Our Twelfth MRC PPU alumni interview is with ex-PPU PhD student Alban Ordureau.
Click the MRC PPU Alumni Interview icon at the top of our website, or here to read the interview.
We are delighted to announce that from December 1, Mahima takes up a Programme Leader position in the MRC PPU to better study the function and regulation of intraepithelial lymphocytes. These are a class of poorly characterised innate-like T cells that reside in the intestinal epithelium. An important focus of Mahima’s laboratory will be to understand how intraepithelial lymphocytes are activated and to define which signalling pathways are involved.
Mahima studied Biological Sciences and Biotechnology at the Birla Institute of Technology and Sciences (BITS), Pilani in Rajasthan, India. She moved to the Max-Planck Institute of Immunobiology (MPIIB) in Freiburg, Germany to do her PhD. In Wolfgang Schamel’s lab, Mahima solved the long-standing questions of the stoichiometries of the T cell antigen receptors (TCR) complexes, and defined a novel paradigm for how the TCR is activated.
For her postdoc, Mahima moved to CRUK’s London Research Institute, where she commenced her research on intraepithelial lymphocytes in Adrian Hayday’s lab. Funded by both EMBO (2009) and Marie-Curie (2010-12) postdoctoral fellowships, she worked to dissect the role of an intestinal epithelial surface molecule, Btnl1, in immune surveillance. During this time, she also showed a major role for intraepithelial lymphocytes in promoting resistance to intestinal viruses.
In 2013 Mahima moved to the School of Life Sciences at the University of Dundee to focus on her two research interests – signal transduction and immunology. As an independent investigator with Doreen Cantrell, she uncovered roles for the novel post-translational modification, O-GlcNAc, in T cell lymphomagenesis and T cell development. In parallel, she continued to explore the interactions between intraepithelial lymphocytes and intestinal epithelial cells, using powerful mass-spectrometry based approaches to identify novel molecular interactions.
Mahima is very excited about setting up her own laboratory and commented “It’s a great opportunity to begin my lab in this dynamic and stimulating research environment.” Dario Alessi, Director of the MRC-PPU stated “we are delighted that we have been able to attract Mahima to the MRC-PPU. Understanding the function of intraepithelial lymphocytes which continuously patrol our intestinal cells will reveal new biology of relevance to better understanding and treating diseases such as intestinal immune disorders and cancer. Mahima also has exciting plans to dissect the signalling pathways that control intraepithelial lymphocyte immune responses that tie in nicely with a lot of other research that is on-going in the MRC-PPU.”
For more information on Mahima and her research interests check out her webpages on the MRC-PPU website by clicking here.
Understanding the signalling mechanisms of Parkinson’s disease is a major area of research for researchers at the MRC PPU. Over recent years several projects have received major funding from Parkinson’s UK, the biggest charity for Parkinson’s in the country. A group of Parkinson’s patients and carers visited the unit last week to explore ways to best share information about the on-going PPU research. This meeting moderated by Hazel Lambert, who contributes to Public Engagement activities at the PPU, essentially asked the Parkinson’s Group how they’d like to hear more about research.
The meeting was attended by two of the PPU’s clinical researchers who are also hospital consultants specialising in movement disorders, Group Leader Dr Miratul Muqit and Consultant neurologist and AHSP Clinical Fellow, Dr. Esther Sammler. Mary Ellmers, Parkinson’s UK’s Service Improvement Advisor for Scotland, and Professor Dario Alessi also attended.
Topics for discussion included the value of laboratory tours and of visiting spaces where research takes place; the opportunity to ask scientists questions and also to learn about wider research areas, not just those related to Parkinson’s. The group were positive about the PPU video on signalling that explains the Unit’s research in a nutshell and remarked that the graphic illustrations made a huge difference in their ability to follow discussions about complex science.
Given the expertise in Parkinson’s related research present in the PPU and more widely in Dundee it was decided to form a new Research Interest Group for Tayside (Tayside-RIG), supported by the Unit. The aim would be for the Tayside-RIG to meet several times a year and invite scientists from MRC PPU and the wider research community to share their science. The PPU very much looks forward to working with Tayside-RIG in the future.
We are organising a 'Dundee-Crick' Signalling meeting on 12-14 November 2017, that will take place at the Francis Crick Institute in London.
In addition to speakers from MRC PPU, Dundee School of Life Sciences, and the Crick Institute we will have a number of other renowned speakers including Vishva Dixit (keynote Genentech), Henning Walczack (UCL), Mike Dustin (Oxford), Gillian Griffiths (Cambridge), John Bertin (GlaxoSmithKline), Darren Cross (AstraZeneca), Fiona Watt (KCL), Richard Marais (CRUK Manchester) and Ultan McDermot (Sanger)
We will also have a number of short talks that will be selected from submitted abstracts.
For further information of how to register for meeting, submit an abstract and programme click here.
Tom Deegan, who has been working as a postdoctoral fellow in Karim Labib's laboratory since May 2015, has been awarded a Sir Henry Wellcome Postdoctoral Fellowship (£250,000 over 4 years).
This is one of the most highly sought after postdoctoral fellowships in the UK. It offers the most talented recently qualified postdoctoral researchers a unique opportunity to start an independent research career, “working in some of the best research environments in the world”.
Tom undertook his PhD research in John Diffley’s group at the Clare Hall Laboratories of Cancer Research UK’s London Research Institute. Whilst here, he utilised a novel reconstituted DNA replication system that uses 42 purified proteins from budding yeast to define the role of the Sld3 protein during the initiation of DNA replication.
Tom is now taking steps to extend the in vitro DNA replication system in new directions, to dissect the mechanism and regulation of DNA replication termination. Tom also plans to collaborate with Steve Kowalczykowski in California and Ian Hickson in Copenhagen during the course of his project.
Tom stated "I am delighted to have been awarded this fellowship from the Wellcome Trust, and am excited by the challenge of pursuing an independent research programme during my postdoc. I would especially like to thank Karim Labib for his support during the application process, and look forward to continuing to work with him on this project.”
Dario Alessi commented “the research that Tom is undertaking that involves reconstituting a complex biological system requiring over 60 recombinant proteins, is at the cutting edge of biological investigation. I am delighted that Tom was able to secure this very prestigious fellowship that required him to defend his proposed work at an interview in front of an illustrious panel of scientists. I am confident that Tom’s unique experience and ability will help him drive our understanding of the fundamental mechanism of how DNA replication termination is controlled.”
Our eleventh MRC PPU alumni interview is with ex-PPU postdoc Mabi Jaleel.
Click the MRC PPU Alumni Interview icon at the top of our website, or here to read the interview.
Our tenth MRC PPU alumni interview is with ex-PhD student Guadalupe Sabio.
Click the MRC PPU Alumni Interview icon at the top of our website, or here to read the interview.
Targeted destruction of specific target proteins in cells is extremely desirable in research and facilitates investigations into their functions. The Affinity-directed PROtein Missile (AdPROM) system, described by the Sapkota lab in Open Biology, combines the CRISPR/Cas9 genome editing technology and proteolysis to achieve a robust degradation of potentially any endogenous protein in cells. The AdPROM system for proteolysis consists of two simple steps: i. generation of cells in which the target protein is knocked in with a Green Fluoroscent Protein (GFP) tag on both allelles using CRISPR/Cas9; and ii. expression in these cells of the AdPROM proteolytic system, which consists of an anti-GFP nanobody tethered to the Von Hippel–Lindau (VHL) protein, a substrate receptor of CUL2 E3 ubiquitin ligase machinery. The expression of the AdPROM proteolytic system into cells causes the selective degradation of the target GFP-tagged endogenous proteins through the ubiquitin proteasome machinery. This elegant AdPROM system is very simple in that it can be packaged onto a single plasmid vector, easily adaptable into an inducible-system, and robust at achieving protein degradation.
Luke Fulcher, PhD student in the Sapkota lab, demonstrated the efficacy of AdPROM proteolytic system for the destruction of GFP-VPS34 in HEK293 and PAWS1-GFP in U2OS cells, which were generated by Annika Hornberger (Alessi lab) and Polyxeni Bozatzi (Sapkota lab) respectively. Thomas Macartney (DSTT) was instrumental in designing strategies, developing methodologies and rapidly generating all the constructs needed for CRIPSR/Cas9 knockins and the AdPROM system.
The AdPROM system provides a solid and complementary platform for drug discovery aimed at exploiting proteolysis, such as proteolysis targeting chimeras (PROTACs), as means of targeting protein function. It can also be adapted to modulate endogenous protein function in other ways, such as changing their sub-cellular localisation and capturing protein complexes.
