NIHRDataInsights/HRCSData huggingface dataset:
Title: The organisational structure of class A GPCRs: Implications for function and drug design
Funder: Medical Research Council

Abstract: G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and the most successfully exploited for the development of therapeutically active, small molecule medicines. All of the currently employed medicines that target GPCRs have been identified and developed based on the presumption that GPCRs exist and function as monomeric species. However, although it has been established that some rhodopsin-family GPCRs can function as monomers, in recent times it has also been established that most, if not all, GPCRs exist in cells as dimers or higher-order complexes. As well as self-associations that generate homo-dimers/oligomers, there is also the potential for interactions between certain pairs of co-expressed GPCRs to generate hetro-dimers/oligomers. As a number of these display distinct regulation and pharmacology, they may represent novel sets of drug targets. A large number of key questions remain, however, before this potential may be realised. This application will employ both current and developing methodologies that require substantial optimisation to explore the quaternary structure of both homo-and hetero-dimers of class A GPCRs at distinct points in their life cycle. Central points that I will adress include: (1) the molecular basis of alpha1-adrenoceptor homo-dimerisation and the minimum size of the complex, (2) if activation of class A GPCR homo-dimers results in signal generation via ?trans-activation? between the component protomers,(3) the allosteric regulation of GPCR hetero-dimers/oligomers, (4) the pharmacological basis of hetero-dimer/oligomer selective ligands and (5) the molecular basis and size of GPCR hetero-dimers/oligomers.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: The structural basis of cell surface receptor signalling mechanisms
Funder: Medical Research Council

Abstract: I propose to dissect the signalling mechanisms of a set of type 1 single membrane spanning receptors. The programme is sub-divided into three project areas each of which addresses aspects of cell adhesion/signalling and the mechanisms by which cell surface receptors can deliver the exquisitely fine local control of information transfer required for embryonic development and tissue morphogenesis. ? Cell guidance: Sema6 ? Plexin A interactions ? Cell adhesion: the FLRTs ? Crosstalk in receptor tyrosine kinase signalling: FLRT ? FGFR, FGFR ? N-cadherin Detailed structural information for each of these systems is of biomedical relevance and in combination they exemplify an interconnected series of questions pertinent to the mode of action of single membrane spanning receptors in general. What mechanisms control signalling through the cell surface membrane? How is bi-directional signalling orchestrated? What is the role of receptor homo- or hetero- dimerisation verses higher order oligomerisation? How does the interplay of cis interactions, providing crosstalk between receptors on the same cell, and trans interactions, mediating cell-cell communication, function? To date studies by us and others have provided substantial insight into extracellular receptor architecture and ligand-receptor specificity, the challenge now is to link extracellular recognition with intracellular signalling and to address not just the isolated molecules but rather the interplay of cell surface receptors relevant to function in the cellular context. Based on the methodological developments we made during the previous quinquennium of MRC (and CR-UK) funding it is now timely for us to tackle this next level of complexity.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Molecular Cell Biology of Post-Golgi Membrane Traffic Pathways
Funder: Medical Research Council

Abstract: We will continue to investigate the molecular mechanisms of protein sorting and organelle biogenesis/maintenance in post-Golgi membrane traffic pathways. Our principal aims during the next five years are (i) to understand the sorting and trafficking of post-Golgi SNAREs and (ii) to understand the coordination and integration of protein machineries required for the delivery of endocytosed proteins to lysosomes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Elucidating the molecular mechanism of drug transport through structural studies of peptide transport proteins.
Funder: Medical Research Council

Abstract: The proton oligopeptide cotransporter family, SLC15, is a family of integral membrane proteins that utilise the proton motive force to power the uptake of short chain peptides and peptidomimetics into a variety of cells. These proteins are the major routes of dietry nitrogen absorption from the small intestine and nitrogen re-absorption from the glomerular filtrate in the kidney. They are of broad pharmaceutical interest due to their ability to actively uptake a number of pharmaceutically important drugs, such as the beta-lactam antibiotics, certain antivirals and HIV protease inhibitors. The aim of this proposal is to elucidate the molecular mechanism by which the SLC15 family of integral membrane proteins function and to gain a deeper understanding of drug transport and oral bioavailability. This research has significant clinical utility, as oral administration is currently the most successful route for drug administration. An understanding, at the molecular level, of how drugs are both recognised and transported across the gut epithelium will aid in more rational drug design and should have significant translational potential. I plan to achieve these goals principally through the insights gained from X-ray crystallography. X-ray crystallography is particularly powerful in this regard as it allows currently unparalleled insights into molecular form and function. Functional studies will also be undertaken and directed by the insights obtained from the structural models. This will be a collaborative project with both eukaryotic and prokaryotic transporters being studied. Using the expertise I have gained in the laboratory of Prof. So Iwata I am currently in the process of determining the first crystal structure of a peptide transport protein. The insights gained from this structure will make a significant contribution to our understanding of drug uptake in the human gastrointestinal tract. Through collaborations with medicinal chemists and physiologists I plan to extend this research to include the human members of this transport family. During my post-doctoral research I helped develop a rational platform for optimising membrane protein transporters for structural studies. This included screening for solubility, monodispersity and thermal stability in different detergents using small quantities of protein to allow rapid and cost effective identification of suitable targets. These methods have worked successfully for the bacterial peptide transport proteins and I now plan to utilise these methods to work on the mammalian SLC15 family members.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Common epigenetic pathways regulating ageing and circadian clocks: implications for therapeutic intervention
Funder: Medical Research Council

Abstract: Abstract The rapid increase in the ageing population represents both a major biological and social challenge. Among these are changes in circadian rhythms, the endogenous 24 hour cycles governing nearly all aspects of mammalian physiology and behavior, including sleep-wake cycle. During ageing, one of the most common and troublesome events are sleep disorders, implying fundamental changes within the circadian pacemakers that gate sleep patterns. It is unclear how ageing impacts on the molecular clockworks and what the underpinning mechanisms are. Epigenetic modifications have recently been implicated in the regulation of circadian clocks. Epigenetic processes are also recognized as a hallmark of ageing. Remarkably, sirtuins are implicated in both processes through their actions on the interface between energy metabolism and cellular senescence. These findings implicate common epigenetic changes in ageing process and altered circadian pacemaker function. The primary aims of the proposal are: 1) To define how ageing affects the overall organization of the molecular clock machinery in animals and man. 2) To test the hypothesis that ageing may impact on the balance of acetylation/deacetylation activity or histone methylation state in pacemaker cells, which leads to alterations in overt output rhythms 3) To probe the possibility that pharmacological/genetic manipulation of the epigenetic-regulated pathways may counteract age-related changes in circadian rhythms In this proposal, I will use young and aged transgenic reporter mice to define changes within circadian clocks, including period, amplitude, resetting response and clock gene activity in neural and peripheral pacemaker cells using real-time bioluminescent imaging and biochemical approaches over multiple circadian cycles. I will study age-related changes in expression and activity of CLOCK and SIRT1, acetylation or methylation state of histones and key clock proteins in tissues and primary cells, complemented by the use of an oxidative stress-induced cellular ageing model. The impact of manipulating SIRT1 activity on circadian clocks will also be examined. Translational studies using human fibroblasts isolated from young and elderly subjects will be undertaken using clock gene reporter technologies. The molecular rhythms thereby revealed will be correlated with age and chronotype, as assessed by standard questionnaire. Circadian clocks have pleiotropic effects and disrupted circadian rhythms correlate with age-associated sleep disorders and other pathologies. This project aims to define the mechanisms underlying age-associated changes within circadian system in animals and man. Long term, outcomes from this work could aid therapeutic drug design against age-related ailments, for which targeting SIRT1 is already an appealing candidate.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Investigating a role for the cohesin complex in chromatin looping, gene regulation and development
Funder: Medical Research Council

Abstract: Cohesin proteins have a well established role in sister chromatid cohesion. In addition to their structural role, new evidence implicates cohesins in the regulation of gene expression. Cohesin has been shown to colocalise with CTCF on mammalian chromosome arms CTCF is a sequence-specific DNA binding protein known to function as a transcriptional regulator, chromatin insulator and boundary element. The functional association between CTCF and cohesin provides one way by which cohesins could influence the expression of genes. Both inter- and intra-chromosomal CTCF-dependent chromatin loops have been described at several loci. It is likely that cohesin proteins could mediate CTCF-dependent chromatin loops due to their unique ability to establish links between chromatin fibres, as well as their colocalization to CTCF sites reported to be involved in chromatin looping. By constraining the conformation of chromatin, cohesins could affect the probability with which distal regulatory elements interact and thus gene expression. The first aim of this proposal is to address whether cohesin proteins, rather then simply behaving as architectural elements, can re-use their ability to tether chromatids to mediate chromatin loop conformations and if these conformations are important for gene function. RNAi and genetic methods will help to establish if these interactions are functional. The binding of CTCF to chromatin is sensitive to DNA methylation and recent reports indicate that DNA methylation profiles change extensively during development. Indeed, cohesin mapping data reveals cell-type specific patterns of cohesin binding that correlate with differential methylation. The second aim of this proposal is to address whether CTCF-dependent cohesin recruitment to specific sites changes from pluripotent to committed cells. The differential localization of cohesin and CTCF proteins could function to regulate the expression of these genes, possibly through the formation of cell-type specific chromatin loops or through the recruitment of regulatory complexes whose composition changes during development. I will use readily available molecular and computational biology techniques to study chromatin topology and cohesin localization during development, at both specific loci and genome-wide to gain insight into the role of cohesins in gene regulation. Cohesins play a key role during cell division to prevent anueploidy. Normal cells are able to sense DNA damage or abnormal chromosome segregation and will cease to divide whereas cancer cells can override these checkpoints and accumulate abnormal DNA. Thus, having a basic understanding of the mechanisms and proteins which facilitate normal chromosome transmission and function will be directly relevant to an understanding of cancer.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Molecular pathways that coordinate telomere maintenance and DNA repair machineries
Funder: Medical Research Council

