor (RAR) gene resulting in the generation of a PML-RAR fusion protein which acts as a potent transcriptional repressor, blocking differentiation and allowing continued proliferation of the leukaemic cells. SUMO modification of PML-RAR is required to maintain transcriptional repression and for the development of disease. Treatment of patients with arsenic is an effective therapy for APL and the arsenic achieves this by inducing degradation of PML. Our preliminary evidence indicates that this is mediated by RNF4. Thus we will seek to prove that RNF4 is recruited to SUMO modified PML after arsenic treatment and that once bound it ubiquitinates PML-RAR and targets it for proteasomal degradation. We will study this process in vivo using siRNA ablation of RNF4 expression and in vitro using well defined recombinant modification components and SUMO modified PML substrates.

Publications

10 25 50
 
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 Project grant
Amount £400,000 (GBP)
Funding ID BB/JO16004/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2012 
End 11/2015
 
Title Database containing over 12000 sites of aspirin-induced lysine acetylation from cultured human cells. 
Description The mass spectrometry proteomics raw data files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/) with the dataset identifier PXD003530 for the label-free site ID analysis, and PXD004995 for the occupancy and SILAC half-life analysis. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Our work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences of nonspecific chemical acetylations. 
URL https://www.ncbi.nlm.nih.gov/pubmed/27913581
 
Title Identification of SUMO-2 modification sites 
Description We used max spectrometry to identify more than 1000 SUMO-2 modification sites in human cells. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Our database provides a list of SUMO modification sites that other researchers can access. This allows them to identify the site of modification and eliminate the site by mutagenesis and then determine the functional consequences. The accession number is PXD001281 
URL http://proteomecentral.proteomexchange.org
 
Description Collaboration with researchers in IFOM Milan 
Organisation European Institute of Oncology (IEO)
Department Department of Experimental Oncology
Country Italy 
Sector Academic/University 
PI Contribution We have had a long term collaboration with the group of Susanna Chiocca where we have carried out in vitro experiments on SUMO and ubiquitin modification. We also carried out adenovirus infections to establish the impact on cellular protein modification
Collaborator Contribution They had an interesting biological problem relating histone acetyl transferases that are modified by SUMO and ubiquitin and altered in activity after viral infection
Impact BC-box protein domain-related mechanism for VHL protein degradation. Pozzebon ME, Varadaraj A, Mattoscio D, Jaffray EG, Miccolo C, Galimberti V, Tommasino M, Hay RT, Chiocca S. Proc Natl Acad Sci U S A. 2013 Oct 21. PMID: 24145437 A role for paralog-specific sumoylation in histone deacetylase 1 stability. Citro S, Jaffray E, Hay RT, Seiser C, Chiocca S. J Mol Cell Biol. 2013 Sep 25. [Epub ahead of print] PMID: 24068740 A mechanism for inhibiting the SUMO pathway. Boggio R, Colombo R, Hay RT, Draetta GF, Chiocca S. Mol Cell. 2004 Nov 19;16(4):549-61. PMID: 15546615
 
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