The paper can be accessed here.
Miratul Muqit, a Wellcome Trust Clinical Investigator and PI based at the MRC-PPU has been selected for the prestigious European Molecular Biology Organisation Young Investigator Programme (EMBO YIP).
Every year EMBO go to great efforts to identify the brightest young Life Sciences researchers working in Europe, Israel, Turkey and Singapore and award them with an EMBO YIP Prize. In addition to the prestige, the EMBO YIP Prize also provides awardees with significant academic, practical and financial support to help them realise their potential as world-class researchers.
Miratul combines his cutting edge research into better understanding how mutations in the PINK1 protein kinase and the Parkin E3 ligase cause Parkinson’s disease with being a Consultant Neurologist treating patients with Parkinson’s and other movement disorders. In 2004 Miratul was a key member of the team that discovered that mutations in the PINK1 gene caused Parkinson’s whilst working as a PhD student with Nick Wood at the UCL Institute of Neurology. Miratul joined the MRC-PPU in 2008 to decipher the molecular mechanism by which the PINK1 kinase is regulated and to uncover its downstream functions. This has led to a series of significant discoveries that include showing that chemical agents, which, induce mitochondrial depolarisation stimulate PINK1 catalytic activity by an as yet unknown mechanism. He also found that PINK1 once activated phosphorylates both Parkin and ubiquitin at an analogous conserved residue termed Serine 65. Miratul’s work further demonstrated that PINK1-phosphorylated ubiquitin binds to Parkin promoting the phosphorylation of Parkin by PINK1 resulting in maximal Parkin activation. Recently Miratul has also discovered a new pathway by which PINK1 regulates the phosphorylation of a family of Rab GTPases, which is the focus of much current research in his laboratory.
Upon receiving news of the prize Miratul said, “I am absolutely delighted to be joining the EMBO YIP programme and engaging with researchers from all across Europe that will bring new ideas to enhance our research efforts. The award is a reflection of the many talented students and post-docs that have worked in my lab and also the outstanding research environment and world-class facilities at the MRC unit. It has also been a privilege to interact with wonderful colleagues and supportive mentors within the MRC unit and I am looking forward to unlocking further new knowledge on the PINK1 pathway. The future of clinical neurology research is to understand the signal transduction pathways that drive these diseases and the MRC-PPU is the perfect environment for Clinician Scientists like myself to tackle these challenging problems.”
Dario Alessi, Director of the MRC PPU was equally pleased by this award “This is fantastic recognition for the ground breaking work that Miratul and his lab are undertaking to better understand signaling components relevant to Parkinson’s disease such as the PINK1 kinase and Parkin E3 ligase. The work that Miratul has performed has made a huge contribution to the uncovering of the mechanism by which PINK1 recruits and activates Parkin at the surface of damaged mitochondria. In particular the finding that PINK1 phosphorylates both ubiquitin and parkin at an analogous Serine residue was totally unexpected and reveals one of the most striking examples of the interplay between phosphorylation and ubiquitylation known in biology. The research that Miratul has been able to perform is even more remarkable given that he is also a Clinician and spends a significant amount of his time away from the lab bench treating Parkinson’s patients. It is indeed very rare for a clinician to receive an EMBO-YIP award. Miratul is the sixth PI from the MRC-PPU to be selected for the EMBO Young Investigator Programme after Daan van Aalten (2002), Karim Labib (2004), John Rouse (2006), Helen Walden (2011) and Yogesh Kulathu (2015) previously receiving this honour. This is a great reflection of the strength and calibre of our Unit’s researchers.”
Two Sapkota lab PhD students, Luke Fulcher and Luke Hutchinson, won prizes for best posters at the sixth annual PiCLS symposium held at the West Park Conference Centre on 17th October, 2016.
Luke Fulcher presented his recent work on the use of Affinity-directed PROtein Missile (AdPROM) system to degrade endogenous target proteins. This methodology has enormous potential for functional modulation of any expressed proteins in cells.
Luke Hutchinson presented his work on novel regulators of the TGF-beta pathway. He has employed endogenous transcriptional reporter systems for TGF-beta and BMP signalling to uncover novel regulators, some of which have shown tremendous promise for therapeutic intervention in pathologies associated with abnormal TGF-beta signalling.
Two previous winners from the Sapkota lab include Polyxeni Bozatzi (2015) and Lina Herhaus (2013).
The PhD in College of Life Science (PiCLS) annual symposium is organised by the PiCLS committee composed of PhD students within the School of Life Sciences in Dundee, who invite eminent scientists to speak from around the world. Some of the speakers this year included Prof. Wendy Bickmore (Edinburgh) and Prof. Markus Aebi (Zurich), among others. Almost all the PhD students from the School of Life Sciences attend the symposium and majority of the present posters
We are delighted to announce that Helen Walden has been promoted to a chair at the University of Dundee where she will become its Professor of Protein Structure and Function
This promotion recognises the ground-breaking research that Helen and her laboratory have undertaken on unravelling the fundamental mechanisms of how ubiquitylation is conjugated to its target proteins. Helen’s recent work has made huge contributions to our understanding of the Parkin and FANCL E3 ligases that are mutated in Parkinson’s and Fanconi Anemia.
Helen commented “I am delighted to be promoted to Professor, this is a great reflection of the hard work and dedication of the lab members over the years. I'm looking forward to the challenges and opportunities the promotion will bring.”
Dario Alessi, Director of the MRC PPU was equally pleased by this promotion “This is fantastic recognition for the vital work that Helen has undertaken to better understand fundamental questions underpinning ubiquitylation biology. This research is providing us with deep insights into how genetic lesions in the ubiquitylation system cause diseases such as Parkinson’s and cancer. It’s been a tremendous past couple of years for Helen with a series of important publications coupled with prestigious awards such as the Colworth Medal and an ERC Consolidator Grant.”
Our ninth MRC PPU alumni interview is with Beatrice Maria Filippi, ex-postdoctoral researcher in the Alessi group.
Click the MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
A vital asset of the MRC-PPU is the Division of Signal Transduction Therapy (DSTT). This division operates as a unique collaboration between world leading pharmaceutical companies and scientists in the MRC-PPU and signalling researchers at the University of Dundee’s School of Life Sciences.
We are delighted to announce the renewal of this collaboration in which Boehringer Ingelheim, GlaxoSmithKline and Merck will provide support of £7.2 million until 2020, enabling our scientists to engage in exciting collaborations with companies in multiple therapeutic areas.
Founded in 1998, expanded in 2003 and renewed in 2008 and 2012, the DSTT is the world’s longest running collaboration between academic research laboratories and the pharmaceutical industry. The latest renewal means the consortium has attracted £58 million in funding since its inception. It is widely regarded as a model for how academia and industry can interact productively.
Professor Dario Alessi, Director of the MRC-PPU, said, “I am absolutely thrilled that we have been able to renew this remarkable 18-year flagship collaboration with our pharmaceutical partners. This alliance has never been more important as our researchers are making such tremendous progress in better understanding human diseases such as Parkinson’s, immune conditions and cancer.”
“The DSTT collaboration provides a unique platform through which our Dundee investigators and pharmaceutical companies can work together in order to launch and accelerate the early stage development of new drugs. It also provides our students and postdocs an opportunity to gain vital experience working closely with pharmaceutical companies that stimulates some of them to embark on a lifelong career of drug discovery to develop better treatments or even cures for human disease.”
Dr Malcolm Skingle, Director of academic liaison at GSK, said, “At GSK, we believe that collaboration is key to helping convert groundbreaking science in to medicines. Working with experts outside our own labs enables us to benefit from each other’s skills and experience, as well as sharing risk – which makes all partners well placed to pursue the most promising avenues of research.
“We’re delighted to be renewing our long-lasting alliance with Dundee, which exemplifies this collaborative approach. Working alongside scientists from Dundee, we’re making inroads in our understanding of a broad range of chronic diseases, and we believe that by continuing our work together we’ll be able to accelerate the translation of this knowledge in to new treatments for patients.”
Dr Clive Wood, Corporate SVP of Discovery Research at Boehringer Ingelheim, said, “We have been delighted to be a member of the consortium and work with the outstanding scientific teams in Dundee. We have gained early insights that have helped to spark in-house discussions, ideation for new therapeutic concepts and generate better understanding of new cellular mechanisms of disease. We look forward to our future work together.”
The MRC Protein Phosphorylation and Ubiquitylation Unit has initiated a search for a new PI working in the area of protein phosphorylation and/or ubiquitylation. Click here for more details and how to apply
Our eighth MRC PPU alumni interview is with Alberto Vitari, ex-PhD student who was supervised by Dario Alessi. Alberto now works for Verily, google’s major new Life Sciences company. Alberto discusses the fascinating highly multidisciplinary research that he and his colleagues at Verily are undertaking to discover what keeps us healthy and how non-invasive technology and data analysis can be exploited to detect emergence of human disease at the earliest pre-symptom stage, where disease progression is going to be most easily prevented or treated.
Click the MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
We are seeking to appoint a highly motivated and experienced scientist with considerable expertise in mass spectrometry to take charge of running major research collaborative projects with the group leaders in the MRC PPU.
The successful applicant will play a major role in working together with our group leaders and staff within their laboratories to design and undertake collaborative mass spectrometry studies. Analysing and interpreting the data that emerges from this work will also play an important part of this role. They will also be responsible for writing up the data for publication and preparing mass spectrometry sections for grant applications.
This position provides an exciting opportunity to be involved in numerous important research projects and for the successful applicant to develop a major international reputation.
The post holder will also be involved in the long-term development of mass spectrometry systems and methodologies in the MRC PPU, and in liaising effectively with the other research staff within the MRC PPU mass spectrometry facility.
For further information and how to apply click here.
Dr Sambit Nanda, a senior research scientist in the Medical Research Council’s Protein Phosphoryation and Ubiquitylation Unit (MRC-PPU) at the University of Dundee, has been awarded a research grant of £15,000 from Tenovus, Scotland to continue his exciting new research on inflammasomes.
Inflammasomes are large multiprotein oligomers, which play important roles during microbial infections or when tissues are damaged. They produce potent pro-inflammatory cytokines that fight infection and also eliminate damaged cells. On the other hand, if inflammasomes are too active or are not switched off quickly enough, this can cause serious diseases ranging from auto-inflammatory and chronic disorders including atherosclerosis, type 2 diabetes, rheumatoid arthritis, chronic obstructive pulmonary disorder (COPD) and neurodegenerative diseases. There is therefore a need to develop new drugs to switch off the inflammasome when it is activated too strongly.
Sambit has recently identified an enzyme which plays an essential role in switching on one type of inflammasome, called the NLRP3 inflammasome and the aim of the Tenovus-funded project is to discover how this enzyme exerts its effect. If successful, the research could eventually lead to the development of improved drugs to suppress inflammasomes.
The research will be carried out in Sir Philip Cohen's lab in the MRC-PPU, which is based in the School of Life Sciences at the University.
Commenting on the award Sambit said:- “ I am very excited to receive the Tenovus award as this grant will provide financial support to carry out detailed biochemical analysis to understand how the NLRP3 inflammasome is activated. I hope that my research will identify new candidate proteins that can be targeted to develop drugs to treat inflammatory diseases.
Embryonic Stem Cells (ESCs) have the potential to provide tissue replacement therapies for a number of debilitating diseases. This is because ESCs have the capacity to differentiate into any cell type in the adult body, a property known as pluripotency.
A major research effort is to identify new signaling pathways which control ESC pluripotency and differentiation. To tackle this problem, members of the Findlay lab teamed up with Nathanael Gray’s lab at Harvard Medical School.
In a high-throughput screen of selective small molecule kinase inhibitors, Charlie Williams, a PhD student in the Findlay lab, discovered that ERK5 inhibitors promote transition of so-called naïve ESCs into the primed state, where they acquire the ability to differentiate.
Charlie Williams and the team used an elegant combination of chemical engineering and genome editing strategies to confirm that the ERK5 signaling pathway maintains ESCs in a state of naïve pluripotency. Rosalia Fernandez-Alonso, a postdoctoral fellow in the Findlay lab, then showed that ERK5 specifically regulates differentiation of ESCs to cardiac muscle cells, or cardiomyocytes.
Greg Findlay, who led the research, said “Our findings have significant implications for tissue regeneration. Most excitingly, our results suggest that Erk5 inhibitors may instruct ESCs to form cardiac cells, which can be exploited to help repair damaged tissue following a heart attack.” In future, the lab will explore this possibility and investigate the mechanisms by which Erk5 controls ESC pluripotency and differentiation.
Professor Dario Alessi, Director of the MRC unit stated, “Congratulations to Greg, Charlie and Rosalia for this very important paper that provides significant new insights into the role that the ERK5 signaling pathway plays in biology. The ERK5 pathway has been a bit of a mystery at least for me, and this study, which is also the first publication from the Findlay laboratory, defines a clear-cut important role for the ERK5 network in controlling differentiation of stem cells. It has ramifications for how differentiated stem cell-derived lineages such as cardiomyocytes might be better produced in the future. I am also excited about the area that this opens up to better understand the molecular mechanism by which ERK5 controls these pathways in future research”.
To read Charlie and Rosalia’s paper, which is published in Cell Reports today, click here.
Mitochondria are the essential energy-generating powerhouses that provide our cells with the energy of life. However, their malfunction has a dark side. Damaged mitochondria have the potential to release destructive reactive oxygen species that have serious and deleterious consequences for the cell. To cope with this, our cells have evolved a protective mechanism to prevent such a “mitochondrial meltdown”, by eliminating damaged mitochondria through a process termed mitophagy. Mitophagy is a cellular waste disposal pathway that delivers faulty and superfluous mitochondria to lysosomes for degradation and recycling. Over the past decade, landmark studies in cultured cells have hinted that mitophagy may be a vital “stress response” that sustains the health of the mitochondrial network or “cellular power grid” when damaged. However, it has been very difficult to visualise this process in tissues and thus, little is known about the true nature of mitophagy in whole organisms. Several important questions remain unanswered - do our cells perform mitophagy all the time, or only under conditions of stress? Likewise, in what tissues and in what kind of cells within our organs does mitophagy proceed? It is vital our knowledge of this process extends beyond the Petri dish to a more biomedical context, especially as mitophagy may be critical in human disease progression. It has recently been proposed that impaired mitophagy may be instrumental in triggering neurodegeneration in some forms of hereditary Parkinson’s disease.
Pioneering research from the Ganley Lab has now shed light on the physiological instances of mitophagy. In a cover article published by the Journal of Cell Biology, research spearheaded by postdoctoral fellow Tom McWilliams describes a fluorescence-based mitophagy mouse model, which they have called mito-QC (for mitochondrial Quality Control). mito-QC allows the visualisation of mitochondrial morphology and turnover in defined cells within tissues for the first time. The work, which was a collaborative effort between the Ganley and Muqit Labs in the MRC-PPU as well as Alan Prescott and the SLS Dundee Imaging Facility, has really opened up the field of mitophagy. This work clearly shows that mitophagy is a physiological process and even in healthy animals, mitophagy is occurring in multiple cell types within diverse tissues. Interestingly, the amount of mitophagy appears to vary between specific cells within tissues. For example, proximal tubule cells within the adult kidney exhibit an extremely high level of mitophagy while the related distal tubule cells, in the same organ, do not. It is of note that impaired proximal tubule function is involved in renal failure and kidney disease and, given its protective function, it is possible that mitophagy plays an important role here. Likewise, Tom was able to show that mitophagy is happening in distinct neurons of the brain and by crossing mito-QC with multiple mouse models of Parkinson’s disease, it is the hope that mitophagy can be finally studied in brain regions that are relevant to disease pathology. Knowing when and where this pathway is disrupted may provide critical knowledge in developing treatments for this currently incurable disease, as well as for other diseases where mitophagy has been implicated.
A major aim will now be to determine the molecular cues that trigger and regulate mitophagy in tissues, in order to understand how disrupting this quality control pathway could lead to disease onset and progression.
Nicola Heser and her son Andrew recently visited the MRC PPU laboratories to learn about the research being carried out by the groups of Miratul Muqit and Dario Alessi to better understand the causes of Parkinson’s disease. In particular they heard about the role of the PINK1 and LRRK2 kinases respectively including recent work from their labs pinpointing their regulation of a family of small second messenger molecules known as Rab GTPases. This discovery represents the first ‘hub’ molecule that can be regulated by different Parkinson’s genes. It is also likely to lead to new methods to monitor the activity of these kinases that will facilitate the development of drugs targeting these important enzymes in patients with Parkinson’s.
Nicola, who herself has been diagnosed with Parkinson’s and is under the care of Miratul at Ninewells Hospital, then donated a cheque arising from a collection raised at her mother’s 80th birthday to support research into the disease.
Miratul said “we are extremely grateful to Nicola’s mother and all the family for this generous donation which will be extremely important for our research studies into Parkinson’s at the MRC unit”.
Our seventh MRC PPU alumni interview is with Elton Zeqiraj, ex-PhD student who was jointly supervised by Dario Alessi and Daan van Aalten.
Click the MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
PI3K and Akt inhibitors are being evaluated in clinical trials for the treatment of human tumours including breast cancer that display driver mutations that inappropriately elevate the PI3K/Akt signalling pathway. Despite initial promise, the majority of these tumours rapidly evolve to resist the PI3K/Akt pathway therapy.
Ruzica Bago, a postdoc based in the Alessi working in close collaboration with researchers at Memorial Sloan Kettering Cancer Centre, AstraZeneca, and the University of Liverpool, set out to decipher intrinsic signalling mechanisms that account for adaptive resistance to PI3K/Akt pathway suppression.
Building upon work carried out by a previous MRC-PPU PhD student Eeva Sommer, Ruzica showed that prolonged treatment of a panel of breast cancer cells lines (ZR-75-1, CAMA-1,T47D and BT-474 ) for over 2-days with with PI3K or Akt inhibitors, led to a marked increase in the expression and activity of a poorly studied protein kinase termed the serum and glucocorticoid-regulated kinase-3 (SGK3), that is closely related to Akt and also activated by the same upstream kinases (PDK1 and mTORC2).
Akt kinase possesses a PH domain at its N-terminus that binds to the lipid second messenger PtdIns(3,4, 5)P3 product generated by PI3K, which induces a conformational change promoting phosphorylation and activation of Akt by PDK1 and mTORC2.
In contrast, SGK3 instead of a PH domain at its N-terminus possesses another lipid interacting motif termed a PX domain, that binds to PtdIns(3)P-and not PtdIns(3,4, 5)P3. SGK3 is the only known protein kinase to possess a PtdIns(3)P binding domain.
Ruzica discovered that SGK3 was activated in an analogous mechanism to Akt namely that PtdIns(3)P-binding to the PX domain promoted PDK1 phosphorylation and hence activation of SGK3. This effect is prevented by introducing a mutation within the PX domain that prevents SGK3 from binding to PtdIns(3)P.
These findings raised the question as to what was the identity of the lipid kinase in the cell that generates the PtdIns(3)P that stimulates the activation of SGK3 in breast cancer cell lines treated with PI3K or Akt inhibitors. Employing structurally diverse highly selective inhibitors, Ruzica experiments strongly point towards an enzyme termed hVps34, which is one of the major lipid kinase in the cell that generates PtdIns(3)P that is located at endosomes of cells at the same location SGK3 residues.
To better dissect SGK3 signalling, Ruzica also characterized a recently reported Sanofi SGK1 inhibitor termed 14h that she observed potently inhibited SGK3 with an IC50 of ~ 3nM. Although 14h was reported to act as an ATP completive inhibitor, Ruzica noticed that in addition to suppressing SGK3 activity, 14h also prevents the phosphorylation of SGK3 by PDK1 and mTORC2 pathway and hence its activation of SGK3 in cells. Consistent with 14h inhibiting the activation of SGK3 in cells, Ruzica demonstrated that in vitro 14h prevents PDK1 from phosphorylating and activating SGK3 in the presence of PtdIns(3)P in vitro.
Given the similarity between Akt and SGK3, Ruzica speculated that these kinases could phosphorylate an overlapping set of substrates. Her data suggested that this seems to be the case, as ether prolonged inhibition of PI3K/Akt enabled SGK3 to fully re-activate the mTORC1 signalling pathway by phosphorylating TSC2. Under these conditions mTORC1 activation is now blocked by 14h SGK inhibitor.
Employing an Akt phosphorylation motif antibody-Ruzica found that out of 9 Akt substrates identified in a cell extract 6 of these were likely to be phosphorylated by both Akt and SGK3
Lastly, Pau Castel working in the laboratory of José Baselga working at the Memorial Sloan Kettering Cancer Center demonstrated that a combination of Akt (MK-2206) and SGK (14h) inhibitors induced marked regression of a breast cancer (BT-474) cell derived tumours in a nude mouse xenograft model, under conditions where either inhibitor administered individually had minimal effects.
These results highlight the importance of the hVps34-SGK3 pathway and suggest it represents a major mechanism, which cells utilise to counteract inhibition of PI3K-Akt signalling. They also provide novel mechanistic insights of how PtdIns(3)P can stimulate activation of SGK3.
The characterisation of the 14h SGK inhibitor suggests that it will become a useful research tool to probe biology controlled by SGK isoforms. 14h represents a valuable addition to our growing arsenal of signal transduction inhibitors to dissect functional roles of protein kinases.
Finally, and perhaps most importantly findings described in Ruzica’s paper highlight the therapeutic potential of a strategy targeting both the Akt and SGK kinases for the treatment of cancer.
To read a copy of Ruzica’s paper published in the EMBO J. click here.
There has been considerable interest in understanding the function of the leucine-rich repeat kinase-2 (LRRK2) since the discovery in 2004 that autosomal dominant mutations that activate this protein kinase cause inherited Parkinson's disease. Work in the Alessi lab in the MRC PPU has been geared towards understanding how mutations in LRRK2 impair its biological functions and cause Parkinson’s.
Earlier this year in a major collaborative work with Matthias Mann’s group and researchers at GlaxoSmithKline (GSK), Merck, and The Michael J Fox Foundation for Parkinson's Research (MJFF), we discovered that LRRK2 directly phosphorylates a conserved Thr/Ser residue in the effector-binding switch-II motif of a number of Rab GTPase isoforms, including Rab10.
The data indicated that Parkinson’s causing mutations stimulate LRRK2 to directly phosphorylate a small subgroup of Rab GTPases proteins including Rab 8A and Rab10. Phosphorylation of Rab proteins by LRRK2 suppressed their interaction with GDIs as well as guanine nucleotide exchange factors that are required for membrane delivery, recycling and activation.
This led to the hypothesis that Parkinson’s mutations in LRRK2 result in inappropriate phosphorylation and inhibition of a sub group of Rab GTPases and this might impair vesicular trafficking and processes such as autophagy.
An important next goal of the research was to develop a robust method to rapidly assess LRRK2 phosphorylation of endogenous Rab isoforms in samples where material may be limiting without the need to use state of the art mass spectrometry to assess Rab protein phosphorylation.
To address this question Genta Ito, a postdoc in the Alessi lab collaboration with other researchers from the University of Dundee, MJFF, GSK, and the University of Hong Kong, elaborated a new procedure exploiting an agent (1,3-bis[bis(pyridin-2-ylmethyl) amino]propan-2-olato dizinc(II) complex) commonly referred to as “Phos-tag” that binds to phosphate ions with very higher affinity.
Genta found that when polymerised into SDS-polyacrylamide gels the Phos-tag reagent dramatically retarded electrophoretic mobility of LRRK2 phosphorylated Rab10, resulting in substantial mobility shifts (see figure). Using this approach, Genta was able to demonstrate that ablation of LRRK2 catalytic activity in a novel, kinase inactive LRRK2[D2017A] knock-in mouse model developed by Alastair Reith’s group at GSK, blocked Rab10 phosphorylation in mouse embryonic fibroblasts (MEFs) as well as lung, demonstrating that LRRK2 is indeed the major Rab10 kinase in these cells and tissue.
Genta also established that the Phos-tag assay can be used to monitor the impact of LRRK2 inhibitors, as well as pathogenic knock-in mutations (G2019S [kindly provided to us by GlaxoSmithKline] and R1441G [provided to us by Philip Wing-Lok Ho and Shu-Leong Ho, University of Hong Kong) on Rab10 phosphorylation (see Fig). Interestingly, Genta found that treatment of cells with LRRK2 inhibitors induced almost complete dephosphorylation of LRRK2 phosphorylated Rab10 with 1 min-indicating that the phosphatase activity acting on Rab10 must be very active in cells.
We hope that the Rab10 Phos-tag assay will aide with the assessment LRRK2 signalling pathway activity in cells and to establish the impact that inhibitors, mutations and other factors have. The prediction is that elevation of LRRK2 activity leads to Parkinson’s disease and the expectation is that if a sub-group of patients can be identified with elevated LRRK2 activity, it would be important to explore whether these individuals might benefit most from LRRK2 inhibitors that are being developed.
Therefore, a major aim of our future work will be to investigate whether this Phos-tag technology could be exploited to assess LRRK2 activity in Parkinson’s patients.
Also congratulations to Gento Ito for securing a group leader position at the University of Tokyo (Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences). Genta will continue working on better understanding LRRK2 and how this is linked to Parkinson’s.
To read the paper describing these findings click here and for the press release accompanying this paper click here.
Esther Sammler, a former PhD student with Dario Alessi and consultant neurologist in Dundee has been awarded an Academic Health Sciences Partnership in Tayside (AHSP) Clinical Fellowship which is aimed at promoting translational research. Esther will set up a translational link between the movement disorder service at NHS Tayside and the MRC PPU, looking at signalling pathways of Parkinson's disease associated genes in biosamples from healthy controls and patients with Parkinson's disease. Esther’s initial aims will be to set up quantitative assays to determine LRRK2 activity in human blood cells, based on the ability of LRRK2 to phosphorylate Rab GTPases. This work will enable Esther to explore whether a subgroup of Parkinson’s patients that display elevated LRRK2 activity can be detected. If this was the case this would open the door to test whether these patients would benefit from LRRK2 inhibitors.
Yosua Adi Kristariyanto, a 4th year PhD student in Yogesh Kulathu’s lab, won the EMBO Journal Poster Award at the recent FASEB Conference on Ubiquitin and Cellular Regulation. The six-day conference held at Big Sky, Montana, USA was a platform for scientists across the world to meet and share recent findings in the ubiquitin field. The poster prize was chosen on the basis of outstanding science and presentation of the poster.
In his poster, Yosua presented exciting new data that provides novel concepts on how polyubiquitin chains are recognized by a small ubiquitin binding domain called the motif interacting with ubiquitin (MIU). Yosua investigated polyubiquitin binding in the MIU motifs of MINDY, a new family of deubiquitinating enzymes that was recently discovered in the Kulathu lab. Congratulations Yosua on this achievement!
Syed Arif Abdul Rehman, Yosua Kristariyanto and Soo-Youn Choi, the first authors on the recent Molecular Cell paper from the lab were interviewed in the Meet the Author section. Read what they had to say here.
The lab came up with artistic ideas for a cover page and Yosua’s sister Gratia Fidelina, a graphics illustrator blended them to produce a great picture that showcases the main findings of the paper.
The American Society for Biochemistry and Molecular Biology (ASBMB) have conducted a major in-depth interview with Davie Douglas: honorary member of the MRC PPU who for nearly the last 20 years has been the main airport taxi driver for our research staff and their guests.
In a wide ranging interview Davie discusses the roles he plays in acting as an ambassador for Life Science research in Dundee, and talks about his famous guestbook that contains signatures and messages from many of the world’s leading researchers, including many Nobel laureates, who Davie has driven over the years.
Please click here to read the article, and here to read the Editor's cover note.
Researchers in the laboratory of Dr. Satpal Virdee have published the first example of genetically encodable aminooxy functionality, which can be installed site-selectively into recombinant proteins using genetic code expansion technology. The findings were published in Chembiochem this week.
Site-specific modification of proteins is a challenging and hotly pursued topic within the chemical biology community. The ability to direct site-selective chemical bond formation between proteins in the presence of chemically diverse functionality within biological samples is non-trivial but advantages and insight gained from such methodologies are indispensible to modern biology. Current focus is on developing ever faster chemistries whilst retaining biorthogonality.
One example of a bioorthogonal reaction is the oxime ligation reaction between an aldehyde (or ketone) with an aminooxy (-ONH2) functional group. However, methods that allow installation of the prerequisite chemical handles for oxime ligation at the genetic level into proteins are limited.
The installation of this aminooxy functional group into ubiquitin, and into the ubiquitin-like protein SUMO2, enabled researchers to carry out site-specific “chemical ubiquitylation” of full-length proteins to produce diubiquitin conjugates and a ubiquitylated substrate (ubiquitylated SUMO2) as proof-of-principle. Importantly, the oxime linkage produced between the protein molecules as a result of the ligation reaction is hydrolytically stable to deubiquitinase (DUB) proteolytic activity whilst being an exquisite structural mimic of the native isopeptide bond which it successfully substitutes.
The technology has enabled Virdee lab researchers to generate a valuable protein toolkit, including protein-based nanomolar DUB inhibitors and non-hydrolysable extended ubiquitin polymers which now provide researchers with a means to probe the cellular roles of Ub linkages without the intrinsic sensitivity of native ubiquitin linkages to DUB activity and to potentially identify hitherto unknown proteins that interact with ubiquitylated substrates and distinct ubiquitin chain types.
The first author of the study, Mathew Stanley, describes the potential use of these tools developed in the Virdee lab. “The protein conjugates produced using this method are structurally highly similar to native conjugates as determined by biophysical techniques and X-ray crystallography. Coupled with the fact that they are stable to the hydrolytic activity of DUBs make these invaluable tools for studying ubiquitin linkage specific processes in cells and for the study of DUB biology, which has become a significant area of interest for many pharmaceutical companies. Looking beyond the ubiquitin system, because of the modularity and versatility of the approach, the methods described will have wide ranging utility in other areas, for example, the biochemical and structural study of other families of proteolytic enzymes and in the production of antibody-drug conjugates and biomaterials.”
A light activatable variant of the aminooxy functional group that the research team present have the group particularly excited about the future applications of the technology.
“With recent advances in ultra-fast oxime labeling reactions and with the existing ability to genetically encode aldehyde functionality into proteins, our contribution now paves the way towards a strategy for light-dependent ultrafast protein labelling and site-specific protein–protein tethering in live cells”.
Satpal Virdee has been awarded a BBSRC Responsive Mode award of £650,000 allowing his lab to further develop technology they pioneered that enables the activity-based profiling of E3 ligases.
The ubiquitin system is involved in many aspects of cellular biology and defects within this system often give rise to diseases such as cancer, neurodegeneration and autoimmune disorders. E3 ligases confer substrate specificity and have become attractive drug targets. These enzymes demonstrate regulated activity but tools for the multiplexed profiling of their activity on a proteome scale, across broad cell and tissue types, are absent.
Furthermore, 1000’s of proteins are known to be ubiquitinated at distinct sites yet our understanding of which of the >600 RING E3 ligases might be responsible is often poor.
This BBSRC award will help Dr Virdee’s lab to establish a chemical proteomic platform for profiling E3 ligases, and thereby assign them to diverse (patho)physiological processes. A novel class of probe will also be developed that will covalently crosslink substrates to their cognate RING E3s.
Upon receiving news of the prize Dr Virdee said, “We, and others, are very excited about the proof of concept work my lab has already carried out and this award will allow us to develop it further. Some aspects of this proposal were high risk and I am delighted that the BBSRC were still prepared to back us.”
Professor Dario Alessi, Director of the MRC unit stated, “Fantastic news that Satpal has been awarded this highly sought after BBSRC grant to develop new classes of probes to unbiasedly analyse the activity state of E3 ligases. This is an important and innovative project that has the potential to transform our understanding of the regulation and function of E3 ligases in health and disease. I am confident that many future breakthroughs will be made possible by work such as this, which is focused on crafting cutting-edge chemical tools to better interrogate biological pathways”.
Satpal is advertising for PhD students and Postdocs to work on this and other projects in his lab. For enquiries please contact Satpal: firstname.lastname@example.org.
Our sixth alumni interview is with Anna Zagorska, ex-MRC PPU PhD student in Dario Alessi's group. Click the MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
In ground-breaking new work published in Molecular Cell, Yogesh Kulathu and colleagues report the discovery of a completely new family of Deubiquitinating enzymes (DUBs).
Ubiquitin signals impact a wide range of eukaryotic biology. It is therefore very important that these signals are tightly regulated. This layer of regulation is provided by dedicated proteases called deubiquitinating enzymes (DUBs), of which there are ~100 encoded in the human genome that can be classified into five families.
In a team effort, Syed Arif Abdul Rehman, Yosua Adi Kristariyanto and Soo-Youn Choi in the Kulathu lab identified and characterized deubiquitinating activity in a completely unstudied protein (FAM63A). When analysing the sequence of FAM63A, Dr. Kulathu noticed that it had conserved residues in a signature typically found in cysteine proteases. Since FAM63A also has a ubiquitin binding motif that selectively binds to K48-linked ubiquitin chains, they wondered if FAM63A could be a DUB.
“To our surprise, we found that FAM63A was indeed a DUB with high selectivity at cleaving K48-linked polyubiquitin chains”, Dr. Kulathu said. Through further sequence analysis, they narrowed down the catalytic activity to be encoded within a previously unannotated domain of unknown function (DUF544). Syed Arif then determined the crystal structure of the catalytic domain which revealed a distinct fold with no homology to any of the known DUBs. This makes FAM63A a prototype of a completely new family of DUBs that the team named MINDY (MIU containing Novel DUB family).
With the help of Prof. Kay Hofmann at the University of Cologne, they identified distantly related FAM188 members to also form part of the MINDY family of DUBs. MINDY DUBs are found in all eukaryotes and intriguingly they are all highly selective at cleaving K48-linked chains, the signal that targets proteins for proteasomal degradation. When proteins aren’t degraded properly, misfolded and aggregated proteins can accumulate. This is a common contributing factor to age-related diseases such as Alzheimer’s disease and Frontotemporal dementia (FTD).
Dario Alessi, Director of the MRC PPU noted “I congratulate Yogesh and his team for this stunning piece of research. This discovery was totally unexpected as I thought that all the DUBs had been identified and catalogued. It emphasises that we don’t know everything and there is still lots of important biology out there just waiting to be uncovered. Above all, this work illustrates the importance of curiosity driven research that one is able to undertake in the core-funded environment of an MRC Unit. It is going to be so exciting to see what roles the MINDY DUBs play in regulating biology in future research.”
This work was funded by the Medical Research Council. The Kulathu lab has recently obtained further funding from Tenovus Scotland, and is looking to hire a postdoctoral researcher hire a postdoctoral researcher to unravel roles for this new family of enzymes in maintaining protein homeostasis. Informal enquiries may be addressed to Yogesh.
On May 20th, Philip Cohen was awarded an honorary doctorate by the Universidad di Autonoma di Madrid. After receiving the award, Philip then gave a 30 minute talk about his career to an audience consisting of members of the University and the general public. The yellow cape and gown that Philip is wearing are the colours of the Medical faculty of the University. Three of Philip's former postdocs who now run their own research teams in Madrid (Susana Alemany, Ana Cuenda and Guillermo Velasco) were in the audience and the pictures were taken by Ana.
Christophe Lachaud has won a Principal Investigator position from the Centre National de Recherche Scientifique (CNRS), the premier research funding agency in France. Each year, the CNRS elects a small number of researchers, from many applicants, who have achieved research excellence within a wide variety of disciplines including physics, mathematics, biology or economics. In 2015, the CNRS topped the Nature Index, an international ranking of scientific institutions by the journal Nature. It is placed ahead of the Chinese Academy of Sciences, Germany’s Max Planck Institutes, Harvard University in the US and the Spanish National Research Council. Being elected to the CNRS is a major achievement, reflecting research at the very top level.
Christophe carried out his post-doctoral work in John Rouse’s lab. He joined the MRC PPU in October 2011 to work on the mechanism of action of the FAN1 DNA repair nuclease, which was very poorly characterised at the time. During that time Chris defined the role of Fan1 in the repair of DNA inter-strand cross links and pinpointed new unanticipated functions of Fan1 including a role as a tumour suppressor. Chris will establish his lab at the Cancer Research Center of Marseille in France, and he will focus on defining mechanisms of DNA repair in cancer. For further information about Christophe’s research programs or open positions please contact him at email@example.com.
Commenting on his award Christophe said, “I am now really excited about the idea of managing my own group. I would like to thank everyone in the MRC PPU, especially John and the people in his group, for all the support they gave me during the last 4 years.”
Congratulations Chris – very best of luck for the future!
Our fifth alumni interview is with Kei Sakamoto, ex-MRC PPU postdoc (in Dario Alessi's group) and group leader, and now Head of Diabetes and Circadian Rhythms at Nestlé Institute of Health Sciences, Switzerland.
To learn more about Kei’s aims to translate his research into food you can buy in the supermarket please click the MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
Tom McWilliams, a postdoctoral researcher with the Ganley and Muqit labs, won the poster prize for Best Basic Science Poster (sponsored by Khondrion) at the recent Wellcome Trust Conference on 'Mitochondrial Medicine: developing new treatments for mitochondrial disease'. The 3-day meeting was held at the Wellcome Genome Campus at Hinxton in Cambridge, and showcased a broad spectrum of research in the field of mitochondrial biology. Tom presented exciting data on his characterisation of an innovative in vivo model developed in the Ganley lab to visualise a form of mitochondrial turnover, termed mitophagy.
Tom's work will give us important insights into the physiological role of mitophagy, a process that has been linked to many diseases ranging from cancer to neurodegeneration. Recently, Tom was also awarded a travel grant from The Guarantors of Brain, who provide funding for clinical and academic neuroscientists to attend conferences and present original research. Tom will use this to attend the prestigious 2016 Gordon Research Conference on Lysosomes and Endocytosis in the United States next month, and give a talk at the Gordon Research Seminar on his recent work on mitophagy.
Congratulations Tom for this achievement!
Miratul Muqit, a Wellcome Trust Senior Clinical Fellow in the MRC PPU, has been elected Fellow of the Royal College of Physicians. Founded in 1518, the Royal College of Physicians is England’s oldest medical institution and Fellowship is “held by some of the most innovative and exceptional physicians in the world”.
Miratul's research group has been studying the PINK1 protein kinase that is mutated in families with heritable forms of Parkinson’s. His research has uncovered the regulation and downstream role of PINK1 including the discovery that PINK1 can phosphorylate ubiquitin in response to mitochondrial damage to activate the Parkin E3 ligase. This has led to new strategies to potentially better treat Parkinson’s.
Miratul, a Consultant Neurologist, is one of several clinicians that have undertaken molecular research at the MRC PPU to address how defects in cell signalling pathways are linked to human diseases. This includes former Clinical PhD Fellows who have successfully completed projects probing the mechanisms of cancer, inflammatory and neurodegenerative diseases.
The Unit currently has two Clinical PhD Fellowships available for clinical trainees in any branch of medicine – click here for more information.
Our fourth alumni interview is with Mirela Delibegovic, ex-MRC PPU PhD student in Tricia Cohen's group. Click the MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
Our third alumni interview is with David Komander, ex-MRC PPU PhD student in Dario Alessi's group. Click the new MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
Bruker engineers are currently installing a novel, very fast Rapiflex MALDI TOF mass spectrometer in the facility of the MRC PPU. The Rapiflex mass spectrometer has an exceptional height of 294 cm and the facility’s ceiling had to be raised to fit in the instrument. It is only the second instrument of this kind with a TOF/TOF configuration in the world.
This mass spectrometer will be able to analyse well over 100,000 samples per day and thus will allow researchers to significantly increase the throughput of drug discovery assays developed in the Unit. In collaboration with Bruker, Matthias Trost’s group will develop and apply novel work flows and methods for high-throughput MALDI TOF screening.
Congratulations to Karim Labib, who has been elected to the Royal Society of Edinburgh, Scotland's National Academy. The Royal Society of Edinburgh each year elects a select number of Fellows who have achieved “excellence within a wide variety of disciplines, spanning the arts, business, science and technology sectors”. Being elected to the Royal Society of Edinburgh is a very prestigious and a major achievement, illustrating that your research is highly recognised and regarded.
Karim joined the MRC PPU in October 2013 and his research group studies how the eukaryotic replisome is regulated by protein phosphorylation and ubiquitylation and the role that this plays in allowing cells to preserve their genome integrity and epigenetic information.
Commenting on the award Karim said, “I’m delighted and honoured to become a fellow of the Royal Society of Edinburgh, and look forward to contributing to the future of scientific research in Scotland and the public’s understanding of it.”
Dario Alessi, Director of the MRC Unit added "I am thrilled that Karim’s research has reached the level to merit Fellowship of the Royal Society of Edinburgh. The work that Karim is undertaking on understanding the molecular mechanism that controls the replication of DNA in Eukaryotes is an amazingly intricate and fundamental. It has great potential to reveal critical insights relevant to better understanding and treating diseases such as cancer".
E3 ligases are one of the largest enzyme families and they confer substrate specificity in the ubiquitin conjugation process. E3 activity is often stringently regulated and aberrant activity is the hallmark of many diseases including neurodegeneration, cancer and autoimmune disorders. However, methods for studying E3 activity are limited therefore new tools that directly measure E3 activity, and that are compatible with endogenous enzymes, would serve as powerful probes for providing insight into the underlying pathology of many diseases. This could also lead to the identification of novel therapeutic targets.
Pioneering chemical probe technology developed at the MRC Protein Phosphorylation and Ubiquitylation Unit is giving scientists the clearest insight yet into E3s that are active in many diseases, including Parkinson’s disease.
Dr Satpal Virdee, leader of the research team, said their findings could "revolutionise" research capability.
Working in collaboration with Dr Miratul Muqit, the team have used their newly developed probes to make novel discoveries relating to the activity of Parkinson’s disease-associated E3, Parkin.
The results of the research are published in the journal Nature Chemical Biology.
The group have reengineered the native substrate of E3s, a ubiquitin-charged E2 conjugating enzyme, such that it covalently labels E3s belonging to the HECT and RBR subfamilies, in an activity-dependent manner. These “activity-based probes” allow not only a single E3’s activity in a cell to be measured directly, but in principle, the activity of dozens of E3s to be measured simultaneously.
“This technology should revolutionise our ability to understand the roles of E3s and protein ubiquitylation in both normal and diseased cells with immediate translational potential to address diseases,” said Dr Virdee.
Dr Virdee’s team have tested the technology by looking at an E3 enzyme, Parkin, which when faulty can give rise to Parkinson’s disease.
“We made a number of novel findings around how Parkin is activated in the cell.”
“For the first time our probes enable direct and quantitative measurement of endogenous Parkin activity. Our probes have given new insights into the pathogenic basis for the many patient mutations that are found within Parkin. We have also shown that our technology can potentially be used as an urgently needed clinical tool to assess the functionality of the Parkin pathway in patients.”
The technology breakthrough could offer multiple benefits for researchers including discovery of novel E3 biology, new therapeutic targets and as a clinical diagnostic tool.
The research was carried out in collaboration with Dr Miratul Muqit’s group in the MRC PPU at Dundee and with colleagues at Sorbonne Universités in Paris.
We have also initiated a search for two postdoctoral researchers to work in this area - for further information please contact Satpal Virdee (s.s.virdee@Dundee.ac.uk).
Our second alumni interview is with Claire Eyers, an ex-MRC PPU PhD student in Philip Cohen's group. Click the new MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
We have overhauled the International Centre for Kinase Profiling website which lists all of the kinase profiling services that the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC PPU) offers to enable researchers in both academic institutes and pharmaceutical companies to profile the specificity and potency of protein kinase inhibitors.
We would invite you to have a look at the revamped site using the below link:
Kinase profiling services include
· MRC-PPU Premier Screen - Test your compounds against an extensive panel of 140 enzymes. This screen is undertaken once every 6 weeks and the data is provided 2 weeks from the screen start date.
· MRC-PPU Express Screen - Compounds can be screened against a subset of 50 kinases representing all areas of the human kinome. This screen is undertaken once every 3 weeks and the data will be provided 1 week from the screen start date.
· Lipid Kinase Screen - A panel of 16 lipid kinases undertaken once every 3 weeks with the data provided 1 week from the screen start date.
· IC50 Determination - If you have a compound you know inhibits a kinase or would like to follow up on a hit from a previous screen you can submit it to determine the IC50 value.
· ATP Competition Assay - Do you need to know if your compound of interest is a type I ATP competitive inhibitor?
· Substrate Screen - Do you have a purified kinase but no substrate to assay its activity?
· Custom Screening - Your compound(s) against your choice of kinase proteins available from our Premier Kinase Panel
The website also includes our widely used "Kinase Profiling Inhibitor Database”. This is a searchable database of specificities of nearly 250 commonly used signal transduction inhibitors screened against a panel of up to 140 kinases frequently at multiple concentrations. We strongly recommend undertaking kinase profiling of any kinase inhibitor to be used in a biological experiment. It is essential to have a good feel for the specificity of the kinase inhibitor you are working with in order to be able to properly interpret your data.To view and peruse data stored in the kinase profiling database click here.
The MRC PPU pioneered analysis of the selectivity of protein kinase inhibitors by setting up the first service to tackle this problem in 1998. This procedure, termed "kinase profiling" proved to be of great help to the pharmaceutical industry, speeding up the development of specific protein kinase inhibitors with therapeutic potential.
Protein kinases are one the pharmaceutical industry's most important class of drug target. Over 30 protein kinase inhibitors have been approved for clinical use and nearly 200 others undergoing clinical evaluation.
The International Centre for Kinase Profiling is operated by the MRC PPU and makes use of all of our huge in-house expertise in studying activity and function of protein kinases. We have been working with industrial partners since 1998 and this has translated into a level of rigor and attention to detail that makes The International Centre for Kinase Profiling a key asset to academia and industry-based scientists - at an affordable price.
We welcome your comments and suggestions on how to improve the site further. Please send all of your suggestions, no matter how minor, to firstname.lastname@example.org
Dr Helen Walden has been awarded the European Research Council (ERC) Consolidator Grant. The highly competitive awards are given to the best and most creative researchers working in Europe.
The funding will create four new research posts at the University’s Medical Research Council Protein Phosphorylation and Ubiquitylation Unit (MRC PPU). Dr Walden and her team will look to discover how to repair the damage caused by two strands of DNA becoming chemically bound during replication, a process known as interstrand crosslinking.
DNA exists as a double-stranded structure coiled together to form a double-helix and replication is the process of producing two identical replicas from the original DNA molecule. This process occurs in all living organisms but crosslinking occurs in the human body around twice a day as a result of damage by chemicals, light and other causes. The body is normally able to undo the bind but in some instances this doesn’t happen, leading to cellular death and diseases such as cancer.
The pathway to repair this type of damage relies on the attachment of a signal to a protein called FANCD2. This signal is then read, translated, and ultimately removed. The Dundee team want to understand how the signal is created, what and how reads it, and how it is removed.
“Every time a cell divides it copies all genetic material,” said Dr Walden. “DNA replication is a fundamental process of the human body but the DNA helix needs to be unwound for it to take place. Interstrand crosslinking means this can’t take place and what we want to do is understand how this sort of DNA damage can be fixed.
“The body can normally clean up errors like crosslinking and we know modification of FANCD2 is a key signal to fix this particular type of DNA damage. What we don’t know is how it works, what switches it on and off and why it works more effectively in some people than others.
“We want to identify the various components of its pathway to understand how it chemically alters the DNA molecule. This is just one of a number of ways in which DNA can become damaged or mutate but if we can understand how the signal is turned on and off then it potentially allows us multiple points on which to interfere to prevent this type of disruption. I am delighted to have received this award, it will enable us to address some challenging and ambitious questions, and am hugely grateful to the ERC for funding this project."
Dario Alessi, director of the MRC PPU added, “I am delighted for Helen that she has received this prestigious award-which is well deserved and testament to the very important research that her laboratory is undertaking. I am confident that the work Helen is embarking on with the ERC funding will provide fundamental knowledge on how DNA integrity is maintained that will be relevant to better understanding and treating human diseases such as cancer”.
Scientists at the MRC Protein Phosphorylation and Ubiquitylation Unit have discovered that “molecular scissors” that repair damaged and abnormal DNA are critical for keeping cancers at bay.
The laboratory of Professor John Rouse first discovered that a protein called FAN1 was important for cutting and repairing damaged DNA in our cells in 2010.
Recent work carried out by Dr Christophe Lachaud under the direction of Professor Rouse have shown that FAN1 carries out another important task separate from repairing DNA, but which is vitally important for preventing cancers.
The team showed that FAN1 plays an important role during the copying of chromosomes that occurs whenever cells divide. In particular, the ability of FAN1 to recognise and cut special types of abnormal structures inside cells during the copying of DNA is important for preventing cancers, particularly of the lungs, liver and pancreas. Moreover, it appears that certain cancers may be caused by failure of FAN1 to cut DNA in the way it is supposed to.
“The DNA in our cells is like an instruction manual for the proper working of each cell,” explained Professor Rouse. “Every time cells divide they need to make a perfect copy of all of their chromosomes, collectively called the genome, so that the next generation of cells also have a proper instruction manual.
“In the process of copying DNA, the machinery responsible for doing this often encounters roadblocks – obstacles that stop the progression of the copying machinery and result in abnormal, potentially toxic DNA structures.
“We showed that Fan1 can recognise these dangerous structures, and uses its cutting activity to makes them less toxic. We found that when we switch off the ability of Fan1 to cut these structures, the genome starts to become abnormal and breaks apart which means the instruction manual has become corrupted. Switching off the cutting ability of FAN1 leads to cancers, such as liver cancer and lung cancer.
“Other scientists have reported that Fan1 is mutated in pancreatic cancers, and we showed that in these cancers Fan1 is not able to recognise the abnormal structures that need to be cut. This leads the genome of these cells to become abnormal accounting for the cancers.”
As the findings have implications for treating cancers, Professor Rouse and his team will now try to discover the ‘Achilles heel’ of cancers in which FAN1 doesn’t work properly. If they are able to find a drug which is only toxic to cells that are defective in Fan1, these drugs might be effective in killing the cancer cells caused by mutations in Fan1 without affecting the normal cells.
Most of the work on FAN1 was carried out by Dr Lachaud, with help from Dr Alberto Moreno in the laboratory of Professor Julian Blow, a world-renowned expert on the process of copying DNA during cell division also based in the School of Life Sciences at Dundee.
The research is published in the latest online edition of the journal Science. The work was funded by the Medical Research Council.
MRC PPU Lab Manager, Allison Bridges, today celebrates 15 years service having joined the Unit on February 1st, 2001 as support technician.
Presenting Allison with a bottle of 15-year old rum to mark the occasion, Unit Director Dario Alessi noted that "Allison is a highly respected, trusted and loyal member of staff, whose experience and knowledge are invaluable to the smooth running of the Unit".
There was a lot of excitement 11 years ago when it was discovered that autosomal dominant missense mutations within the gene encoding for a previously unstudied protein kinase termed LRRK2 (leucine-rich repeat protein kinase 2) predispose humans to develop Parkinson's disease. Mutations in LRRK2 account for 4% of familial Parkinson’s, and are observed in 1% of sporadic PD patients, making it one of the most commonly mutated genes linked to Parkinson’s.
Importantly, the most common Parkinson’s- pathogenic mutation is found within the catalytic domain (G2019S) and activates protein kinase activity suggesting inhibitors might offer benefit for the treatment of Parkinson’s.
However, a very major stumbling block has been that despite nearly 1500 papers being published to date, no one has this far been able to pinpoint a clear-cut physiological endogenous substrate of LRRK2 that can be validated in independent laboratories. This has greatly hindered our understanding of how LRRK2 is linked to Parkinson’s disease as well preclinical evaluation of LRRK2 inhibitors that have been developed by pharmaceutical companies.
In order to identify the key LRRK2 substrates Dario Alessi with generous support from the Michael J Fox Foundation (MJFF) was able to put together an international team of scientists involving researchers in Matthias Mann's laboratory at the Max Planck Institute in Martinsried, the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit at the University of Dundee (MRC PPU), GlaxoSmithKline and Merck in addition to Marco Baptista and Brian Fiske at the MJFF. The key aim of this collaboration was for all teams to work together and make use of each other complementary expertise to exploit state-of-the-art mass spectrometry, genetic and pharmacological approaches to define clear-cut endogenous direct target(s) of LRRK2.
After nearly four years of intensive work our team is delighted to announce that we have at long last identified the first genuine physiological substrate of LRRK2, which comprises a subset of Rab GTPase isoforms including Rab7L1, Rab8A, Rab10 and Rab12. The data indicates LRRK2 phosphorylates these Rab isoforms at a conserved Thr or Ser residue lying in the middle of the key business effector-binding Switch-II motif of Rab GTPases (Thr72 in Rab8A). We show that Parkinson’s causing mutations in LRRK2 including the G2019S mutation as well as others such as the R1441G/C (located within the ROC GTPase domain) or Y1699C (located within the COR domain) markedly enhanced LRRK2 phosphorylation of Rab8A and Rab10 isoforms in vivo.
There are ~70 Rab GTPases encoded by the human genome that play central roles in regulating all aspects of membrane dynamics in eukaryotic cells and are master regulators of cargo collection, vesicle formation, vesicle motility, vesicle docking and vesicle fusion. Although 41 Rab isoforms possess a potential LRRK2 phosphorylation site in the middle of the switch II effector binding domain, our initial data indicates that only a subset of these are likely phosphorylated by LRRK2 in cells.
Our results suggest that LRRK2 mediated phosphorylation of Rab isoforms is inhibitory as phosphorylation of Rab isoforms by LRRK2 prevents them from interacting with known effects such a GDIs (required for the insertion of Rab isoforms into membranes) and GDP/GTP exchange factors (e.g. Rabin-8 for Rab8A).
Therefore the key conclusion from our work is that Parkinson’s mutations in LRRK2 stimulate the phosphorylation of a subset of Rab isoforms and this inhibits their biological function.
This is an enormously exciting result that we believe is relevant to better understanding Parkinson’s as previous work has also linked Rab GTPases to Parkinson’s as inherited mutations in two Rab isoforms (Rab7L1/Rab29 and Rab39B) cause Parkinson’s–like disease in humans. Furthemore, recent work from MRC-PPU laboratory of Miratul Muqit has found that the PINK1 protein kinase that is also mutated in Parkinson’s, indirectly controls the phosphorylation of certain Rab GTPases including Rab8A at a distinct site to LRRK2 (Ser111 on Rab8A). Finally, the work of Susan Lindquist has also linked the binding of alpha-synuclein to Rab8A with Parkinson’s disease. Taken together these findings provide mounting evidence that disruption on Rab GTPase biology could at the heart of better understanding and treating Parkinson’s disease.
We anticipate that our work on elucidating the LRRK2-Rab signalling network could open up a new era of research which could lead to improved opportunities for better understanding LRRK2 biology and how mutations cause Parkinson’s. Reagents we have elaborated to monitor LRRK2 phosphorylation of Rab GTPases could aide pharmaceutical companies advance LRRK2 inhibitors into the clinic as it should now become possible to readily assess the impact that LRRK2 inhibitors have by assessing the level of LRRK2 phosphorylated Rabs in vivo. It would be fascinating to explore whether monitoring Rab phosphorylation could also be used as a diagnostic method to assess whether Parkinson’s patients would benefit from drugs that targeted LRRK2. Finally tools to study Rab phosphorylation will help address the question of whether Rab GTPases are hyper-phosphorylated in Parkinson’s patients that do not have LRRK2 mutations and whether such patients would benefit from LRRK2 inhibitors.
Read the full article here: http://elifesciences.org/content/early/2016/01/28/eLife.12813
We have also initiated a search for two postdoctoral researchers to work in this area for further information please contact Dario Alessi (d.r.alessi@Dundee.ac.uk) or click here.
We want to capture some of the major achievements of students and postdocs who trained at the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC PPU), and have created a new feature on our website the 'MRC PPU Alumni Interview'. These interviews, conducted by Hazel Lambert a well-known science writer, will reveal what our alumni are doing now, what makes them tick, and they’ll share their insight and reflections regarding their time in the MRC PPU.
One of the most important missions of the MRC PPU is to recruit the most driven, talented, creative researchers in the world and provide them with an extraordinary research training experience while deciphering phosphorylation and ubiquitylation biology. The success of our alumni suggest we do just this.
In the first interview of this series Darren Cross an ex-MRC PPU PhD student in the 1990’s now a Principal Scientist at AstraZeneca, discusses the key role he recently played in initiating and leading the discovery phase of a new anti-cancer drug called Tagrisso. This project broke AstraZeneca records, with Tagrisso being the fastest drug to go through clinical evaluation.
Click the new MRC PPU Alumni Interview icon on the top of our website, or here to read the interview.
We have completely overhauled the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC PPU) reagents and services website which lists all of the MRC-PPU's, cDNA clones, antibodies and recombinant proteins that have been generated over the years in the MRC PPU and enables researchers worldwide to request these for use in their own experiments.
We would invite you to have a look at the revamped site using the below link:
You just need to type in the name of the reagent that you are looking for and if we have it in our collection you will get the list of all of the different versions of cDNA clones sorted by vectors and epitope tags, antibodies and recombinant proteins we have for this reagent. These can then be easily requested through the website.
The website currently lists 24868 cDNA clones, 440 purified recombinant proteins and 412 antibodies. Our website is continuously updated with new reagents as an when they are generated.
The new website also lists collections of reagents available for different signalling pathways that our Units work on and that can easily be perused including:
PI3K/PDK1/mTOR/AGC kinase Pathway,
TGFbeta and BMP Pathway
ERK Signalling Pathway
Components of the Ubiquitin Signalling Pathway (E1’s, E2’s, E3’, DUBs and Ub’s)
Interferon Signalling Pathway
DNA Replication Termination Signalling Pathway
Autophagy/Mitophagy Signalling Pathwa
Parkinson's Disease Pathways
Innate Immune Signalling Networks
There should be a comprehensive data-sheet for each reagent. If there isn’t a data-sheet, for example for some constructs that were made many years ago we will re-sequence these constructs and generate a new data-sheet for these constructs as and when they are requested.
In addition, the website describes services that our Unit offers namely antibody generation (including antigen preparation and affinity purification of antibodies), recombinant protein expression and purification from both bacteria and insect cells as well as cDNA cloning services that includes generating constructs for CRISPR/CAS9 knock-out and knock-in projects
Finally, please feel free to forward suggestions to us on ideas for improvements that we can make to improve functionality or fix glitches.
If you have any suggestions for improvement please don’t hesitate to e-mail your suggestions, however minor, to Matthew Elliott: email@example.com
We look forward to your continued support in 2016.