NIHRDataInsights/HRCSData huggingface dataset:
Title: Small G proteins and the organisation of the Golgi apparatus
Funder: Medical Research Council

Abstract: All eukaryotic cells have a set of internal compartments that act together to synthesise lipids and membrane proteins, to release secreted proteins, to take up nutrients, and to degrade membrane-bound and internalised molecules. Proteins move between these compartments by means of transport vesicles that bud from, and then fuse with, specific organelles. The accurate organisation of membrane traffic requires that these vesicles find and fuse with only the correct destination organelle. Vesicles are initially 'tethered' to the correct membrane, and then fusion is mediated by a set of fusogenic proteins called SNAREs. However, the function and even identity of many of the components involved in tethering remain unclear, as do the mechanisms by which they are accurately recruited to the correct vesicles and organelles. We address these questions in the Golgi apparatus, an organelle that has a central role in the sorting and modification of proteins in the secretory pathway. The Golgi apparatus contains a number of long coiled-coil proteins called ‘golgins’ that have been suggested to act in vesicle tethering, and our recent work has show that this is correct for at least some. We are applying biochemical and genetic methods to identify interacting partners for these golgins, as well as using fluorescent microscopy in living cells to follow their dynamics. Most of these golgins are peripheral membrane proteins that are recruited to the Golgi directly from the cytoplasm. As with such proteins on other organelles, this recruitment is usually mediated via recognition of the active form of specific GTP-binding proteins. These ‘G proteins’ thus contribute to organelle identity, and the spatial accuracy of their activation underlies the organisation of membrane traffic. Therefore we are examining how the relevant G proteins are activated only on Golgi membranes, and what proteins they are recognising. One long term aim is to understand the fundamental principles by which the cell establishes a network of distinct but interconnected organelles. For much of our work we use either mammalian tissue culture cells or the fruit fly Drosophila melanogaster to investigate the function of the proteins we identify in the different tissues of a multicellular organism.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE
Title: Membrane protein sorting
Funder: Medical Research Council

Abstract: Proteins in cells are regulated in their abundance, and sometimes need to be removed entirely. A major challenge for cell biology is to understand how these processes are controlled. Proteins are also subject to quality control, with abnormal or misfolded proteins being degraded. Such mechanisms protect cells from the damage caused by misfolded proteins, and are closely related to those used for specific regulatory purposes where proteins need to be removed, such as the downregulation of signalling receptors. We wish to understand how proteins are recognised and destroyed, and how these processes can be regulated. The key event is ubiquitination, and we have focussed on the HECT family of ubiquitin ligases in mammalian cells. We have shown that aggregation or polymerisation of proteins can greatly enhance their ubiquitination by HECT ligases, and this may be used as a regulatory mechanism during signal transduction, notably through the polymerisation of the Dvl proteins in the Wnt pathway. It may also be used as a way of detecting defective proteins, which may aggregate following a heat shock, for example. The presence of many weak binding sites in close proximity has a dramatic effect on attracting and activating the ligases. We have previously shown that multiple binding sites are a common and necessary feature of natural activators of these enzymes, even though a single PPxY motif is sufficient for interaction. Alpha synuclein, the causative agent of Parkinson’s disease, forms pathogenic beta sheet filaments. We have found that these, but not the monomeric protein, are ubiquitinated by the Nedd4 family of HECT ligases, apparently by direct recognition of the beta sheet form. Subsequent degradation may limit toxicity. A point mutation in alpha synuclein that leads to early onset of disease largely abolishes ubiquitination, which may contribute to its damaging effects.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Mechanisms of mRNA localisation and cytoskeletal transport
Funder: Medical Research Council

