Analysis of a WRN-like mitochondrial protein (WLMP) - identity and role in mitochondrial DNA damage responses

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

Understanding the biology of normal human ageing is key to improving health during later life. Because ageing is a gradual, long-term process, it is very hard to study in the laboratory. However, by looking at premature ageing diseases, we can start to identify key molecules involved in ageing. The best model system we have is the premature ageing disease called Werner's syndrome. People with this syndrome first show signs of early ageing such as grey hair, wrinkled skin and age-related diseases including hardening of the arteries, cancer and diabetes in their teens or twenties, and have a much shorter than average lifespan. Scientists have identified a single region of the genetic blueprint (a gene) which when lost causes Werner's syndrome. This is particularly exciting as it is possible to work out the function of the gene, which is called WRN, in the test tube and to examine the effect of losing this gene on the ageing of human cells grown in dishes in the lab. We now know from these studies that the protein made from the WRN gene acts to cut or unwind unusual DNA structures, preventing such DNA from rearranging itself - a dangerous event that can lead to the cells ageing prematurely or even leading to cancer. Another key aspect of normal ageing is the accumulation of damage in our DNA throughout our lives, so that older people have high levels of DNA damage. Many things cause damage, but perhaps one of the most important is a dangerous form of oxygen made as a by-product of producing the energy needed for life. This energy production happens in the cell's powerhouses, called mitochondria, which have their own DNA. Because the dangerous form of oxygen is made in the mitochondria right next to mitochondrial DNA, it is widely believed that the DNA in mitochondria is very prone to damage. We have discovered that the WRN protein, or a protein that looks very similar to it, is present in the mitochondria. This is a very exciting new finding that suggests a direct link between what we know about premature ageing in people and ageing due to mitochondrial DNA damage. We want to carry out a series of experiments designed to find out whether the protein we see in mitochondria really is WRN. We shall also see whether more WRN goes into mitochondria when their DNA gets damaged, and we shall try to discover the signal that is responsible for sending WRN to damaged mitochondria. If we find a direct role for WRN in mitochondrial DNA repair, this will open up a whole new research field that may in the longer term help in the development of drugs to slow down mitochondrial DNA damage, prevent premature ageing and enhance the quality of life of older people.

Technical Summary

Werner's syndrome (WS) is the best current model of human ageing, and is caused by mutation of WRN, which encodes a helicase/exonuclease. WRN resolves aberrant DNA structures arising during DNA replication, suppressing illegitimate recombination. Whilst WS is useful in analysing cellular senescence, it has not until now been possible to link cellular senescence with the mitochondrial theory of ageing. Accumulation of ROS-induced DNA damage within mitochondria is thought to be a major contributor to ageing, but the mechanisms by which the cell deals with mtDNA damage are at best unclear. Our novel and exciting preliminary data suggest localisation of WRN to mitochondria: the presence of WRN in organelles whose DNA is subject to high levels of damage is suggestive that WRN may play a role in mtDNA repair. Our data are based upon immunofluorescence (IF) and immunoblotting (IB) studies, but we cannot yet be sure that the protein detected is genuinely WRN. We therefore term the protein WRN-like mitochondrial protein, or WLMP. In this one year 'proof of principle' project, we shall establish the identity of WLMP. Using a panel of anti-WRN antibodies, we shall confirm our IF/IB data, then sequence immunoprecipitated WLMP from isolated mitochondria, and in parallel, generate peptide fingerprints by mass spectroscopic analysis. If WLMP is identified as WRN, we shall attempt to localise a mitochondrial targeting sequence by generating a series of GFP-tagged WRN deletions and assaying mitochondrial uptake. The role of the electrochemical gradient in WLMP accumulation will be assessed using uncoupling agents such as rotenone. To analyse a possible role of WLMP in mtDNA repair, we shall perform co-localisation studies of WLMP with mtDNA by confocal and electron microscopy. WLMP association with mtDNA will be determined in cells with endogenous DNA damage (mutant for POLG, twinkle helicase or p53), or those damaged by ddC and other drug treatments.
 
Description We have identified a novel protein EXDL2 in the cell's energy producing centres, the mitochondria, with a different form found in the nucleus. EXDL2 is highly related to WRN, a protein important in preventing premature ageing. This is a very exciting new finding that suggests a direct link between what we know about premature ageing in people and ageing due to mitochondrial DNA damage. Experimentally decreasing levels of EXLD2 protein in cells makes them very sick, and we see a preferential loss of the mitotic form of EXDL2.

We believe that this discovery of EXDL2 as an important in mitochondrial protein possibly involved in mitochondrial DNA repair, this may open up a whole new research field that may in the longer term help in the development of drugs to slow down mitochondrial DNA damage, prevent premature ageing and enhance the quality of life of older people.
Exploitation Route We have applied for further finding to explore the importance of EXDL2 in mitochondrial DNA stability and in the nuclear genomic response to interstrand DNA crosslinks. This has relevance for treatment of age-related disease, mitochondrial disease and genomic DNA damage.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Our findings have not yet been published, though have been presented formally at a mitochondrial research workshop and will now be written up for publication. We have used the results as preliminary data in subsequent grant applications, and one aspect of the data has been taken forward in two undergraduate research projects.
First Year Of Impact 2013
Sector Other
 
Title Monospecific antibodies against human WRN and DmWRNexo 
Description Rabbit polyclonal antibody against short peptide of human WRN (KLH-coupled); similar antibody generated against DmWRNexo 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact Supplied anti-DmWRNexo Ab to collaborator for use in DPhil project - paper reporting immunofluorescence staining of DmWRNexo during embryonic development is nearly ready for submission for publication. 
 
Description Kazakhstan project 'Small molecules' 
Organisation Cardiff University
Department Cardiff University School of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution One of a consortium of 4 institutions (Nazarbayev University, Kazakhstan; Cardiff University; University of Brighton and University of Oxford)
Collaborator Contribution We jointly contributed scientific research to a collaborative project. Nazarbayev provided funds and two short term researchers to expedite the research.
Impact Publication Kenessary et al 2012 N Biotech. originated from this, though included some input from my ongoing ESRC work. Two more papers are in preparation.
Start Year 2011
 
Description Kazakhstan project 'Small molecules' 
Organisation Ministry of education and science of the Republic of Kazakhstan
Country Kazakhstan, Republic of 
Sector Public 
PI Contribution One of a consortium of 4 institutions (Nazarbayev University, Kazakhstan; Cardiff University; University of Brighton and University of Oxford)
Collaborator Contribution We jointly contributed scientific research to a collaborative project. Nazarbayev provided funds and two short term researchers to expedite the research.
Impact Publication Kenessary et al 2012 N Biotech. originated from this, though included some input from my ongoing ESRC work. Two more papers are in preparation.
Start Year 2011
 
Description Kazakhstan project 'Small molecules' 
Organisation University of Brighton
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution One of a consortium of 4 institutions (Nazarbayev University, Kazakhstan; Cardiff University; University of Brighton and University of Oxford)
Collaborator Contribution We jointly contributed scientific research to a collaborative project. Nazarbayev provided funds and two short term researchers to expedite the research.
Impact Publication Kenessary et al 2012 N Biotech. originated from this, though included some input from my ongoing ESRC work. Two more papers are in preparation.
Start Year 2011
 
Description OxAgeN - establishment of the Oxford Aging Network (L. Cox) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Established a network of scientists and clinicians with an interest in ageing (research and clinical practice) to foster closer collaboration and accelerate discoveries from bench to clinic. Representatives from commercial organisations and third sector (U3A, Age UK) are also members.
Impact: decision to hold scientific conference on ageing in Oxford.
Year(s) Of Engagement Activity 2015
URL http://www.imm.ox.ac.uk/ageing
 
Description Progeroid WRN in DNA replication 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited one hour lecture at UCL on my lab's research

We have forged closer academic ties with UCL and the director of the Healthy Ageing Institute is coming to Oxford to viva one of my DPhil students - we hope to pursue this to form a more direct scientific collaboration if appropriate.
Year(s) Of Engagement Activity 2012
 
Description Talk, MitOX Dec 2015 (L Cox) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited talk at mitochondrial conference on a WRN-related protein in mitochondrial DNA maintenance. outcome: request to publish sceintifc research paper in Biochemical Society Transactions.
Year(s) Of Engagement Activity 2015
 
Description WRN in DNA instability and ageing 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Invited 1 hour lecture on my lab's reserch to the Genome Stability Unit, University of Sussex

There was excellent feedback and discussion as a consequence of the talk. No direct collaborations have yet resulted but ideas for future projects (eg using a novel recombinogenic cell line) were discussed.
Year(s) Of Engagement Activity 2012