NIHRDataInsights/HRCSData huggingface dataset:
Title: The Structural Mechanism of K2P Channel Gating
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Two-pore-domain potassium (K2P) channels comprise a major and structurally distinct subset of the mammalian K+ channel superfamily, and underlie the background, or leak, currents that regulate the resting membrane potential and excitability of many mammalian cells. However, in contrast to many other classes of K+ channel, relatively little is known about the structural mechanisms of K2P channel gating. In particular it is unclear how different regulatory stimuli open the channel pore - do they open the channel at the helix bundle crossing or modify the proposed 'inactivation gate' at the selectivity filter? Also, it is unknown how these two gates are affected by the binding of different activatory ligands. We have recently identified a range of pharmacological tools which can be used to probe the structure and gating mechanism of the K2P channel pore. Using these tools our initial studies of TREK-1 indicate that the primary gating mechanism resides close to or within the selectivity filter and does not involve closure of the channel at the helix bundle crossing. We have also obtained evidence which indicates that a structural and functional asymmetry exists within the K2P channel pore, and that fundamental differences between the role of the pore-lining helices in TREK-1 and TRESK gating will provide an important insight into how the binding of regulatory ligands affects channel activity. This proposal aims to exploit these recent advances in order to probe the fundamental molecular mechanisms of K2P channel gating. To help with achieving these objectives the project involves an industrial partnership with Pfizer which provides unique access to a wide range of resources, expertise and new molecular tools.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mitochondrial Thiol Regulation and Programmed Cell Death in Yeast
Funder: Biotechnology and Biological Sciences Research Council

Abstract: All organisms contain complex regulatory machinery to maintain the redox status of -SH groups in proteins and low molecular weight sulphydryls. Glutaredoxins and thioredoxins are key oxidoreductases that constitute the main cellular redox systems Whilst there has been extensive research on the cytoplasmic systems, little is known regarding the mitochondrial systems This work programme will define the thiol regulatory systems which maintain the mitochondrial thioredoxin system and determine the role of mitochondrial thioredoxin (Trx3) in programmed cell death (PCD). The starting point for this project will be to determine the absolute cellular concentrations of the mitochondrial redox proteins. The GSSG/2GSH redox couple will be measured as a general indicator of the mitochondrial redox state and the redox state of Trx3 will be measured directly. These data will be used to establish how the mitochondrial thiol regulatory network is altered during conditions which induce PCD. We will test the hypothesis that oxidation of Trx3 plays a key role in regulating PCD by examining the requirement for Trx3 and its cysteine residues. The central question to be addressed is whether oxidation of Trx3 is a common marker of PCD and the availability of trx3 cysteine mutants will allow us to directly confirm that oxidation of Trx3 is mediating any effects. The mechanism of Trx3-induced PCD will be addressed by examining mitochondrial events which have been linked to apoptosis including changes in the mitochondrial membrane potential, respiratory activity and cytochrome c release. A particular focus will be to identify potential interactions with the yeast Yca1 metacaspase and Aif1 Apoptosis-inducing factor and other redox-dependent interactions which might be important for promoting PCD. We have preliminary data that the mitochondrial 1-Cys peroxiredoxin (Prx1) interacts with Trx3 and the final part of the project will examine the role of Prx1 in regulating Trx3-mediated PCD.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mitochondrial- and lysosomal-targeted iron chelators for the monitoring and adjustment of cellular labile iron pools
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Recent cellular studies have indicated that mitochondrial and lysosomal redox active and chelatable labile iron (LI) plays a key role in conferring cellular sensitivity to oxidative damage and the resulting pathologies and cell death. The design of organelle-targeted iron sensors for sensitive evaluations of LI distribution in lysosomal and mitochondrial subcellular compartments appears therefore to be an essential prerequisite for development of highly targeted iron chelators as preventive agents for iron-related oxidative injuries. Recently we have developed a promising HPO-based fluorescent iron III sensor (HPO-20) that exclusively monitors LIP in the lysosomal compartment. However based on current literature, there is no such exclusive equivalent compound for monitoring LI in mitochondrial compartment. This proposal responds primarily to the urgent need to develop highly specific mitochondria-targeted fluorescent iron chelators to be used both as biological probes to evaluate intra-mitochondrial LI and as mitochondria-specific iron chelators to selectively remove potentially harmful LI from mitochondria. To achieve high specificity, we plan to modify a series of fluorescent iron chelators by linking them to specific mitochondrial-homing peptides that can effect targeted delivery directed to mitochondria. The specificity, mode of action and protective properties of these mitochondrial iron sensors/chelators will be evaluated in a series of specific cell lines exposed or not to oxidising agents. The subcellular LI distribution in mitochondria, lysosomes and cytosol and subcellular LI trafficking under normal and oxidative stress conditions will also be evaluated in these cell lines. With no adequate organelle-specific iron sensors/chelators available, our compounds are expected to be of significant value to both biological and pre-clinical research as powerful biological probes and as highly specific preventive agents against iron-related oxidative injuries.


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES;5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE
Title: Studies of cellular processes involved in the control of nutrient signalling and their relevance to ageing
Funder: Biotechnology and Biological Sciences Research Council

Abstract: mTOR and the degradative process autophagy, which it regulates, are the key cellular nutrient response mechanisms They are vital for cellular physiology and are promising targets to extend healthy lifespan. Despite being in the spotlight of research in the last decade, the molecular details of the mechanisms regulating mTOR and autophagy in response to nutrients are still incomplete. We will build on our recently published observations showing that mTOR and autophagy are sensitive to nutrient-driven changes of intracellular pH (pHi). The project will aim to identify membrane transporters affecting mTOR activity and processes downstream of mTOR in response to nutrients. We will identify transporters that are required for the observed by us changes in pHi. We will also test the hypothesis that changing pHi alone is sufficient to control mTOR activation independently of nutrient status. The role of identified in cell culture membrane transporters in the nutrient signalling regulation will be assessed on the organismal level using drosophila as a model organism. The changes in drosophila TOR (dTOR) activity will be correlated with pHi changes and with the consequences for cellular physiology and lifespan. As one of the targets of this project, we hope to be able to identify (a) long-living mutant(s) in which dTOR activity is uncoupled from the upstream nutrient signalling and is controlled by changes in pHi alone. This integrative approach will not only expand our knowledge of the molecular mechanisms controlling TOR signalling and downstream processes but will also test the role of thus identified processes for organismal physiology. In addition to providing fundamental knowledge about nutrient-sensing mechanisms, our results may inform studies directed towards identification of new ways to correct perturbations of nutrient response control.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Investigating the role of inosine-containing double stranded RNA in mammalian cells
Funder: Biotechnology and Biological Sciences Research Council

Abstract: We have previously shown that the presence of inosine-containing dsRNA (IU-dsRNA) in mammalian cells suppressed interferon (IFN) induction and apoptosis in response to long dsRNA. Moreover, both effects could be accounted for by our observation that IU-dsRNA inhibited activation of IRF3 (IFN regulatory factor 3), a key component in the pathway by which long dsRNA induces IFN and apoptosis. In addition, our data suggested that IU-dsRNA acts at an early step in the pathway by inhibiting MDA5 (melanoma differentiation association protein 5) or RIG-I (retinoic acid-inducible gene I), which act as cytosolic sensors for dsRNA. These observations together lead us to propose that any IU-dsRNA generated by ADAR1 can directly inhibit IFN induction and apoptosis. We now aim to examine the molecular mechanism underlying the observation that IU-dsRNA suppresses IFN induction and apoptosis. We will use a combination of cell biology, molecular biology and biochemistry to investigate the specific interaction between IU-dsRNA and RIG-I or MDA5. Structural analyses will also be used to examine the molecular detail of this interaction. We will use quantitative proteomics to determine whether IU-dsRNA interacts with additional components of the IFN pathway, which may also be important for inhibiting IFN. The observation that IU-dsRNA inhibits IFN suggests editing may be pro-viral in mammalian cells. We will explore this idea using influenza A virus (IAV) as a model system. IAV triggers an IFN response via activation of RIG-I, and undergoes hyper-editing by ADARs. Finally, we aim to identify cellular IU-dsRNA(s) that suppress IFN induction and apoptosis. CLIP-Seq (Cross-Linking/Immunoprecipitation/Sequencing) will be used to identify edited RNAs interacting with RIG-I or MDA5, and the effect of these RNAs on IFN induction in mammalian cells will be validated using qPCR. In undertaking this analysis we will better understand the critical role(s) of RNA editing in mammalian cells.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Peptide-based solutions for light-triggered delivery of macromolecular therapeutics and nanoparticles
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The use of several important biopharmaceuticals is severely hampered by their limited ability to reach intracellular targets. This is either due to poor diffusion across the cell membrane or endosomal/lysosomal sequestration upon uptake . Photochemical internalisation (PCI) is a promising solution which exploits the photodynamic action of sub-toxic doses of photosensitisers to promote rupture of lysosomal vesicles, so that entrapped drugs can reach their targets. PCI is designed to use red light where tissue absorption is relatively weak, unlike blue or UV light, so light-triggered, site-specific drug release can be effected at therapeutically useful depths in tissue. We aim to develop innovative solutions to 3 key challenges in the PCI approach, using peptide-based delivery systems 1) Photosensitisers for PCI must be lysosomotropic, to localise in the same vesicles as administered drugs. This makes many photosensitisers clinically used for photodynamic therapy unsuitable for PCI. We aim to overcome this by conjugating photosensitisers to cell-penetrating peptides (CPPs) to both improve their uptake and control sub-cellular localisation. 2) PCI requires the photosensitiser and drug to localise in the SAME intracellular vesicle. We can achieve this by covalently attaching a drug cargo to a peptide carrier, from which it may dissociate and reach its target post-PCI. Alternatively, a peptide carrier, with the photosensitiser attached, may be used to generate a drug-encapsulating nanoparticle, such as a liposome. 3) For maximum efficacy, the combination of photosensitiser and drug should be delivered only to specific tissues. A CPP construct that is activated for uptake by disease-dependent levels of a protease activity is an ideal way to achieve this. We will characterise our constructs in detail with respect to their photophysical properties and efficiency at delivering diverse drug molecules in a variety of in vitro cell models and validate them in vivo.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE
Title: A novel ageing-related pathway regulating ROS homeostasis
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Reactive oxygen species (ROS) are a major cellular stress that is increased when mitochondrial function is compromised. ROS can damage cell components including DNA, proteins, and lipids, and contribute to cellular ageing and ageing-related disorders such as cancer, heart disease and neurodegeneration. It is therefore crucial that mitochondria communicate with the nucleus to promote the reprogramming of gene expression to combat the increased oxidative stress. We have uncovered a novel nuclear function for Clk-1, a mitochondrial hydroxylase involved in the electron transport chain. Genetic studies have demonstrated that reduced function of Clk-1 increases lifespan in both mice and the nematode C.elegans, but the underlying mechanisms are poorly understood. Our data show that increased ROS levels can direct a pool of Clk-1 to the nucleus where it associates with chromatin and regulates gene expression. We have demonstrated that nuclear Clk-1 is required for maintaining ROS homeostasis and for protecting cells from oxidative stress. The project will focus on key questions that remain including how ROS control Clk-1 nuclear localization, how Clk-1 coordinates gene expression to regulate ROS-sensitive signalling pathways, and establishing the biological significance of nuclear Clk-1. We will use cultured cells to uncover the mechanisms underpinning Clk-1 action and the model organism C.elegans to determine the importance of nuclear Clk-1 in vivo for regulating development, oxidative stress responses and longevity. The diversity of mechanisms controlling ROS homeostasis are only beginning to be characterised, so our finding of a novel function for Clk-1 as a ROS-dependent mediator of mitochondrial-nuclear communication will advance our understanding of how cells manage ROS levels during development and ageing, and also in ROS-related diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Combining Genomics and Phosphoproteomics to Identify Kinase-Substrate Networks that Promote Mitochondrial
Funder: Biotechnology and Biological Sciences Research Council

Abstract: In almost all cells insulin is a key factor in promoting mitochondrial biogenesis and function. In this proposal we aim to use a combination of functional genomic and phosphoproteomic technologies in order to describe, on a systems-level the key kinase-substrate reactions that promote mitochondrial homeostasis. Specifically we aim to implement a recently developed quantitative image-based readout to perform a high-throughput RNAi screen for regulators of mitochondrial morphology in drosophila S2R+ cells. This screen uses automated image processing algorithms to extract dozens of different features that describe mitochondrial shape in single-cells. We have already demonstrated that we can use this screen to identify genes, such as InR, Mef2, and p38, that contribute to regulating mitochondrial shape - and thus function. In parallel we will determine all proteins that are phosphorylated following insulin stimulation using phospho-proteomics. We have established a protocol for purifying phosphorylated peptides from trypsin digested samples of S2R+ cells using a combination of Immobilised Metal Affinity Chromatography (IMAC) and Titanium Dioxide (TiO2). Phosphorylated peptides are separated by hydrophilic interaction liquid chromatography (HILIC) and identified by mass spectrometry. The completion of these two aims will thus lead to the identification of proteins that are both required for mitochondrial morphogenesis and phosphorylated following insulin stimulation. These proteins are thus excellent candidates for proteins that link insulin to mitochondrial homeostasis. In order to determine the kinases responsible for phosphorylating these proteins following stimulation we will then perform a systematic screen using in vitro phosphorylation of substrate peptides mixed with cell-lysates prepared following RNAi of all drosophila kinases (KAYAK-RNAi screen). We will use classical methods such as combinatorial gene inhibition and genetic rescue to validate our findings.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Quantitative mapping of the proteomes of therapeutic stem cells.
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The key objective of the project is to develop tools for rapidly identifying when ES cell cultures acquire, during scale up for biomedical applications, genetic or epigenetic changes that might compromise their safe and effective use. We will provide to the consortium cultured adapted human ES cell lines exhibiting altered growth and differentiation patterns, and their early passage non-adapted counterparts. These lines will include already well characterised sets including normal/abnormal pairs of the human ES cell lines Shef4, Shef5, H1, H9 and H14. These typically have karyotypic changes but we also have stocks of late passage ES cells that have acquired altered growth patterns but without overt karyotypic change, perhaps due to sub-karyotypic genetic change or to epigenetic change. These and other lines provided by Neusentis will be assessed for various genotypic and phenotypic parameters of culture adaptation: genotypic change will be assessed by G-banding karyology and the presence of small populations of cells with gains of specific chromosomal regions will be assessed by interphase FISH. Analysis of copy number variation will also be performed when appropriate. Phenotypic analyses will include, as appropriate, the use of high content clonogenic assays to test for changes in the capacity of cells for survival, proliferation and differentiation, and time lapse microscopy will be used to assess cell motility parameters. Cell extracts will be prepared from normal and culture adapted sublines from the panel and provided to Cellzome for proteomic analysis. For specific experiments, cells will be sorted for expression of key stem cell markers such as SSEA3, SSEA4 and TRA-1-60, before sample preparation, to address the potential problem of cell heterogeneity caused by spontaneous differentiation. Proteomic differences that are then detected in culture adapted pairs of cells will be correlated with other geneotypic or phenotypic changes that are identified.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.2 CELLULAR AND GENE THERAPIES
Health categories: GENERIC HEALTH RELEVANCE
Title: A study of the role of anionic lipids in cell signalling: a mechanism for the modulation of ABC multidrug transporters.
Funder: Biotechnology and Biological Sciences Research Council

Abstract: There is evidence that the activities of many membrane proteins are modulated by anionic phospholipids in the bilayer. The levels and distribution of anionic lipids are modified in response to cellular events and this effect on membrane proteins provides a largely unexplored mechanism of cell signalling. We will characterise the binding of anionic lipids to ABC multidrug transporters SAV1866, from S. aureus, and mouse P-glycoprotein. Potential anionic lipid binding sites will be revealed by inspection of their crystal structures and coarse-grain molecular dynamic simulations of anionic lipids with these proteins. Anionic lipid binding sites will be established by replacing the three Trp residues in SAV1866 with single Trp residues at positions adjacent to potential anionic lipid binding sites. These single Trp mutants will be reconstituted into defined lipid bilayers containing brominated lipids that will quench Trp fluorescence by contact quenching. An analysis of the fluorescence quenching profiles in the presence of zwitterionic and anionic brominated lipids will identify the location of specific anionic lipid binding sites. Anionic lipid binding will also be correlated with effects on SAV1866 multidrug transport by measuring drug transport and ATPase activity in defined lipid bilayers and we will use mutagenesis of positively charged residues associated with the anionic lipid binding sites to further characterise these sites. We will also extend our studies to examine potential anionic lipid binding sites on P-glycoprotein. These studies will demonstrate how alterations in lipid composition and distribution (as occurs during the transition from log to stationary phase in bacteria or in lymphocyte signalling) can act to alter membrane protein function. In addition the characterisation of anionic lipid binding sites will identify targets that could be exploited in reducing drug resistance encountered in the treatment of cancer, microbial infections, etc.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE