Congratulations to Mirela Delibegovic, a PhD student in Tricia Cohen's laboratory from 1999 to 2003, on her recent promotion to Professor in the School of Biological Sciences, University of Aberdeen, UK! Mirela, a lively member of the MRC PPU 15 years ago, has a long-standing interest in research related to the causes and possible treatments for Type II Diabetes. Her PhD studies focussed on the hormonal control of glycogen metabolism, and by employing gene ‘knockout’ technology she showed that one form of glycogen-targeted protein phosphatase 1 has the potential to contribute to obesity, glucose intolerance and insulin resistance in later life, and that a novel form of glycogen-targeted protein phosphatase 1 was regulated by insulin. Before leaving Dundee, Mirela married another member of the MRC PPU Nimesh Mody who also was awarded his PhD in 2003.
Mirela was appointed as a postdoctoral scientist with Professor Benjamin Neel at the Harvard Medical School in Boston, USA, where she continued research in the field of diabetes investigating protein tyrosine phosphatase1B in peripheral and hypothalamic metabolic signalling pathways, supporting its potential as a good target for the treatment of Type II Diabetes. In 2007 Mirela moved to the University of Aberdeen and was awarded a group leader fellowship from Research Councils UK for investigation into molecular aspects of ageing and obesity in animal models and later many other grants. Her studies have focussed mainly on tissue specific models of protein 1B and provided excellent data on the role of protein tyrosine phosphatase 1B in metabolic changes, such as obesity, cardiovascular disease, and inflammation, supporting the notion that inhibition of PTP1B may be beneficial in these disorders, sometimes associated with Type II Diabetes.
On being asked how she managed all her research with two young children, Mirela replied “I have to say I couldn’t have done it all without Nimesh... I was a partner in a big EU project so I’ve had to be away a lot and he’s been holding the fort!”
The MRC PPU is proud to announce that Dr. Yogesh Kulathu has been recognized yet again for his outstanding work by receiving a highly sought after ERC Starting Grant. This €1.5 million grant will allow Yogesh to initiate an ambitious new project to understand how T-cell biology is regulated by the ubiquitin system.
Every year, the European Research Council (ERC) selects and funds the very best, creative researchers of any nationality and age, to run projects based in Europe. With a success rate of ~10%, ERC Starting Grants are very competitive and are awarded to the “best up-and-coming research leaders working in Europe” to carry out pioneering frontier research. These grants come with significant support to enable awardees to pursue ground-breaking ideas and create excellent new research teams to tackle important research questions.
T lymphocytes are key cells of the adaptive immune system that protect us against pathogens and malignant cells. T cell activation, differentiation and immune responses are tightly controlled processes that are orchestrated by complex biochemical pathways. Deregulation of these biochemical pathways can result in lymphomas, autoimmune diseases and inflammation. Hence, understanding the biochemical events regulating lymphocyte biology have long been a topic of intense research.
As Dr. Kulathu explained “Past efforts in this area have largely focussed on protein phosphorylation and we know little about the roles of other posttranslational modifications (PTMs) such as ubiquitin and ubiquitin-like modifiers. The ERC grant will allow my group to investigate how T cell function and immune responses are regulated by ubiquitin signalling networks.”
Professor Dario Alessi, Director of the MRC PPU expanded: “This research will provide novel mechanistic insights into signal transduction in lymphocytes. Moreover, Yogesh’s work has the potential to uncover T cell specific signalling nodes that can be manipulated to reengineer T cells for immunotherapy to treat cancer. We are all thrilled for Yogesh and his team.”
Kashyap Patel, a former clinical PhD student in Kei Sakamoto’s lab in the MRC PPU, has been awarded a highly competitive Wellcome Trust Clinical Postdoctoral Fellowship to undertake research at the University of Exeter into the genetic basis of Diabetes.
The Wellcome Trust Postdoctoral Fellowship scheme was established to support the most talented clinicians wishing to pursue a scientific career path towards independence.
During his PhD studies, Kash discovered a critical role for the salt-inducible kinase (SIK1, 2 and 3) as suppressors of gluconeogenesis in the liver that was subsequently published in Nature Communications.
He then moved to the University of Exeter where he was appointed Clinical Lecturer in the world-leading laboratory of Andrew Hattersley FRS and completed his clinical training in Diabetes and Endocrinology. Over the last 18 months he has developed genetic methodologies that have transformed understanding of diabetes occurring before the age of 6 months. With support from the Wellcome Fellowship, Kash will now build on this advance to identify novel genes that cause Mendelian-inherited forms of Diabetes.
On 5 November, 2015 Sam Strickson, who has been supervised by Philip Cohen, has successfully defended his PhD. Over the past 4 years Sam has performed groundbreaking research into the role of the ubiquitin system in regulating the Interleukin 1 signalling network.
His examiners were Mads Hansen from the Ludwig Institute of Cancer Research, University of Oxford, and Gopal Sapkota of the MRC Protein Phosphorylation and Ubiquitylation Unit at the University of Dundee.
Darren Cross who undertook is PhD training within the MRC-PPU working in Philip Cohen’s laboratory (1993-1997) has played a major role in developing a new drug termed TAGRISSO™ (AZD9291) that was recently approved (13 November 2015) by the US Food and Drug Administration for the treatment of metastatic non-small cell lung cancer.
AZD9291 is a novel EGF Receptor (EGFR) inhibitor that does not significantly affect the wild type but potently inhibits the common cancer T790M mutation that renders the EGFR resistant to many of the other EGFR inhibitor drugs that are on the market.
Darren Cross led the group in AstraZeneca that initially developed AZD9291 and published the first paper on this compound in 2014:
Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, Spitzler PJ, Orme JP, Finlay MR, Ward RA, Mellor MJ, Hughes G, Rahi A, Jacobs VN, Red Brewer M, Ichihara E, Sun J, Jin H, Ballard P, Al-Kadhimi K, Rowlinson R, Klinowska T, Richmond GH, Cantarini M, Kim DW, Ranson MR, Pao W. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov. 2014 Sep;4(9):1046-61. Click here for copy of paper
The structure of TAGRISSO™ (AZD9291) is shown in the above Figure. It works by forming a covalent bond to a kinase domain Cys797 residue in mutant T790M-EGFR. The property of TAGRISSO™ (AZD9291) not significantly inhibiting wild type EGFR is very favourable and makes it possible to achieve a therapeutic dose of compound in patients that inhibits the cancer driving function of the T790M-EGFR mutant, without causing side effects that result from inhibition of wild type EGFR.
TAGRISSO™ (AZD9291) displays a response rate of 59% in patients with T790M EGFR metastatic non-small cell lung cancer and duration of response of 12.4 months.
TAGRISSO™ (AZD9291) was one of fastest development programmes in the history of pharmaceutical research – from start of clinical trials to approval in just over two and a half years.
For more information click here
Agne Kazlauskaite, who has been supervised by Miratul Muqit and Dario Alessi, has successfully defended her PhD. Over the last 4 years Agne has undertaken groundbreaking research into the function of the PINK1 kinase including the discovery that PINK1 phosphorylates ubiquitin.
Her examiners were David Komander of the MRC Laboratory of Molecular Biology in Cambridge who is a former MRC PPU PhD student himself (supervised by Dario Alessi and Daan van Aalten) and Calum Sutherland of the School of Medicine in Dundee.
Scientists within the halls of the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC PPU) have been focused on elucidating the mechanisms underlying two of the most prolific post-translational modification events that occur in cells for a number of years. These events are critical for the normal function of cells and signalling within each of our bodies – and indeed, when things go wrong (as in disease), abnormal signalling events by way of phosphorylation or ubiquitylation, are often the culprit. But how best to convey the importance of these events to the world at large?
Working with a team of scientists, writers, and animators – along with a Hollywood icon – the MRC PPU is pleased to share a new video that describes one of the most significant components of the research carried out in the Unit – communication between cells. Entitled ‘The Heart of Research and Discovery’ and narrated by the stage and film actor Brian Cox (also Rector of The University of Dundee!), the animation conveys the history and significance of one type of signalling event – phosphorylation. It further goes on to illustrate the impact of phosphorylation on basic biology, health and disease, and culminates with describing the role the MRC PPU plays in the development of therapeutics focused on the enzymes that regulate phosphorylation. It is hoped that this animation (made in collaboration with the company Vivomotion) will be helpful for the general public – from patients and caregivers, to students – so that they can gain insight into the implications of the Unit’s research for diseases such as cancer, hypertension and neurodegeneration.
To view the video please click here.
The MRC Protein Phosphorylation and Ubiquitylation Unit (MRC PPU) is proud to announce a newly re-designed website for DNA Sequencing and Services facility. The facility’s customer-centric approach is reflected in this new site which streamlines information access, sample submission as well as access to resulting data. Moreover, customers can now more easily keep track of their accounts, all in one place.
With Dr. Nicholas Helps at the helm, the facility has long offered a range of services extending beyond DNA sequencing – all at competitive pricing and efficient turnaround times – for investigators’ research needs including:
• DNA Sequencing
• Minipreps (+/- Sequencing)
• Maxipreps (+/- Sequencing)
• Fragment Analysis
Given customer feedback about the new site in recent months, additional improvements have been incorporated into the site – further improving usability.
But with all these updates, the staff have not lost their personal touch and still welcome all queries, suggestions and direct email submissions as well! We hope that the research community, near and far, will continue to take advantage of all the great quality of services that the facility has to offer.
The MRC PPU has a long-standing history of developing tools, reagents and services for the greater scientific community. The MRC PPU DNA Sequencing and Services facility, has been a mainstay for investigators not just at The University of Dundee or Scotland, but the United Kingdom and beyond.
More information about on the MRC PPU DNA Sequencing and Services facility can be found here.
MRC PPU programme leader, Yogesh Kulathu, has been elected into the EMBO Young Investigator Programme for three years, starting on 1 January 2016.
Every year, EMBO selects some of the best young scientists in Europe to join this prestigious programme through which they are provided a range of benefits and support to help them realise their potential as world class researchers.
“I am very pleased to be recognised for my work and and honoured to be selected into this program” said Yogesh. “I look forward to interacting and collaborating with the vast network of Young Investigators – past and present.”
This year 23 young researchers have been elected as EMBO Young Investigators. Yogesh joins a network of 365 Young Investigators who represent some on the best young group leaders from all over Europe.
Yogesh’s laboratory studies how ubiquitin signals are decoded and regulated to produce distinct cellular outcomes. Using a range of techniques, from structural and biochemical methods to genetic approaches in model organisms, they elucidate the regulation of ubiquitin signaling and how perturbations result in diseases.
Dario Alessi, Director of the MRC PPU was equally pleased by the election “This is indeed a great recognition and speaks to the caliber of scientific research that Yogesh is undertaking in his lab.”
A major focus of research at the MRC PPU is directed at understanding the molecular basis of neurodegenerative diseases such as Parkinson’s since there are no available disease-slowing therapies for these devastating diseases.
To date ~20 genes have been identified in familial Parkinson’s patients including mutations in the mitochondrial protein kinase, PINK1 (PTEN-induced kinase 1).
The Muqit lab has been investigating the regulation and downstream signalling of PINK1 and previously discovered that PINK1 is activated upon mitochondrial depolarisation and phosphorylates Serine 65 of the N-terminal ubiquitin-like domain of Parkin as well as ubiquitin itself to maximally stimulate Parkin ubiquitin E3 ligase activity.
A major question in the field was whether PINK1 had additional targets and in a joint collaboration, the Muqit lab teamed up with the laboratory of Matthias Trost to employ state-of-the-art subcellular phosphoproteomics to address this important question.
Amongst nearly 15,000 phosphosites isolated from cells expressing active PINK1, Chandana Kondapalli (a former PhD student in the Muqit lab) and Matthias excitingly discovered three members of a sub-family of Rab GTPases, namely Rab8A, 8B and 13 that were all phosphorylated at a highly conserved residue, Serine111, in response to PINK1 activation.
Post-doctoral scientist, Yu-Chiang Lai, next undertook a series of a biochemical experiments to validate the physiological relevance of these novel PINK1 targets. Using phospho-specific antibodies raised against Ser111, Yu-Chiang demonstrated that Rab Ser111 phosphorylation is absolutely PINK1 dependent and abolished in HeLa PINK1 knockout cells. Furthermore, using human patient derived fibroblasts obtained from Olga Corti’s lab in Paris, he further showed that Rab phosphorylation is totally disrupted in patients harbouring PINK1 mutations.
To investigate the impact of phosphorylation on Rab GTPase function, Aymelt Itzen’s laboratory at the TUM in Munich undertook biophysical analysis using phosphomimetic versions of Rab8A. Strikingly the Itzen lab found that Rab Ser111 phosphorylation dramatically impaired Rab8A activation by its physiological guanine exchange factor, Rabin8 suggesting that PINK1 activation leads to inhibition of Rab function.
Yu-Chiang’s new findings suggest that monitoring phosphorylation of Rabs at Ser111 may represent novel biomarkers of PINK1 activity in Parkinson’s patients. Yu-Chiang’s analysis has also revealed that PINK1 does not directly phosphorylate Rabs suggesting the existence of an intermediate kinase or phosphatase that is regulated by PINK1 and in future work it will be exciting to identify the Rab Ser111 kinase.
To read a copy of Yu-Chiang’s paper published in The EMBO Journal click here.
Research undertaken by the laboratory of Miratul Muqit has featured in a two part exhibition of contemporary art, artefacts and scientific research.
The Hearts & Minds exhibition opened on the 18th September and has run for a month at the LifeSpace gallery in Dundee. The exhibition showcases research at the University of Dundee to better understand changes in the brain during neurodegeneration.
In 2014 Miratul envisaged bringing to the public an event that highlighted the intricacies of the brain with a focus on neurodegeneration. Working with Sonal Das at the MRC PPU and artist Caitlin Monney, he and his team collaborated with centres across the UK to source Magnetic Resonance imaging (MRI) brain scans of adults that Ms. Monney then illustrated – paying special attention to highlight those areas most affected in a variety of neurodegenerative disorders. The dramatic alterations exhibited in these illustrations convey the impact of the disease process and the importance of basic research in finding cures for these devastating illnesses.
A recent paper from the Walden and Shaw labs, published in the EMBO Journal, reported the mechanism of inhibition and allosteric activation of Parkin, an important ubiquitin ligase mutated in Parkinson's disease. When the activation signal phosphoubiquitin binds to Parkin, the inhibitory domain is ejected from a distant site to reveal a surface required for Parkin to engage with components of the ubiquitin-conjugating machinery. Many of the disease-causing mutations in Parkin disrupt this fine balance between inhibited and activated Parkin.
The work was published online in EMBO Journal in August (followed the next month by a News & Views piece discussing the findings) and published in print (vol.34 issue 20) today, with an image depicting the dynamic rearrangements that occur to transition Parkin from an inhibited to active state chosen as the cover image.
Anetta Härtlova from the Trost group won the poster prize at the 2015 TOLL meeting in Marbella, Spain. The prize was given to her by Jules Hoffmann who was the 2011 Nobel Prize winner for the discovery of the Toll-like Receptor.
Congratulations to Anetta for this great achievement!
Saturday, September 19th marked Doors Open Day at the School of Life Sciences and the MRC PPU was in full force participating with two events geared at engaging the public and informing them about the research we do.
In The Wonder of the Brain, visitors of all ages were encouraged to learn more about the inner workings of the brain and had the opportunity to take part in numerous games. These included matching neurotransmitter ‘vesicles’ with the proper receptor, making thinking caps that reflected regions of their brain or neurons out of pipe-cleaner and included ‘train their brain’ exercises focused on memory and motor skills. This event was in conjunction with the Hearts and Minds exhibit occurring in the LifeSpace Gallery that included example of normal and diseased brains through Magnetic Resonance Images and illustrations that highlight changes that occur during neurodegeneration.
Help the Scientist Beat Cancer! was another event organized by the unit that encouraged children to learn about different kinds of cells (which they could make and take home) before moving on to discover what made normal cells different from cancerous ones – the latter which they worked diligently to ‘destroy’. This culminated in youngsters identifying creative ways to stop the ‘bad proteins’ that often turn healthy cells into cancer cells.
The goal was for visitors to come away with a better understanding and appreciation for the research that many of the volunteers are currently pursuing to help in the fight against cancer, neurodegeneration and other diseases. We undoubtedly achieved these goals many times over given the fact that both events had numerous visitors throughout the day and all had great questions about science.
This event would not have been possible without the help of the following volunteers and staff from the MRC PPU: Nicola Darling, Sonal Das, Federico Diez, Luke Fulcher, Juanma Ortiz-Guerrero, Katie Mulholland, Michael Munson, Angie Nicoll, Catherine Rodger, Laia Pedro-Roig, Grant Ross and Hannah Tovell.
Greg Findlay, Programme Leader at the MRC-PPU and Soo-Youn Choi, a postdoctoral researcher in the lab of Yogesh Kulathu have been awarded research grants from Tenovus Scotland.
Greg’s group has been investigating mechanisms controlling pluripotent stem cell (PSC) differentiation. This grant will enable a collaborative project between Greg’s team and the Dundee human PSC facility to investigate how small molecule inhibitors of BET bromodomains can be exploited to improve PSC differentiation protocols and elaborate new strategies for cell replacement therapy.
The Kulathu lab is interested in understanding how lymphocyte biology is regulated by ubiquitylation. This grant will enable Soo-Youn to investigate ubiquitin-dependent regulation of B cell proliferation. By analyzing B-cell specific knockout mice, Soo-Youn will study the role of ubiquitin networks in regulating B cell development and lymphomagenesis.
Agne Kazlauskaite, a PhD student in Miratul Muqit and Dario Alessi's labs, has been awarded the 2015 Dundee Prize for Cell and Molecular Biology for her seminal contributions to the mechanistic understanding of how the Parkinson’s disease associated kinase PINK1 activates the Parkin E3 ubiquitin ligase. In particular Agne’s work highlighted the critical role for PINK1-dependent phosphorylation of Parkin at the highly conserved residue, Serine 65 that lies within the N-terminal Ubiquitin-like domain and the phosphorylation of ubiquitin at the equivalent Serine 65 residue.
The Dundee Prize for Cell Biology is awarded for excellence in basic research recognising novel high impact discoveries of basic mechanisms or new methods that advance the field. Previous MRC-PPU recipients of the prize include Lina Herhaus (2013) and Anna Zagorska (2010) who were former PhD students in Gopal Sapkota and Dario Alessi’s labs respectively.
To read a copy of Agne’s paper describing the work for which the prize was awarded click here.
Hypertension is a major public health problem with an estimated 30% of the adult population in the UK suffering from the disease. If left untreated, hypertension turns into a “silent killer”, as it strongly increases an individual’s risk of heart disease or stroke.
Some forms of hypertension are hereditary and in recent years it was uncovered that mutations in enzymes of the ubiquitin-proteasome system cause a sub-type familial hypertension called Pseudohypoaldosteronism type II (PHAII). Defects in an E3 ubiquitin ligase complex consisting of the Cullin CUL3 and the substrate adaptor KLHL3 lead to a misregulation of a signaling pathway in the kidney that causes excessive retention of salt, which results in an increase in circulating blood volume and consequently high blood pressure.
Earlier work by the Kurz laboratory had established that the critical substrates of this ligase in regulating salt uptake are kinases of the WNK family. These enzymes are normally ubiquitylated by CUL3/KLHL3, which leads to their degradation by the proteasome. This regulation is critical to maintain salt homeostasis. Hypertension-causing mutations in KLHL3, the substrate adaptor of the complex, were previously shown by the Kurz group to lose binding to WNKs, leading to their ectopic stabilization. Some patients, however, carry mutations in CUL3 and the molecular mechanism of how these mutations lead to hypertension was unresolved.
Work by Frances-Rose Schumacher, a postdoc in the group of Thimo Kurz, now uncovered the molecular defects caused by these patient mutations. The mutations in CUL3 lead to increased structural flexibility that prevents the E3 ligase from directing ubiquitin towards its bound WNK substrates. Ubiquitin is instead erroneously linked to CUL3, causing its ectopic degradation. CUL3 furthermore loses interaction with important functional regulators, the COP9 Signalosome and CAND1.
Frances’ work further showed that mice carrying the human CUL3 mutations develop hypertension through the stabilization of WNK kinases, but the analysis of the mouse phenotype also revealed that there is likely an additional vascular contribution to hypertension. This novel finding may explain why patients with CUL3 mutation develop a more severe form of hypertension than patients carrying KLHL3 mutations.
This work was executed in a very fruitful collaboration with Keith Siew and Kevin O’Shaughnessy at the University of Cambridge and is reported in the most recent issue of EMBO Molecular Medicine.
Prof. Daan van Aalten, Programme Leader at the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU) based at The University of Dundee, has been focused on the function of a novel post-translational modification, O-GlcNAcylation, for the past decade. His group’s recent finding concerning the protein sequences targeted by this modification represent a significant advance in this field.
O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Dyregulation of this modification leads to defects during embryo development and has been linked to diabetes, cancer and neurodegenerative disease. Although the OGT enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, Shalini Pathak, a postdoc in Daan’s lab, used a high-throughput OGT assay on a library of peptides to demonstrate that the enzyme possesses sequence specificity. Jana Alonso, another postdoc in Daan’s lab then mapped the sites of O-GlcNAc modification by ETD-mass spectrometry. Using X-ray crystallography, Marianne Schimpl, another postdoc in Daan’s lab, then showed how these acceptor peptides bind to human OGT. Together this work suggests that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites, and defines an approximate “sequon”. This work has several implications. Understanding the specificity of the enzyme will help researchers in the field develop more potent inhibitors that will in turn help probe the role of O-GlcNAcylation in a number of processes and disease models. Furthermore, it remains challenging to detect O-GlcNAcylation by mass spectrometry and this work paves the way for more accurate bioinformatic prediction of O-GlcNAc sites.
The results of this study have been published in Nature Structural Biology in August 2015 (click here).
Dr. Helen Walden, Programme Leader of the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC PPU) based at The University of Dundee, has been focused on understanding the intricate molecular architecture of proteins for over 10 years. Her group’s recent finding around one particular protein – Parkin – is especially critical for the Parkinson’s disease research field.
Parkinson’s disease is a neurodegenerative disorder affects close to 5 million individuals worldwide, with the numbers set to increase as the population ages. Whereas the cause of Parkinson’s disease is uncertain in most cases, in approximately 5% of all cases, mutations in particular genes have been identified as playing a causative role in disease development. Since 1997, close to 20 genes have been found as playing a role in PD. Parkin is a protein encoded by the PARK2 gene and manifests as an autosomal recessive form of PD with a profile of younger onset. It is thought that research on the targets encoded by these genes may lend additional insight into those who suffer from PD where there is no known genetic link.
While it is important to understand the biological ramifications of any protein, without understanding the subtle alterations that are occurring on an atomic level, it can prove difficult to fully understand the biological findings. It is this intricate task that Dr. Walden and her lab has undertaken with regard to Parkin. And in her new publication, Dr. Walden has lent keen insight into with a high-resolution structure of the whole human form of the Parkin protein – an accomplishment that has to-date eluded other investigators who have largely focused on smaller ‘pieces’ of Parkin.
Past results from several laboratories around the world had demonstrated that Parkin activation relied upon two factors: 1) a special type of chemical modification called ‘phosphorylation’ and interaction with another small protein called ubiquitin, that is in turn phosphorylated.
But how this occurred and how the different parts of the Parkin protein contributed to these interactions was not well delineated. Until now, that is. Dr. Walden’s new findings lend insight into not only how Parkin is activated, but suggest a sequence of events that are critical to that activation.
Dr. Walden’s work has far reaching implications for the pharmaceutical industry as they work towards identifying a target that may be amenable to small molecular manipulation. Parkin further represent a potential target that if successfully activated, could alter the course of progression of Parkinson’s disease. This work was done jointly with Gary Shaw's Lab at the University of Western Ontario.
The final results of these studies have been published in EMBO Journal in August, 2015.
Luke Fulcher, who recently graduated from University of Dundee with a BSc in (1st class) in Physiological Sciences, is due to start a PhD in the MRC PPU this coming August. Luke’s has just been awarded The Queen’s College Scholarship Award from University of Dundee, a prestigious postgraduate research scholarship awarded once every three years to a student of exceptional academic excellence. The award provides Luke’s academic fees along with an annual stipend. Luke will start his PhD with a rotation in the Ganley Lab to study how the protein kinase ULK1 regulates autophagy initiation. Ian said: “We are delighted that Luke chose to do his PhD in the Unit. It was clear from meeting Luke during his interview that he is a very talented individual with a keen and dedicated interest in the biochemistry of how cells function. He is fully deserving of this award. I’m excited that he chose to do his first rotation in my lab and am looking forward to working with Luke to decipher the mechanisms of autophagy”.
The lysosome acts as the stomach of the cell in that it digests and recycles unwanted, damaged or toxic cellular components. This is a vital process for the cell and prevents accumulation of “junk” that can have devastating consequences as is becoming evident in diverse diseases that encompass neurodegeneration and cancer. Material is constantly being delivered to the lysosome by fusion with membrane compartments such as autophagosomes and endosomes, yet in-spite of this, the lysosome retains its identity and does not increase dramatically in size due to all this membrane fusion. Therefore there must be a mechanism whereby membranous material is transported back out of lysosomes. Recent work from the Ganley lab has helped shine light on the signals that allow this process, termed Autophagosome-Lysosome-Reformation (ALR), to proceed.
Michael Munson, during his PhD studies in the Ganley Lab, uncovered that the lipid kinase VPS34 plays a vital part in allowing tubules, which transport out the excess membranes, to break away from the lysosome during ALR. Michael was able to show that not only was VPS34 important, but that it was in turn activated by another kinase, the master growth regulating mTOR protein kinase. This regulation involved direct mTOR phosphorylation of the protein UVRAG, present in a subset of VPS34 protein complexes, and loss of this phosphorylation only seemed to alter ALR. Given that VPS34 is involved in a myriad of other processes, this highlights the specificity that can be achieved in signalling.
The mTOR signalling pathway is often upregulated to drive cell growth in carcinogenesis, so Michael next asked if disruption of ALR, through loss of the UVRAG mTOR phosphorylation sites and concomitant block in lysosomal tubule scission, could alter cell growth. Surprisingly, loss of this phosphorylation resulted in rapid death when cells were stressed by starvation, likely by destabilization of the lysosome and leakage of its destructive hydrolases into the cytosol. Importantly, this suggests that ALR may be a good pathway to target in tumour cells, which are often in a state of starvation caused by their rapid cell growth. Future work will help build on this exciting possibility.
To read a copy of Michael’s paper, published in EMBO Journal, click here.
Alban Ordureau, a former student and postdoc in Philip Cohen's lab in the MRC-PPU (2007-2012) has been awarded a prestigious Lefler Fellowship to continue his research at Harvard medical School, Boston, USA, which is aimed at clarifying the molecular mechanisms underlying Parkinson’s disease.
The Lefler Fellowship was established by the Department of Neurobiology at Harvard Medical School to provide support for the training of the most promising pre-doctoral students and postdoctoral fellows working on projects relevant to neurodegeneration and neurodevelopment.
Over the past two and half years, while working and as a post-doctoral fellow in the laboratory of Wade Harper at Harvard Medical School, Alban has used state-of-the-art mass spectrometry and biochemistry techniques to uncover a feed-forward mechanism that regulates the activation of the E3 Ubiquitin Ligase Parkin by the protein kinase PINK1. This pathway, which is triggered by mitochondrial damage, is defective in Parkinson’s disease. His studies, together that of other laboratories, including those of Miratul Muqit and Helen Walden in the MRC-PPU at Dundee, has shed new light on how the Parkin E3 ubiquitin ligase is activated by PINK1 and how the phosphorylation of ubiquitin chains retains Parkin on damaged mitochondria, allowing it to catalyse the ubiquitylation of mitochondrial outer membrane proteins to promote the removal of damaged mitochondria via mitophagy.
So far, our understanding of the PINK1-Parkin pathway has been based on studies with immortalized tissue culture cells engineered to express the normal and mutant forms of Parkin. There has been little attempt thus far to examine this biochemical pathway in a quantitative manner in primary neurons. With support from the Lefler Fellowship, Alban will pursue his quantitative analysis of the PINK1-Parkin pathway to systematically explore Parkin’s activation mechanisms and the role of ubiquitin phosphorylation in neuronal mitophagy.
Trametinib, a drug that inhibits the protein kinase MEK, has been found to increase the lifespan of fruit flies by 10%. If the drug has a similar effect in homo sapiens then it might prolong life by as much as 10 years, causing this discovery to make the international news headlines a few days ago. The work was carried out in the laboratory of Linda Partridge, which is based at University College London and at the Max Planck Institute for Biology of Ageing in Cologne, Germany. The research was published in the June 25th 2015 issue of the journal Cell.
MEK, the protein kinase targeted by Trametinib, was discovered in Philip Cohen's laboratory in the MRC-PPU, Dundee over 25 years ago, while they were studying the mechanism by which Nerve Growth Factor induces the differentiation of neurons. They found that the mitogen-activated protein (MAP) kinases, termed ERK1 and ERK2, were switched on by another kinase in NGF-stimulated cells, which they purified and characterized and termed MAP kinase kinase1-4. The protein kinase was later renamed MEK and shown to be activated by yet another protein kinase called RAF5,6. The Cohen lab subsequently found that neuronal differentiation required the sustained activation of MEK, ERK1 and ERK2, explaining why it was induced by NGF but not by EGF7,8.
Trametinib was developed for clinical use in cancer by GlaxoSmithKline and approved for the treatment of malignant melanoma in 2013. The combination of Trametinib with the Raf inhibitor Dabrafenib later proved to be even more effective and to delay the onset drug resistance considerably and was approved in 2014. These drugs were key components of GlaxoSmithKline’s oncology drug portfolio, which they sold last year to Novartis for US$9.1 billion.
The story of Trametinib shows once again the importance of carrying out basic fundamental research on important biological questions, and how it can lead to completely unforeseen consequences over a quarter of a century later.
1. Gomez et al, 1991, Nature 353, 170-173; 2. Nakielny et al, 1992, EMBO J. 11, 213-2129; 3.Nakielny et al, 1992, FEBS Lett. 308, 183-189; 4. Ashworth et al, 1992, Oncogene 7, 25555-25556; 5. Howe et al, 1992, Cell 71, 335-342; 6. Kyriakis et al, 1992, Nature 358, 417-421;
7. Traverse et al, 1992, Biochem. J. 288, 351-355; Traverse et al, 1994, Curr. Biol.4, 694-701
On Friday, June 26, 2015 The Academy of Medical Sciences announced its inaugural group of participants for its SUSTAIN programme, a new programme that will work with women researchers to enable them to thrive in their independent research careers. Esther Sammler, a clinical lecturer in Neurology from Dario Alessi’s group is amongst the first 20 participants in SUSTAIN. Esther, and all the participants, will be provided with an innovative programme of training and support to develop their leadership and career potential.
In an endeavour to combat the decline of female researchers progressing to senior roles in science, the Academy is focusing efforts to reverse this trend, through SUSTAIN, and ensure women in research are supported along their career to enable them to secure senior and leadership positions: SUSTAIN aims to provide training, mentoring and peer networking to help facilitate the advancement of women working in this field.
Esther was pleased to be selected: “I am absolutely delighted to be able to participate in this amazing scheme. Success and failure in clinical research are certainly not all about gender, but combining my clinical training in neurology and the demands of a busy clinical service with research in the Alessi group and a family with 2 small children can be quite challenging. I am expecting the SUSTAIN programme to help me gain and keep the momentum I need to succeed as a clinical academic and to remind me that I am not the only one facing these tasks.”
Professor Alessi was similarly enthusiastic: “I have every confidence that given Esther’s dedication and now with her selection in the SUSTAIN programme, and the kind of support it provides, that this will undoubtedly accelerate her advancement in both a clinical and research setting here in Dundee.”
Understanding the mechanisms of Parkinson’s disease remain a major biomedical challenge since the disease is on the rise and there remain no treatments that can cure or slow it down.
Over the last few years research in the Muqit lab has been focused on understanding how mutations in a previously obscure protein kinase PINK1 (PTEN-induced kinase 1) leads to early-onset Parkinson’s disease. Chandana Kondapalli (a former PhD student) made a major advance in this question through her discovery that PINK1 can phosphorylate Parkin at Serine 65 (Ser65) that lies within the N-terminal Ubiquitin-like domain (Ubl) of Parkin.
J. Macdonald Menzies Prize PhD student, Agne Kazlauskaite, (supervised by Miratul Muqit and Dario Alessi) then demonstrated that phosphorylation of Parkin at Ser65 was critical for activation of Parkin E3 ligase activity. Last year Agne made the unexpected finding that PINK1 also phosphorylated ubiquitin at Ser65 and elegantly demonstrated that both PINK1-dependent phosphorylation of Parkin at Ubl Ser65 as well as ubiquitin at Ser65 is required for optimal activation of Parkin E3 ligase activity. Agne was able to publish her findings first ahead of two competing groups – the laboratories of Richard Youle and Noriyuki Matsuda – who independently came to similar conclusions.
However, an outstanding question was how Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributed to Parkin activation? In new work Agne, together with collaborators in the MRC-PPU and Division of Biological Chemistry and Drug Discovery (BCDD) have made fundamental insights that shed light on this question. Agne initially made the exciting discovery that ubiquitinPhospho-Ser65 dramatically increases the rate by which Parkin Ubl Ser65 is phosphorylated by PINK1.
Analysis of the Parkin structure by Daan van Aalten suggested a candidate phosphate-binding pocket via which ubiquitinPhospho-Ser65 might bind Parkin. Through a series of mutagenesis and ubiquitylation experiments Agne was able to determine that two residues, Histidine 302 (His302) and Lysine 151 (Lys151) play a key role in ubiquitinPhospho-Ser65 mediated phosphorylation and activation of Parkin by PINK1.
Next Julio Martinez-Torres and Atul Kumar from Helen Walden's lab and Axel Knebel undertook binding studies to demonstrate that mutation of His302 and Lys151 markedly disrupted binding of ubiquitinPhospho-Ser65 to Parkin suggesting that these residues likely line the ubiquitinPhospho-Ser65 binding pocket. Elegant analysis by Scott Wilkie and Tony Hope from the BCDD demonstrated that binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction of the Ubl domain to the C-terminus of Parkin that would be predicted to make the Ubl Ser65 site more accessible to PINK1.
Finally Agne was able to confirm in cells that a Parkin His302 mutant was unable to be maximally phosphorylated at Ubl Ser65 by PINK1 leading to impaired activation of E3 ligase activity suggesting a critical role of ubiquitinPhospho-Ser65 in priming Parkin for phosphorylation and activation at mitochondria.
Agne’s new findings provide fundamental mechanistic insights into how PINK1 and phospho-ubiquitin activates Parkin. Her findings will also aid in the development of Parkin activators as a promising class of drugs for treating Parkinson’s disease.
To read a copy of Agne’s paper published in EMBO Reports click here.
Congratulations to Julien Peltier and Matthias Trost for receiving the Thermo Scientific TMT Research Award at the 2015 annual meeting of the American Society of Mass Spectrometry (ASMS) last week in St. Louis, Missouri. This prize was awarded for a research proposal led by Julien Peltier in Matthias’ group using the novel Tandem Mass Tag (TMT) for the identification of drug substrates by a new method, called “Cellular Thermal Shift Assay” (CETSA) using mass spectrometry. The award provides $5,000 of consumables and free access to software licenses to the Trost group.
Congratulations are due to Marija Maric, who has just been awarded the Pontecorvo Prize for 2014, for her discovery that the end of chromosome replication is regulated by ubiquitylation and the p97 ATPase. Marija completed her PhD studies last year in Karim Labib's group, and was funded by Cancer Research UK (CRUK).
The Pontecorvo Prize is a prestigious national award for the best PhD thesis supported by CRUK each year. The panel were particularly impressed by Marija’s paper in the journal Science last year, (Maric et al, 2014, Cdc48 and a ubiquitin ligase drive disassembly of the CMG helicase at the end of DNA replication - click here to read), which opens up a whole new area of research into how the final stages of chromosome replication are controlled by ubiquitylation.
Chromosome replication is regulated in an exquisite fashion, in order to ensure that it only occurs once per cell cycle, thus allowing the stable inheritance of the genome from one generation to another. At the heart of this regulation is the DNA helicase that unwinds the parental DNA duplex. The helicase can only be assembled on its DNA substrate once per cell cycle, as cells enter S-phase, and the helicase then remains stably associated with DNA replication forks until the replication process has been completed. Marija found that the helicase is then ubiquitylated on just one of its 11 subunits, leading to a disassembly reaction that requires the Cdc48/p97 segregase. This work raises many important questions for future studies of the mechanism and regulation of chromosome replication in eukaryotic cells. It will be interesting to identify the ubiquitin ligase that controls the DNA helicase in human cells, with the ultimate aim of testing whether inhibition of this ligase might selectively kill cancer cells.
The Pontecorvo prize is associated with an honorarium and a free place at the next conference of the National Cancer Research Institute. Marija will also be expected to give a talk about her work at the next national CRUK student meeting. The funding for the prize was provided by Professor Peter Goodfellow FRS, and the award is named after the geneticist Professor Guido Pontecorvo, who worked at the Lincoln’s Inn Field laboratories of CRUK’s London Research Institute from 1968-1975. Marija will remain with Karim’s group at the MRC-PPU until later this year, before starting a postdoc in Simon Boulton’s lab at the Francis Crick Institute.
Miratul Muqit, Neurologist and Programme Leader at the MRC-PPU, has today been awarded over £90,000 by Parkinson's UK to fund research which could shed light on the molecular mechanisms underlying Parkinson’s.
Across Scotland, Parkinson’s affects about 10,000 people, yet there remains no cure. Advances in genetics have identified nearly 20 genes that are mutated in patients with familial forms of Parkinson’s and this has paved the way for dissecting the crucial pathways disrupted in Parkinson’s.
Miratul’s group has been investigating how mutations in the PINK1 gene contribute to neurodegeneration in Parkinson’s. In the last few years his group has identified the E3 ligase Parkin and ubiquitin as substrates for PINK1 and demonstrated that the phosphorylation of these substrates at an equivalent residue Serine 65 leads to maximal activation of Parkin E3 ligase activity. This signalling pathway appears to play a vital role in protecting cells from mitochondrial damage.
Over the next three years, Miratul’s team will employ state-of-the-art technologies to identify new substrates and cellular pathways regulated by PINK1. It will be exciting to assess whether these are also dependent on Parkin phosphorylation and whether they are disrupted in cells from patients with Parkinson’s.
“In a cell you will see thousands of pathways, but we want to find the crucial ones. PINK1 is a gene that causes changes in the cells and we want to know if these changes disturb an essential pathway. We hope to create a road map of the pathways vital for the survival of brain cells. This way we can better understand how to diagnose and treat the condition.”
Dr Arthur Roach, Director of Research and Development at Parkinson’s UK, said:
“Understanding the role of genes in Parkinson’s could be the key to discovering what causes some people to develop Parkinson’s, something that has remained unknown for so long. Funding projects like this in Dundee is crucial if we are to progress in our research and one day find a cure.”
Please click here for a video interview with Miratul, and here for his interview with Tay2 radio.
A collaborative effort spearheaded by Alejandro Rojas-Fernandez and Lina Herhaus, from the Hay (GRE) and Sapkota (MRC-PPU) labs respectively, has led to the publication of a methodology paper in Scientific Reports for rapidly generating endogenously driven transcriptional reporters in cells through CRISPR/Cas9. Thomas Macartney (DSTT) and Christophe Lachaud (MRC-PPU) also made significant contributions.
CRISPR/Cas9 technologies are increasingly being exploited for genome editing to achieve gene knockouts and knock-ins in somatic cells. Alejandro and Lina chose to employ this technology to engineer a simple, sensitive and robust transcriptional reporter system driven by the endogenous promoter. Such a system is more desirable over the conventional artificial transcriptional reporters that critically lack the endogenous chromatin context and regulatory components.
In the published report, Alejandro and Lina describe a CRISPR/Cas9-based methodology for rapidly integrating the firefly luciferase gene at the locus of TGFβ-responsive gene PAI-1 in somatic cells. For this, they employed a polycistronic cassette containing the luciferase gene and a non-fused green fluorescence protein (GFP) to ensure the detection of transgene delivery and rapid isolation of positive clones. They demonstrate that firefly luciferase can be efficiently delivered downstream of the promoter of the PAI-1 gene in osteosarcoma U2OS cells.
TGFβ signalling pathway controls a plethora of cellular functions during embryogenesis and in adult tissues through regulation of transcription. Using the engineered U2OS cells with firefly luciferase gene integrated at the PAI-1 locus, Alejandro and Lina verify that known chemical and genetic regulators of TGFβ signalling impact the reporter activity in analogous manner to the transcriptional regulation of endogenous PAI-1 expression.
The unique approach described in this study has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.
The paper, entitled “Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9” can be accessed here.
(If you think the methodology described will help your studies, please contact email@example.com or firstname.lastname@example.org for reagents and advice)
The Wellcome Trust has awarded Miratul Muqit an Enhancement Award to support a Postdoctoral Fellowship to investigate molecular mechanisms of Parkinson’s disease.
The 3-year fellowship will build on ground breaking studies from the Muqit lab on the regulation and downstream function of the PINK1 kinase that is mutated in Parkinson’s. This led to the discovery that PINK1 targets ubiquitin to generate a novel chemical messenger molecule phospho-ubiquitin.
The successful candidate will deploy state-of-the-art technologies to investigate how PINK1 activation and phospho-ubiquitin are critical for protecting against Parkinson’s disease.
For information on how to apply please click here.
The deubiquitylase OTUB1 is ubiquitously expressed and is known to impact key cellular processes, from TGFβ and p53 signalling to DNA damage repair, by targeting a multitude of substrates both in the cytoplasm and nucleus. Until now it was not known how OTUB1 navigated around the subcellular compartments.
While working in Gopal Sapkota's lab, Lina discovered that OTUB1 was phosphorylated by casein kinase 2 (CK2) at Ser16. She showed that this phosphorylation did not affect the catalytic activity of OTUB1 and its ability to bind to ubiquitin chains and the E2 ubiquitin-conjugating enzyme UBE2N. Instead she discovered that the phosphorylation of OTUB1 at Ser16 was critical for its localisation to the nucleus. She was able to demonstrate that mutating CK2 phosphorylation site on OTUB1 or pharmacologically inhibiting CK2 caused complete nuclear exclusion of OTUB1.
OTUB1 is known to be involved in the repair of double stranded DNA damage. Together with Ana Perez-Oliva, Lina was able to show that mutating the CK2 phosphorylation site on OTUB1 or pharmacologically inhibiting CK2 impaired DNA repair in cells exposed to ionizing radiation, which causes double stranded DNA damage. Inability of cells to repair DNA damage causes genomic instability, which can lead to cell death or cancer. As ionizing radiation is used therapeutically to kill cancer cells, these findings may potentially be exploited to sensitize cancer cells to ionizing radiation.
These findings are now published in Science Signaling. There is an editor’s summary of the paper here.
Robert Gourlay, Simone Weidlich and David Campbell from the MRC-PPU and collaborators Giorgio Cozza and Lorenzo Pinna from University of Padova (Italy) also contributed to this study.
Lina successfully defended her PhD thesis on 15th October 2014 and was awarded an EMBO fellowship to undertake postdoctoral research in Prof. Ivan Dikic’s laboratory in Frankfurt (Germany), where she is currently investigating the role of ubiquitylation on tumor-stroma crosstalk.
Autophagy is a cell protective pathway that delivers unwanted, damaged or toxic components to the lysosome for degradation and recycling. It essentially prevents the cell from becoming a junkyard. Under most circumstances autophagy is a very beneficial intracellular pathway, however in cancer the situation is more complicated. In tumour cells autophagy is still thought to act in a protective manner, helping the cancer cells to survive the harsh conditions of tumourigenesis and the damage caused by chemotherapy. Therefore inhibiting autophagy in tumour cells is thought to be a promising approach to treat cancer. Unfortunately, there are currently no specific small molecule inhibitors of autophagy to test this hypothesis.
ULK1 is a serine/threonine protein kinase that is essential for autophagy induction. Katy Petherick and Owen Conway, a postdoc and undergraduate student in the Ganley Lab, together with collaborators from MRC Technology, investigated whether ULK1 could be targeted to block autophagy. They found that two compounds, MRT67307 and MRT68921, potently inhibited ULK kinase activity in vitro and blocked autophagy in cells. Importantly, Katy was able to engineer a drug-resistant ULK1 mutant that rendered cells insensitive to inhibitor treatment. This proved for the first time that autophagy can be blocked in cells by specifically targeting ULK1 with a drug.
The study provides a vital step in validating ULK and autophagy inhibition as a potential treatment for cancer. To read a copy of Katy’s paper that is currently in-press, click here
Ubiquitylation regulates diverse cellular processes and this versatility is made possible because polyubiquitin chains of eight different linkage types can be assembled. Differently linked polyubiquitin chains couple to functionally distinct outcomes. For instance, proteins modified with K48-linked polyubiquitin are targeted for proteasomal degradation. Until now, nothing was known about K29 and K33 polyubiquitin chains and there weren’t any methods to study them. Research carried out by Yosua Kristariyanto, a PhD student in Yogesh Kulathu's group, now provides exciting insights into two these two unstudied polyubiquitin modifications.
In two new papers just published in Molecular Cell and Biochemical Journal, Yosua together with Soo-Youn Choi have identified ubiquitin chain editing complexes, that combine E3 ligases with linkage selective deubiquitinases (DUBs), to enzymatically assemble K29 and K33 polyubiquitin chains. Importantly, these methods allow large-scale assembly using wild type ubiquitin yielding pure polyubiquitin chains, which will enable research into these uncharacterized modifications. For the first time, polyubiquitin tetramers of seven different linkage types could be assembled, which enabled Yosua to screen linkage selectivity of ubiquitin binding domains (UBDs). This led to the discovery of the NPL4-like zinc finger (NZF) domains of TRABID as selective binders to K29 and K33 chains.
To understand the structural basis of selective recognition, Yosua with the help of Syed Arif Abdul Rehman, a postdoc in Kulathu lab, solved crystal structures of K29-linked diubiquitin, K33-linked diubiquitin and triubiquitin, and the first NZF of TRABID (NZF1) in complex with K29-linked diubiquitin. Intriguingly, in the crystal structure of the complex, K29 chains adopt a helical filament-like structure and further studies are required to address if such filamentous structures are formed in cells and what roles they may have. The structure of the complex also explains how the tandem NZF domain repeats of TRABID may recognize long K29 and K33 chains. Through detailed biochemical and biophysical analyses, the mechanisms underlying polyubiquitin recognition by NZF domains are revealed and highlights how despite their small size, NZF domains have evolved distinct mechanisms to achieve linkage-selective polyubiquitin recognition.
Taking these findings further, Yosua then developed methods to study K29 and K33 polyubiquitin chains in cells. Using the newly discovered linkage-specific binders as affinity reagents reveals that K29 chains are made in cells as short polymers (2-4 ubiquitin moieties). Intriguingly these short K29 chains are found within polyubiquitin containing other linkages. This is an emerging theme in the ubiquitin field that heterotypic polyubiquitin chains containing multiple linkages can be formed and function as specialised signals. The heterotypic nature of polyubiquitin adds to the complexity of the already information rich ubiquitin system, and will require novel approaches to study them.
To read a copy of Yosua’s papers that have just been published, click here (K29) and here (K33).
Ana Perez-Oliva a postdoc working in the Alessi lab on a collaborative project with Ian Hickson at Janssen Research & Development, was interested to explore the function of an unstudied deubiquitylase enzyme termed USP45 that is overexpressed in a significant number of human cancers.
This led Ana to uncover that the endogenous USP45 robustly co-immunoprecipitated with components known to play a major role in controlling DNA damage responses including ERCC1, XPF and SLX4 that previous work in John Rouse’s lab had revealed form a complex. Ana then collaborated with Christophe Lachaud and Ivan Muñoz, postdocs in John Rouse’s lab, to demonstrate that USP45 interacts directly with ERCC1 and not XPF or SLX4.
Ana also identified a short highly conserved acidic motif lying within the N-terminal non-catalytic region of USP45 that she demonstrated was essential for interaction with ERCC1. Ana pinpointed specific point mutations within this motif that ablated the ability of USP45 to associate with ERCC1, XPF and SLX4.
Ana next found that levels of ubiquitylated endogenous ERCC1 are markedly enhanced in USP45 knock-out cells that were generated by Piotr Szyniarowski. Moreover, in vitro studies, Ana was able to show that wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, could efficiently deubiquitylate ERCC1. This suggests that ubiquitylated ERCC1 comprises a direct physiological substrate for USP45
Ana, Christophe, and Ivan, were then able to demonstrate that cells lacking USP45 are markedly hypersensitive to UV irradiation and other agents that induce DNA interstrand crosslinks, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage was markedly reduced in USP45 knockout cells. The translocation of ERCC1 to sites of DNA damage-induced subnuclear foci was also markedly impaired in USP45 knock-out cells.
USP45 was also found to localise to sites of DNA damage in a manner dependent upon deubiquitylase activity of USP45, but independent of its ability to bind ERCC1-XPF. This suggests that USP45 might play other roles or possess additional interactors or substrates at sites of DNA damage that need to be uncovered in future work.
In future work it will be important to understand how ERCC1 ubiquitylation is controlled and what its function is. It would also be interesting to explore whether inhibiting USP45 could be employed as a therapeutic strategy to induce sensitisation of cancer cells to chemotherapeutic platinum-based therapies that suppress growth and induce death of cancer cells by inducing DNA interstrand crosslinks. Moreover, if cancer patients displaying loss of function mutations in USP45 could be identified, the prediction would be that they would be sensitised to platinum based therapy. It may also be interesting to screen unclassified diseases caused by inefficient repair of interstrand DNA crosslinks where no gene has been assigned, such as Fanconi anemia patients, for mutations in USP45.
These data suggest that USP45 acts an important new regulator of XPF-ERCC1, crucial for efficient DNA repair. To read a copy of the paper describing this work click here.
Research in the Alessi lab has been focused on understanding how mutations that truncate the C-terminal non-catalytic moiety of a protein kinases termed TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, spinocerebellar ataxia type 11 movement disorder.
In the course of the work Noor Esoof (PhD Student) and Ning Zhang (Postdoc) discovered that the Synaptic-vesicle-protein-2A (SV2A) which is a ubiquitous component of synaptic vesicles was efficiently phosphorylated by TTBK2 as well as related Casein kinase-1 family members.
Phosphorylation analysis indicated that TTBK2 and related kinases phosphorylated human SV2A at two constellations of residues; namely Cluster-1 (Ser42, Ser45 and Ser47) and Cluster-2 (Ser80, Ser81 and Thr84). Ning Zhang was also able to demonstrate that residues are also phosphorylated in endogenous SV2A in brain.
Excitingly, work carried out by Noor, Ning and Maximilian Fritsch (Postdoc) discovered that phosphorylation of Thr84 within Cluster-2 functions to trigger binding to the C2B domain of synaptotagmin-1, which is the key Ca2+ sensor for evoked synchronous neurotransmitter release at the synapse.
Working with Daan van Aalten, Maximilian Fritsch solved the crystal structure of the C2B domain of synaptotagmin-1 bound to an SV2A peptide phosphorylated at Thr84. This revealed that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326 and Lys328) on the surface of the synaptotagmin-1 C2B domain.
In a series of very elegant experiments undertaken by Michael Cousin and Sarah Gordon at the University of Edinburgh, they were able to demonstrate that synaptotagmin-1 retrieval during SV endocytosis was markedly dysfunctional when interaction of phosphorylated Thr84 with the C2B domain of synaptotagmin-1 was disrupted in primary cultures of mouse neurons.
This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1, and implicates SV2A as a phospho-dependent scaffold required for the specific retrieval of synaptotagmin-1 during synaptic vesicle endocytosis.
In future work it will be important to establish whether its TTBK2 or a related kinases that phosphorylates SV2A and whether disruption of this pathway is associated with spinocerebellar ataxia or any other movement disorder. It will also be important to understand the roles of cluster 1 and cluster 2 phosphorylation in greater detail. Knock-in mice in which phosphorylation sites have been ablated have been generated and these will be useful in enabling us to address this question.
To read the paper describing this work click here
Marija Maric, who just completed her PhD studies in Karim Labib's group, has received the 2014 Howard Elder Prize for her discovery that the end of chromosome replication is regulated by ubiquitylation. Marija’s award-winning PhD work was supported by Cancer Research U.K. and the MRC, leading to the publication of a research article in Science last October.
The DNA helicase that controls the progression of eukaryotic DNA replication forks is regulated in a highly sophisticated fashion, to ensure that cells can only produce a single copy of each chromosome per cell cycle. Helicase assembly during the initiation of chromosome replication is driven by two protein kinases that phosphorylate conserved assembly factors, but until now very little was known about the opposite process by which the helicase is disassembled at the end of DNA replication. Marija showed that the DNA helicase in budding yeast is specifically ubiquitylated on one of its 11 subunits, leading to a disassembly reaction that requires the Cdc48/p97 segregase. This work is a major step forward for the chromosome replication field and raises many questions for future studies in the coming years. At present it is not known how ubiquitylation of the helicase is restricted to the final stages of chromosome replication. Reconstitution of the disassembly process will also be a major challenge for the future, in order to elucidate each step of the mechanism. Finally it will be of great interest to identify the ubiquitin ligase that might regulate the DNA helicase in human cells, to see whether inhibition of this ligase might selectively kill cancer cells.
The Howard Elder Prize was endowed by Dr Alison Burt 25 years ago, in memory of her father Dr Howard Elder, who was a former medical graduate of the University of Dundee. The prize is awarded to a PhD or postdoctoral researcher who is deemed to have published the most significant paper in an area related to cancer research. Marija will remain in Karim’s group at the MRC PPU until the summer of 2015, before leaving to start her postdoctoral work.
Marija is the 5th MRC-PPU researcher to be awarded the Howard Elder prize. The previous awardees were Xu Huang 2008, Elton Zeqiraj 2009, Craig MacKay 2010 and Kumara Dissanayake 2011.