Abstract: Cytoplasmic transport of organelles and macromolecules by molecular motors is of fundamental importance for the establishment and maintenance of cell polarity. In addition, defective motor transport is implicated in neurological diseases and pathogenic viruses and bacteria frequently exploit cellular transport routes. The challenge of studying microtubule-based transport is that movements are driven by large macromolecular assemblies, with a single cargo often bound simultaneously by multiple opposite polarity dynein and kinesin motors. Using a tractable model system in Drosophila embryos we have shed light on mechanisms responsible for polarised sorting of specific mRNA molecules by microtubule motors. We have revealed molecular links between localising mRNAs and dynein and used novel in vitro motility assays to show that long distance movement of the motor is activated by mRNA localisation signals. Recently, together with Andrew Carter’s lab at the LMB, we have used purified mammalian proteins to show that the adaptor protein BicD is a key factor in switching on dynein processivity. We have also elucidated the mechanisms of dynein regulation by Lissencephaly-1 and mRNA localisation signals. We have also been investigating how cargo transport is orchestrated in neurons using the intact Drosophila nervous system as a model. We recently established a novel imaging assay in the adult wing and are using it to study the relationship between axonal transport and the healthy lifespan of neurons. We are also investigating how specific mRNAs are sorted into axons and dendrites in neurons, and how these processes contribute to neuronal development and function. We have developed and distributed optimised tools for CRISPR genome engineering in Drosophila, and are using these in several of our own projects. Our long-term plans are to combine genetics, cell biology, structural studies and single molecule assays to understand the key processes that control sorting of cargos and pathogens by motors. This should have therapeutic relevance; for example, it may be possible to target drugs to discrete regions of the cell to maximise their efficacy or to develop agents that stimulate motor activity in order to alleviate problems associated with defective transport in neurodegenerative diseases.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Evolution of Novel Biopolymers
Funder: Medical Research Council

Abstract: Synthetic biology seeks to probe fundamental aspects of biological form and function by construction (i.e. resynthesis) rather than deconstruction (analysis). Synthesis thus complements reductionist and analytic studies of life, and allows novel approaches towards fundamental biological questions. We have been exploiting the synthesis paradigm to explore the chemical etiology of the genetic apparatus shared by all life on earth. Specifically, we ask why information storage and propagation in biological systems is based on just two types of nucleic acids, DNA and RNA. Is the chemistry of life’s genetic system based on chance or necessity? Does it reflect a "frozen accident", imposed at the origin of life, or are DNA and RNA functionally superior to simple alternatives. We have developed advanced generation selection strategies, that allow the evolution of polymerases capable of utilizing exclusively unnatural nucleotide substrates enabling the enzymatic synthesis, replication and evolution of entirely synthetic genetic polymers (XNA). Such systems expand the central dogma and will conclusively address questions such as the capacity of genetic polymers other than DNA and RNA for information storage and propagation (i.e. heredity). Beyond heredity, we have demonstrated a capacity for Darwinian evolution by the de novo selection of specific ligands (XNA aptamers) and catalysts (XNAzymes) based on these entirely synthetic backbones. Thus, key hallmarks of living systems, including heredity and evolution are not limited to DNA and RNA but can be implemented in synthetic polymers and are likely to be emergent properties of polymers capable of information storage. The further exploration of the informational, structural and catalytic potential of synthetic genetic polymers, should advance our understanding of the parameters of chemical information encoding, and will provide a source of novel ligands, catalysts and nanostructures with tailor-made chemistries for applications in biotechnology and medicine. We have also leveraged our selection technology to make progress in the engineering and evolution of RNA polymerase ribozymes towards a general polymerase and self-replication capacity We have discovered RNA polymerase ribozymes that are capable of the templated synthesis (i.e. transcription) of another simple ribozyme or RNA oligomers exceeding their own size (>200 nts), a key milestone on the road to self-replication.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.3 CHEMICAL AND PHYSICAL SCIENCES
Health categories: GENERIC HEALTH RELEVANCE
Title: Vertebrate mutagenesis and DNA damage tolerance
Funder: Medical Research Council

Abstract: The group studies how problems encountered during DNA replication lead to both genetic mutation and to epimutation, changes in gene expression caused by local alterations in the chromatin of somatic cells. DNA replication is readily interrupted by DNA damage and by naturally occurring secondary structures in the DNA. DNA damage frequently stalls the replicative DNA polymerases but can be replicated by specialised translesion DNA polymerases, which possess active sites that are capable of accommodating damaged or distorted DNA templates. While the use of these polymerases allows replication to be completed it also increases the chance of mutagenesis. A major goal of the lab has been to understand how and when these enzymes are deployed and how their activity is regulated to allow efficient and safe replication of damaged DNA. We have shown that in addition to assisting in the replication of damaged DNA, some specialised polymerases, acting in concert with specialised DNA helicases, also play a role in the replication of DNA secondary structures. We have shown how one type of DNA secondary structure, the G-quadruplex, stall DNA replication, interrupt histone recycling and locally prevent the proper inheritance of histone modifications through cell division leading to altered gene expression. This observation has linked replication stress to disruption of epigenetic memory and to variations in gene expression, a feature of many cancer cells. We are working to further understand how DNA replication impediments impair epigenetically encoded transcriptional memory and the circumstances in which loss of coupling between DNA replication and the propagation of epigenetic information leads to disease. Finally, our work, which predominantly uses somatic cell genetics, has also led to the identification and development of cell lines capable of continuous mutagenesis which we have used to evolve specific antibody specificities and other proteins of therapeutic interest.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Solution structure by NMR spectroscopy
Funder: Medical Research Council

Abstract: We will use NMR spectroscopy, and where appropriate X-ray crystallography, to determine the structures of a number of proteins and their complexes with DNA, with peptides and with other proteins. Where possible, high-resolution structures of the relevant complexes will be determined directly using extensive NOE measurements; in the cases of some weaker complexes (e.g. those of proteins with small peptides derived from the sequence of a larger binding partner), ligand footprinting will be carried out using a combination of chemical shift titrations, cross-saturation experiments and, where possible, intermolecular NOE measurements. A key component of the work will be a study of human poly-ADPribose polymerase-1 (PARP-1); this protein detects and signals the presence of DNA single-strand breaks so as to initiate repair, and it is an important target in cancer therapy. NMR is particularly well-suited to characterise the intermolecular and interdomain interactions that underpin PARP-1’s molecular mechanism of action upon DNA recognition. Other targets for study will be selected from amongst the projects ongoing in other groups at LMB and CIMR, and will be undertaken as collaborations. In particular, protein structures and protein-peptide interactions relevant to vesicle trafficking and intracellular immunity will be studied, among others. Solution structures of a number of protein domains and their complexes will be determined.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Systematic genetic code reprogramming
Funder: Medical Research Council

Abstract: We will extend our progress towards the synthesis of an orthogonal genetic code. We will evolve the orthogonal ribosome and develop orthogonal aminoacyl-tRNA synthetase tRNA pairs to develop an orthogonal translation system for genetically encoding multiple distinct unnatural amino acids or other monomers. The genetic encoding of multiple distinct unnatural amino acids in combination with exploration and discovery of amino acids that can undergo bio-orthgonal chemical reactions will allow us to genetically encode new covalent constraints in proteins as well as to assemble biophysical probes (such as fluorophores) on intracellular proteins via proximity accelerated chemical reactions. The approaches we develop will also allow us to genetically encode or install post-translational modifications including mono-, di- and tri- methylation, ubiquitylation, and phosphorylation (as well as combinations of these modifications) that will drive a molecular understanding of the role of post-translational modifications in chromatin and non-histone proteins. We aim to extend the chemical scope of the ribosome by extending the orthogonality to the large subunit of the ribosome - where peptide bonds are formed - and evolve the peptidyl transferase center to polymerize non-alpha-L amino acid monomers. Expanding the chemical scope of orthogonal translation, in combination with the ability to encode the incorporation of multiple unnatural monomers will allow us to genetically encode and evolve polymers that may have superior properties (for human purposes) to natural polymers, and to investigate the evolution of new life forms with an additional class of genetically encoded polymer.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Spindle Pole Body duplication
Funder: Medical Research Council

Abstract: We have proposed a molecular model for SPB duplication based on the identification of core SPB components and their localization by immuno-electron microscopy during SPB duplication. A central feature of the model is the half-bridge, a specialized membranous structure attached to one side of the SPB, and the ability of the half-bridge to bind core SPB components at either end. I have identified a new half-bridge component, Sfi1p, which binds multiple copies of the yeast centrin, Cdc31p, through a continuous set of novel repeats. I am investigating the role of Sfi1p in SPB duplication by looking at its interactions with other SPB components and its role in the morphology of the half-bridge. I am also characterizing a potential human homologue of Sfi1p, hSfi1 which localizes close to centrioles, and whether it has a role in centriole duplication.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Structural studies of phosphoinositide signalling and intracellular sorting
Funder: Medical Research Council

Abstract: Eukaryotic cells have evolved a number of mechanisms to respond to environmental stress. These stress responses are tempered by growth factors and other signals from surrounding cells in multicellular organisms. Our research programme involves the mechanisms whereby these cellular signals are coupled to regulation of anabolic and catabolic processes in mammalian cells. Phosphoinositide 3-kinases (PI3Ks) modify lipid membranes and the modified phospholipids produced by the enzymes serve as second messengers and sorting signals in eukaryotic cells. The PI3Ks are essential for cell response to environmental cues. Furthermore, PI3Ks are part of a larger family of kinases known as the PI3K-related kinases (PIKKs). We study the structures, dynamics and functions of PIKKs and related pathways in cellular signalling, sorting and stress responses. This has included almost all of the members of the PI3K family: class I PI3Ks activated by receptor tyrosine kinase and GPCRS, the primordial class III PI3K complex present in all eukaryotes, the golgi-associated PI4K, the PI3K related protein kinases target of rapamycin (TOR) and PIKKs such as ATM involved in DNA repair pathways. Each of these PIKKs is a regulatory nexus, and our work produces variants in which specific regulatory inputs of the large enzyme complexes are ablated without affecting other activities. These modified complexes provide tools to decipher cellular regulation in ways that would not be possible with other approaches. We use X-ray crystallography, cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to marry structures with dynamics. In collaboration with our pharmaceutical partners, we are seeking to develop HDX-MS approaches to give better insight into conformational dynamics that could result in new therapeutic strategies. On-going efforts in the group concern the structural and dynamic basis of regulation of oncogenic PI3Ks, autophagic PI3K complexes, integration of amino acid and growth factor sensing in the PI3K pathway, regulation of ribosomes in response to amino acid starvation and DNA damage repair.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Membrane protein structure by electron cryomicroscopy
Funder: Medical Research Council

Abstract: The overall goal of our work is to determine the atomic structure of interesting or important membrane proteins and membrane protein complexes. We have chosen to work on membrane proteins because many of the most interesting functions in cells involving transport or signalling across membranes are carried out or controlled by proteins in the cell membrane. We are interested in atomic structures because this brings a precise understanding of function at the fundamental level of the chemistry of the interactions. Although much can be done using existing methods and approaches including X-ray crystallography of 3D crystals, electron crystallography of 2D crystals, and single particle cryoEM, we know from direct experience that there are many structures that are still difficult to determine even when significant amounts of pure material have been obtained. There are also many membrane proteins, such as the human voltage-gated sodium channels (VGSCs), which are relatively unstable after purification in detergent: for this class of membrane proteins also, new approaches are required. We are therefore working to improve the power and resolution of single particle electron cryomicroscopy, to allow more efficient determination of atomic structures, with less effort and on more fragile specimens. Our work encompasses the technical improvement of single particle cryoEM as well as analysis of the structure of selected membrane proteins or membrane protein complexes.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Structural and functional studies of the pre-mRNA splicing machinery
Funder: Medical Research Council

Abstract: Pre-messenger RNA splicing is a crucial step of eukaryotic gene expression that removes introns from mRNA precursors (pre-mRNA) to produce mature mRNA with continuous protein coding sequences. Pre-mRNA splicing is catalysed by an intricate molecular machine called the spliceosome and proceeds by a two-step phosphoryl transfer mechanism, analogous to group II intron self-splicing. Our research aims to gain insights into the mechanism of pre-mRNA splicing through biochemical, genetic and structural studies of the spliceosome. The spliceosome’s major components are U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP proteins, including the nineteen complex (NTC) and the nineteen-related (NTR) proteins. The spliceosome is assembled de novo from these building blocks on each intron and disassembled when the splicing reaction is complete. The spliceosome has no preformed active site and it is created only after remodeling of the fully assembled spliceosome. RNA helicases play crucial roles not only in generating the RNA-based active site but also in docking and undocking the substrates (BP adenosine and the 3′SS) together with step-specific factors. We have obtained snapshots of the spliceosome at several key steps during the assembly and catalysis. These structures include the A complex (Plaschka et al. in press), the U4/U6.U5 tri-snRNP (Nguyen et al., Nature 530, 298–302 (2016)) and the pre-catalytic B complex (Plaschka et al., Nature 546: 617-621 (2018). We have also determined the cryo-EM structure of the spliceosome immediately after the first trans-esterification reaction (Galej et al., 537, 197–201 (2016)); the spliceosome stalled after Prp16-mediated remodelling but before exon ligation (Fica et al., Nature 542, 377-380 (2017)); the P complex immediately after exon-ligation (Wilkinson et al., Science 358: 1283-1288 (2017)). These structures have provided new insights into the mechanisms of spliceosome assembly, activation, catalysis and disassembly. In conjunction with the cryoEM studies we carry out biochemical and genetic experiments to understand how this intricate molecular machine is assembled, activated and functions to achieve precise excision of introns from pre-mRNA.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE
Title: Structure and function of the translational machinery
Funder: Medical Research Council

Abstract: The ribosome plays a central role in translation of the genetic code into protein. It is also a major target for many clinically important antibiotics. In higher organisms, the initiation of translation is regulated by the cell and disruption of this process has been implicated in cancer and viral infections. Following on our work on the structure and function of bacterial ribosomes in many states of translation, we are currently focussing on eukaryotic and mitochondrial ribosomes and in particular their regulation and involvement in quality control. We also aim to understand how viruses inhibit ribosome function.


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