Abstract: Double-stranded DNA breaks (DSBs) are normally repaired by ligation or recombination. Aberrant healing of a DSB by telomere addition can cause terminal chromosomal deletions associated with human ageing, genetic disorders and cancer. Previously, I established that activation of the Mec1-Rad53 DNA damage (DD) signalling pathway leads to phosphorylation of the telomerase inhibitor Pif1, which then promotes telomerase activity at telomeres but inhibits telomerase at DSBs. The PIF1 phospho-site mutation pif1-4A specifically abrogates DD-induced effect on telomerase, while keeping the normal Pif1 function intact. Therefore, DD signalling acts via Pif1 to modulate telomerase to become telomere- versus DSB-specific. To establish and define the mechanistic link between telomere synthesis and the DD response and to test the evolutionary conservation of this regulatory pathway in mammals I will 1. Elucidate the mechanistic role of Pif1 phosphorylation in response to DD for its function in telomerase inhibition at DSBs by A) comparing Pif1/Pif1-4A protein-protein interactions and their dynamics in response to DD (protein immunopurification+MassSpec), B) identifying modulators of Pif1 activity at DSBs via genome-wide genetic screens for pif1-4A suppressors and C) defining the role of Pif1 in dynamics of protein composition at DSBs (ChIP of known DSB-localizing proteins). 2. Dissect molecular mechanisms involved in telomere extension in response to DD by investigating the dynamics of the telomere-capping Stn1-Cdc13 complex and telomerase (ChIP and CoIP) and addressing the possible role of previously identified Stn1 posttranslational modifications in this dynamics. 3. Address whether the link between telomere elongation and the DD response plays a key role in telomere integrity and genome stability by assaying PIF1 and pif1-4A for telomere integrity under conditions of limited telomerase (cell viability/senescence test) and for telomere protection upon DSB induction (DSB-telomere fusions quantified by qPCR). 4. Determine the evolutionary conservation of Pif1 function and its regulation in response to DD in mammalian cells by analysing mammalian Pif1 phosphorylation in response to DD (MassSpec) and localization to DSBs (immunofluorescence). Uncovering the mechanistic basics of the Pif1-dependent inhibition of telomerase at broken DNA ends, but not at telomeres, during the DD response opens a gateway to our understanding of how cells coordinate DNA metabolism pathways to promote genome stability. This research not only addresses one of the fundamental questions in basic genome biology but also has important implications for approaching human health problems associated with genome instability, such as normal human ageing, multiple genetic disorders, and especially for understanding cancer cause and therapy.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Development of interventions to enhance health and wellbeing in later life (The LIVEWELL programme)
Funder: Medical Research Council

Abstract: The dramatic increase in human longevity has been accompanied by an increasing burden of frailty and of chronic poor health. Much of humankind?s experience of ill-health and expenditure on medical and social care (especially in Western countries) are concentrated in the later years of life. The challenge is to find ways of improving health and maintaining wellbeing throughout the life-course. In contrast with the wealth of observational data supporting the hypothesis that health and wellbeing in later life are influenced strongly by behavioural factors and social conditions, there is a dearth of evidence about interventions that are effective in promoting improved health and wellbeing. Prerequisites for the development of such interventions include identification of i) intervention modalities that will be effective, culturally appropriate and cost effective; ii) windows of opportunity when interventions are likely to be most effective; and iii) outcomes measures responsive to the interventions that can measure change in health and wellbeing. The LIVEWELL programme will fill this research gap by addressing these prerequisites for interventions. Based on the MRC framework for development and evaluation of complex interventions, we will identify types of intervention that are expected to be feasible, effective, and cost effective i.e. behavioural and social changes that will be attractive to older people. The lack of outcome measures which capture the healthy ageing phenotype, as distinct from measuring disability or ill-health, is a significant impediment to the evaluation of interventions. We will develop tools for this purpose. We will focus on the ?retirement window? i.e. the period before and after the main income provider in a household retires from primary employment. In discussion with older people, and based on both rigorous systematic reviews of the literature and behaviour change theory, we will develop interventions to address each of the main modifiable behavioural and social factors. This will include novel interventions in the areas of diet (encouraging a Mediterranean eating pattern), physical activity (e.g. walking and swimming) and social interactions. We will integrate these new interventions with more established interventions on smoking cessation and moderate alcohol consumption to develop a package of interventions which, when appropriate, can be tailored to the needs of individuals and communities. Further, we will develop novel ways of delivering these interventions which will be feasible and cost-effective and which will encourage sustained behaviour change and improved social circumstances in preparation for later studies to test the intervention package.


Research activity codes: 3.1 PRIMARY PREVENTION INTERVENTIONS TO MODIFY BEHAVIOURS OR PROMOTE WELL-BEING;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE
Title: Dynamics and function of early endsomes
Funder: Medical Research Council

Abstract: Mitogenic growth factors act by triggering the activation of signalling cascades, and early endosomes play a crucial role in this process by providing spatially discrete platforms for the assembly of signalling protein networks. The duration of signalling is controlled by the progress of activated receptors through early endosomes, and this in turn is linked to the maturation of the endosomes themselves. We have shown that early endosomes, identified by the presence of Rab5, are highly motile structures that exhibit very complex dynamics, moving both towards and away from the cell centre, and switching from moving to stationary phases and back. Since we have previously demonstrated that the dynein-driven movement of endosomes at a later stage in the endocytic pathway contributes to their maturation and their ability to sort cargo, we now wish to test the hypothesis that microtubule- and actin-based motility make fundamental contributions to the function and maturation of endosomes shortly after receptor internalisation. The key questions we will address are: 1. Do the dynamics of these early endosomes alter during the passage of cargo and during their maturation? 2. Does endosome motility control endosome maturation, morphology and cargo transit? 3. Does interfering with endosome dynamics influence cell signalling? Our approach will be to image living cells expressing low levels of fluorescent proteins that mark different populations of signalling endosomes, cargo molecules, and the phosphoinositide-modifying proteins that are vital for endosome maturation. We will analyse and compare the motility of these different markers quantitatively using newly-developed algorithms that allow us to automatically track all moving endosomes within living cells and define their motile properties. A key advance in this proposal will be to image two colours simultaneously at ~30 fps, enabling us to analyse the behaviour of pairs of endosomal proteins. These studies will establish whether steps in the signalling and maturation pathways correlate with alterations in motility, such as switching from moving to stationary, or from short range to long range motion. We will determine the contribution that motors make to endosomal maturation and signalling by disrupting the function of key microtubule- and actin-based motors. In addition, we will test whether changes in endosome motility are linked to the flux in phosphoinositides that occurs as endosomes mature. Our extensive background work tracking and analysing the motility of Rab5-positive endosomes makes us uniquely qualified to address these important questions and contribute to the understanding of mitogenic signalling.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Statistical Methods for Pharamacogenetics
Funder: Medical Research Council

Abstract: Pharmacogenetics (PGx) has the potential to generate improved drug therapies for patients, particularly in more accurate dose-finding and avoidance of adverse events, as well as economic benefits to the pharmaceutical industry (possibility to rescue drugs not currently marketable) and to healthcare providers such as the NHS (reduced prescriptions of ineffective or harmful drugs). Broadly speaking, PGx has until now failed to deliver substantial return on investment, but the potential rewards are so great that continuing effort and investment is warranted. Because of small and heterogeneous datasets, appropriate statistical analyses are crucial to maximising the output of pharmacogenetics research, but to date there appears to have been relatively little investment, at least in the public sector, in the development of statistical methods tailored to the needs of PGx research. In this proposal we seek to make several contributions to statistical methodology to enhance the prospects of PGx, focussing on two broad strands: (1) the prediction of drug response from genome-wide genetic data, and (2) Bayesian modelling of population and other sources of heterogeneity in order to extract more information from complex data sets.


Research activity codes: 1.4 METHODOLOGIES AND MEASUREMENTS;5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE