NIHRDataInsights/HRCSData huggingface dataset:
Award Title: Molecular Mechanism of Autophagy – Resolving Autophagosome Formation in Time and Space
Funder: The Francis Crick Institute

Abstract: How does the cell form a customized waste basket when bacteria suddenly invade the cytoplasm, when mitochondria stop functioning threatening the cell by releasing reactive oxygen species or when nutrients become so scarce that the cell needs to reclaim and recycle a diverse range of cellular building blocks? Autophagy holds the answer to all those seemingly unrelated challenges a cell may face throughout its life. Autophagy initiates a fascinating cellular response by converting small vesicles into sheet-like structures which upon closure give rise to double membrane vesicles allowing fusion with the lysosome/vacuole. There the cargo is degraded allowing for the recycling and repurposing of all contained cellular building blocks equipping the cell with a powerful mechanism to adapt to its ever-changing environment by maintaining cellular homeostasis. Additionally, autophagy also equips the cell with the ability to neutralize potentially cytotoxic threads such as invading pathogens, damaged or non-functional organelles or protein aggregates explaining the many beneficial properties attributed to autophagy for human health. Consequently, deregulation of autophagy is associated with many human diseases such as cancer, neurodegeneration and infection. Autophagy is a captivating but also very complex process which requires multiple cellular factors and layers of regulation. The individual steps involved are difficult to study solely in the crowded environment of the cell. Hence, the lab has developed a cell-free system to re-build the process of autophagosome formation in the test tube to understand when, where and how each of the components contributes to autophagosome formation. This system can be manipulated at will, allowing us to dissect and visualize the different steps in order to gain unprecedented insights into the mechanism. In order to understand autophagosome formation at the molecular level we will take advantage of our cell-free system to understand the structural and mechanistic basis of vesicle tethering and fusion – the very first steps during autophagosome formation. We will employ biophysical approaches including single-moleculefluorescence resonance energy transfer (FRET)-based vesicle fusion assays to identify the machinery required for vesicle tethering and fusion. In parallel, we will employ biochemical and cell biology approaches to understand how posttranslational modifications (e.g. phosphorylation) regulate membrane formation. Functional membrane tethering and fusion intermediates will be analysed by cryo-electron microscopy and single particle analysis or by tomography-based approaches. High resolution structures of vesicle tethering and fusion intermediates will significantly advance our knowledge of autophagy guiding future efforts to identify novel strategies for therapeutical intervention.


Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: GENERIC HEALTH RELEVANCE
Award Title: Molecular Mechanisms of Cell Death
Funder: Medical Research Council

Abstract: Cell death is a fundamental cellular response that plays a crucial role both during development and in the removal of unwanted or damaged cells following stress, injury or infection. Inappropriate cell death regulation contributes to many human diseases, including cancer and autoimmune and neurodegenerative disorders. Proteins/pathways that control cell death have also been identified as defined nodes that form key decision points to regulate the response to toxic insult. This Programme aims at understanding the fundamental mechanisms of cell death that regulate life/death decisions at the cellular level. By understanding the underlying molecular and cell biology of these processes, we aim to deliver field-changing mechanistic insights into toxicology and disease. Our research Objectives are to develop a number of strategies including develop a number of strategies including novel ‘in vitro’ reconstituted models which, combined with an integrated molecular, cell biological and proteomics-based approach, place us in a prime position to examine the molecular determinants of cell death.  In particular, we will employ cutting-edge technologies to: 1. obtain novel insights on the regulation and molecular architecture of multiprotein signalling complexes that direct cell fate 2. define signalling networks conferring drug-induced mitochondrial toxicity or cell survival in translational models of hepatotoxicity 3. selectively target nodes of resistance to cell death in patient-relevant 3D tumour models. The data generated through these integrated approaches will provide novel insights into the fundamental mechanisms of cell death that underlie the response to toxic injury, thus informing strategies to mitigate toxicity of existing therapies as well as newer agents under development.


Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health Categories: GENERIC HEALTH RELEVANCE
Award Title: Molecular Oncology Group
Funder: Cancer Research UK

Abstract: Background: Over the last 10 years, our Group has made important contributions to our understanding of the fundamental processes underlying melanoma development and progression.
Our studies have had significant impact on melanoma patient care. We have developed tractable models of melanoma that have allowed us to study gene-gene and gene-environment interactions.
With our move to Manchester, we have established excellent connections to clinical colleagues at the Christie NHS Foundation Trust, and developed a translational programme that aims to inform patient stratification and the clinical management of melanoma and other cancers.

 Aims: Our aims are:
• To investigate how ultraviolet light drives melanoma development.
• To develop new treatments for uveal melanoma.
 • To develop new drugs for melanoma and other cancers.
• To develop new treatments for prostate cancer.
• To develop a precision medicine platform for melanoma and other cancers.

Methods: Underpinning all of our studies are the models of disease we have developed and refined over the past ten years, including genetically engineered mouse models (GEMM) and patient-derived models (cell lines, PDX, CDX) models.
We have extensive expertise in next generation sequencing (NGS) approaches and functional analysis of cancer biology.
We will use our GEMMs to dissect the gene-gene and gene-environment interactions on melanoma development and progression, and to study responses to immunotherapies to guide optimisation of these drugs in the clinic.
We will develop and study GEMMs of uveal melanoma and prostate cancer, which, coupled with analysis of patient samples, will allow us to identify novel targets and develop or optimise much-needed new therapeutic approaches for these cancers. Using patient-derived models, we will identify new therapeutic targets for melanoma and other cancers, and investigate mechanisms of resistance, allowing us to develop precision medicine approaches for melanoma and other cancers, and to develop new anti-cancer drugs.

 Impact: Our philosophy is that patient care should be based on improved knowledge of cancer biology.
Our overarching aim is to understand the processes of cancer to develop better approaches to prevention, to develop new drugs, and to improve the clinical management of cancer patients.


Research Activity Codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;5.1 PHARMACEUTICALS
Health Categories: CANCER AND NEOPLASMS
Award Title: Molecular basis for motor-cargo cooperation in mitosis
Funder: Wellcome Trust

Abstract: Defects in chromosome segregation give rise to aneuploidy and chromosome instability, important hallmarks of cancer. Equal partitioning of sister chromatids to daughter cells requires microtubules to assemble into a mitotic spindle and to power chromosome segregation. Microtubules depend on multiple microtubule motor proteins to assemble into a spindle and segregate chromosomes. However, how motors cooperate with each other and with their cargos to drive mitosis is still virtually unknown. I will use a holistic mechanistic approach to understand how motor cooperation ensures faithful mitosis. We will focus on two key mitotic motor families, the non-motile Kinesin-13 microtubule depolymerases and CENP-E, critical for chromosome alignment, as paradigms for understanding motor cooperation during cell division. 
 
Combining structural and cell biology with single molecule reconstitution, we will define how Kinesin-13 motors cooperate with Kinesin-8 translocating motors for microtubule end targeting and depolymerization, and how CENP-E cooperates with the outer kinetochore of unaligned chromosomes, to achieve chromosome biorientation and segregation. These studies will provide a mechanistic understanding of the emerging role of kinesin-cargo cooperation in ensuring faithful cell division. Understanding these interactions will create new opportunities for the rational design of kinesin inhibitors in the treatment of cancer.

Lay Abstract: It is essential to all living organisms to transmit their genetic information, packed into chromosomes, accurately from one generation to the next. When a cell divides, it distributes the previously duplicated chromosomes to each daughter cell so that they inherit the same genetic message. Defects that result from unequal chromosome segregation can lead to dramatic consequences for an organism. Aneuploidy is frequently observed in cancer and results in genetic diseases such as trisomies including Down’s syndrome.
 
To segregate its chromosomes equally, the cell uses microtubules: these are long dynamic filaments, which store energy to move the chromosomes apart. Multiple nanoscale molecular motors organize the microtubules so that they can assemble to form the chromosome segregation machinery called the spindle. This proposal seeks to understand how these motors coordinate their activities to shape microtubules into a spindle, and attach and move chromosomes along it. We will define how motors achieve movement and transport at the molecular level, and how failure in the process can lead to cancer or cell death. This research is key to understanding the molecular processes of cell division in the eukaryotic kingdom and will create opportunities for the discovery of anti-cancer drugs targeting these motors.

Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Award Title: Molecular basis of chromosome synapsis and genetic exchange in mammalian meiosis
Funder: Wellcome Trust

Abstract: Meiotic cell division is defined by a unique and highly dynamic programme of events that results in homologous chromosome segregation following crossover formation. In mammals, the telomeric ends of chromosomes become tethered to the nuclear envelope by the meiotic telomere complex (MTC), where they undergo rapid movements, driven by microtubule forces transmitted by the LINC complex, that facilitate the identification and alignment of homologous chromosome pairs through recombination. Once established, homologue chromosome pairs become synapsed along their length by the zipper-like assembly of the synaptonemal complex (SC), which provides the unique three-dimensional architecture necessary for recombination intermediate resolution and crossover formation. We will uncover the structure, assembly mechanism and recombination function of the SC, the mechanistic basis of nuclear envelope tethering by the MTC and the mechanism of force transduction by the LINC complex. This will be achieved through a structural biology approach of biophysics, crystallography and cryo-EM, coupled with collaborative structure-directed mutation in mouse meiosis. Our work will result in unprecedented molecular understanding of how the mammalian SC, MTC and LINC complex operate together as an integrated molecular machine to achieve their essential functions of mammalian meiosis, and crucially how their dysfunction leads to human infertility, miscarriage and aneuploidy.

Lay Abstract: Sperm and egg cells each contain 23 chromosomes, which combine upon fertilisation to achieve the full complement of 46 chromosomes that defines human life. These reproductive cells are produced by meiosis, in which the chromosome number is halved and genetic material is shuffled to enhance diversity. We aim to uncover how the elaborate chromosome choreography of meiosis is achieved by a series of integrated protein machines, including ropes and pulleys that drag chromosomes around the cell and a zipper that binds chromosomes together to enable genetic exchange. We will utilise biochemistry and structural biology to reveal the how the ‘cogs’ of these machines work together at the most fundamental molecular level to ultimately achieve their remarkable cellular functions. Through this work, we will uncover how meiosis normally produces healthy sperm and eggs, and how it goes wrong in infertility and recurrent miscarriage, which affect 15% and 5% of couples, respectively.

Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: GENERIC HEALTH RELEVANCE;REPRODUCTIVE HEALTH AND CHILDBIRTH
Award Title: Molecular characterisation of post-translational regulation in offensive Type VI secretion systems
Funder: Wellcome Trust

Abstract: The Type VI secretion system (T6SS) is a nanoweapon used by many Gram-negative bacteria to deliver toxic ‘effector’ proteins into neighbouring cells. The targeted cells are most often rival bacteria, but can also include the host or microbial fungi. Understanding what triggers the T6SS weapon to assemble and fire will improve our knowledge of how microbes compete for resources, and how diverse communities of microbes develop. Two T6SS firing ‘strategies’ have been identified: offensive T6SSs assemble and fire pre-emptively, whereas defensive systems only fire in retaliation upon sensing incoming attacks from neighbours. A signalling system of proteins which mediate both strategies has been identified. However, key molecular details of how this is used to orchestrate ‘offensive’ T6SS firing are lacking. In this study, we will elucidate the molecular mechanism of these offensive T6SS protein-protein interactions, using the model T6SS of the opportunistic pathogen, Serratia marcescens. Using a combination of genetic, biochemical and cell biology approaches, we will determine the trigger that activates the pathway, how this signal is transduced to activate the T6SS, and the conservation of this regulation in clinical isolates. Long-term, understanding how bacteria interact in diverse communities will contribute to novel strategies to tackle bacterial infections.


Research Activity Codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health Categories: INFECTION
Award Title: Molecular characterization in human neurons of genes associated with severe obesity identified from consanguineous pedigrees.
Funder: Medical Research Council

Abstract: Up to 10% of severe early onset obesity has an identified monogenic cause. We showed that studying the disease in consanguineous Pakistani pedigrees enriches for homozygous variations, increasing the likelihood of finding causative mutations. Using this approach, we have identified that homozygous and compound heterozygous loss of function mutations in ADCY3, which encodes for adenylate cyclase 3, and homozygous mutations in the gene ROCK1, which encodes for Rho-kinase 1, are linked to severe obesity. In this proposal, we will study both these genes in detail, as well continue our search for other new obesity candidates.  Given the role of the brain in the regulation of body-weight and food intake, we will molecularly characterize these genes in human neurons.  Using single nucleus RNAseq, we will identify human neurons expressing ADCY3 and ROCK1. We will characterize the heterogeneity of the neurons and define markers for different populations. We will use RNAscope single molecule FISH to map where both of these genes are expressed within the human hypothalamus. Using CRISPR/Cas9 gene editing, we will 'knock-in' the ADCY3 and ROCK1 mutations that are associated with obesity into human pluripotent stem cells and then differentiate them into hypothalamic neurons. In the cells with ADCY3 mutations, we will study the effect on the primary cilia and leptin responsiveness. In the cells with ROCK1 mutations, we will measure leptin and insulin responsiveness. Using single cell RNAseq, we will examine the transcriptomic consequences of the mutations on the neurons.  Using CoDE-seq, an augmented whole-exome sequencing approach that additionally enables the accurate detection of CNVs, we will continue to further identify candidate genes associated with severe obesity. Where the weight of evidence dictates, these will be molecularly characterized as above. Our aim is to uncover novel appetitive control pathways and reveal new potential therapeutic targets to tackle obesity.


Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: METABOLIC AND ENDOCRINE
Award Title: Molecular mechanism of viral transgene silencing by KAP1 (TRIM28)
Funder: Medical Research Council

Abstract: Transcription of transposable elements is tightly regulated to prevent damage to the genome. A key source of regulation of transposable elements is the family of KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1). KRAB-ZFPs recognize specific transposable elements and recruit KAP1, which controls the assembly of an epigenetic silencing complex including histone H3K9 methyltransferase SETDB1. The chromatin remodeling activities of this complex repress transcription of the targeted transposable element and any adjacent genes. We have performed a detailed and quantitative set of biochemical and structural analyses on the tripartite motif (TRIM) of KAP1. Our preliminary data show that KAP1 forms homodimers through a coiled coil domain. Moreover, KAP1 dimers assemble into higher-order oligomers via B-box domains, and KAP1 forms a tight 1:1 complex with the KRAB domain. However, the structure and composition of the KAP1 repressor complex, and how its assembly impacts the structure and physicochemical properties of its chromatin binding site, remain unknown. Here, we propose to identify the molecular mechanisms through which KAP1 recruits and activates chromatin-modifying enzymes at target transposable elements. We will determine three-dimensional structures at near-atomic resolution of a KAP1/KRAB-ZFP retrotransposon recognition complex, and a repressor complex containing a KRAB-ZFP, KAP1 and SETDB1, by X-ray crystallography and cryo-electron microscopy. We will design structure-based mutations to identify functionally important structural features and protein interaction interfaces with biochemical and cell-based functional assays. Our work will provide a detailed mechanistic understanding of how KAP1 represses transcription of transposable elements. Reactivation of transposable elements can cause serious human disease. This work will inform the design of compounds that inhibit pathogenic activation of transposable elements.


Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: GENERIC HEALTH RELEVANCE
Award Title: Molecular mechanisms enabling cDC2s to control Th2 cell priming
Funder: Medical Research Council

Abstract: The molecular control of cDC2 functions is not sufficiently understood. Progress has been hampered by the heterogeneous nature of cDC2s in vivo; the few activated cDC2 needed to prime naïve T cells are outnumbered by non-activated cDCs that also migrate. We have recently shown that, after stimulation with Schistosome Egg Antigen (SEA), cDC2 become able to prime naïve T cells to become Th2 in vivo. But at a molecular level our understanding of cDC2 functions, particularly Th2-priming cDC2s, is still insufficient. The intestine is a productive tissue for the study of cDC2 functions; it contains more cDCs and T cells than any other organ system. We have established techniques for purification and functional analysis of bona fide migrating intestinal cDC2.  We have performed microarray analyses of both types of cDC2s (CD11b+ single-positive cDC2 and CD11b+CD103+ double-positive cDC2). Both cDC2 populations prime Th2 responses in vivo after stimulation with SEA. We found only three genes that significantly change their expression in both SEA-stimulated cDC2 populations. Our RNA sequencing data confirm that these genes are expressed by cDC2s in the small intestine, lymph, and intestinal lamina propria. Two of the three genes (Rasgrp3 and Il1f9) are known to control cytokine production by myeloid cells, but neither has yet been ascribed a function in cDCs. Here we have two aims. First, we will investigate the functions of Rasgrp3 and Il1f9, to understand how they contribute to cDC2s ability to prime Th2 responses, both in vitro and in vivo. Second, we will deliver labelled antigens in vivo and generate single-cell sequencing data to identify clusters of genes expressed by the few activated antigen-carrying Th2-competent migratory lymph cDC2s, among the heterogeneous migratory cDC2. Thus, we aim to uncover novel molecular functions in cDC2, that will enable improved manipulation of the adaptive immune response, both in the intestine and in other tissues.


Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: INFLAMMATORY AND IMMUNE SYSTEM
Award Title: Molecular origin of non-disjunction in human meiotic and early mitotic divisions
Funder: Wellcome Trust

Abstract: In humans, pregnancy with the wrong number of chromosomes (aneuploidy) is common and the risk increases with maternal age, rising from ~2% for women under the age of 25, to around 35% for women over 40. Aneuploidy is a leading cause of early miscarriage, whilst affected full term births exhibit severe medical problems. Most aneuploidies arise from chromosome segregation errors that occur during the meiotic divisions of the oocyte or early mitotic divisions of the embryo. The next challenges are to determine the mechanisms of chromosome segregation in oocytes, to understand how high fidelity is ensured and to identify the origin of aneuploidies found in early human embryos. Work on model systems has identified adaptations to the kinetochore which direct and monitor specialized pathways of chromosome segregation during meiosis. However, the extent to which similar mechanisms function in human oocytes is unknown. Indeed, our recent work revealed unexpected properties of kinetochores in human oocytes, potentially contributing to the high error rate. We will perform a detailed examination of kinetochore behaviour in human oocytes and early embryos to gain the first insights into chromosome segregation at the start of life and uncover potential sources of error.

Lay Abstract: A specialized cell division called meiosis produces eggs and sperm. A high proportion of human eggs are faulty because they have the wrong number of chromosomes, particularly in older mothers. Fusion of such defective eggs with a sperm results in infertility, miscarriage, or diseases such as Down Syndrome. We aim to understand the mechanisms by which chromosomes are distributed into human eggs and to determine if their function is altered in reproductively challenged patients. Our team of clinical reproductive scientists and cell biologists will use advanced microscopy to examine fluorescently-labelled factors in human immature eggs as they undergo meiosis and in the first cell divisions after fusion of the egg and sperm. We anticipate that our work will provide a better understanding of the pathways that sort chromosomes into human eggs and inform future work to identify the causes of infertility.

Research Activity Codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health Categories: GENERIC HEALTH RELEVANCE;REPRODUCTIVE HEALTH AND CHILDBIRTH