NIHRDataInsights/HRCSData huggingface dataset:
Title: Control and enzymatic activation of the APC/C ubiquitin ligase system
Funder: Wellcome Trust

Abstract: CDK1 and APC/C are two key regulatory enzymes controlling the cell division, growth, differentiation and death, through phosphorylation and ubiquitylation, respectively. Although it has long been apparent that phosphorylation modifies APC/C function, the challenges posed by the need for functional assays to study this control puts the elucidation of the molecular basis of phosphorylation control beyond our grasp. We have recently overcome these limitations with a pipeline that uses reconstituted recombinant APC/C in Xenopus cell free extracts to show how CDK1 activates the APC/C through coordinated phosphorylation of Apc3 and Apc1. We will now extend this pipeline with targeted assays that will determine how phosphatases regulate these phosphorylation events. Because we have found that the disordered loop domains of APC/C subunits are targets for both post-translational modifications (PTMs) and interacting partners, including protein phosphatases, we will study how the loop domain controls the APC/C. Cell cycle specific and stress-dependent PTMs and binding proteins will be identified and we will determine their impact upon APC/C-dependent ubiquitylation. This approach of combining high throughput reconstitution mutated apo-APC/C in extracts from which any component of interest can be depleted offers a unique opportunity to gain an unprecedented insight into APC/C function and control.

Lay abstract: We study a cellular enzyme that plays a key role in selective protein destruction; the anaphase-promoting complex/cyclosome (APC/C). The APC/C controls many events including genome DNA duplication and segregation, cell growth, differentiation and death, DNA damage repair, brain and metabolic functions. Greater understanding of APC/C function will therefore be of great benefit to human health. Although 20 years have passed since its discovery, our understanding of APC/C function and control is lamentably limited. This is partly due to the sheer size and complexity of the enzyme. The APC/C comprises 14 subunits, many of which are chemically modified at multiple sites by the addition and removal of phosphate groups. Very little is known about the control of these cycles of chemical modification. We have developed a unique approach that can now be used to study these controls. We reconstitute the entire APC/C enzyme by making the subunits in a surrogate production system. We then study the function of these reconstituted APC/C complexes in highly accessible frog egg extracts. This programme will extend these studies to define more precisely how the APC/C is controlled by phosphorylation, identify new binding partners and map the changes in APC/C structure that accompany activity.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Molecular Mechanisms of Centromere Inheritance and Kinetochore Function
Funder: Wellcome Trust

Abstract: The integrity of life relies on the ability of cells to faithfully pass on their genetic information in the form of chromosomes to daughter cells when they divide. Aberrant chromosome distribution leads to daughter cells with abnormal chromosomal number, a state called aneuploidy. Aneuploidy is often implicated in cancer and causes infertility, miscarriages and birth defects such as Down’s syndrome. Hence, understanding the mechanisms that ensure accurate chromosome separation is crucial in fighting several important human disorders. The key protein machinery that facilitates chromosome distribution to daughter cells is called the kinetochore, which is assembled on a specialised chromosomal site called the centromere. Kinetochores physically attach chromosomes to filamentous structures called microtubules to achieve chromosome separation. A large number of different proteins regulate these biological processes. We specifically aim to understand how protein machineries establish and maintain centromere marks on chromosomes and how kinetochores assembled on them help in distributing chromosomes equally and identically to daughter cells. We will do this by determining and analysing structures of centromere associated molecular machines as they interact with their binding-partners.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Mechanisms of DNA interstrand crosslink repair in humans.
Funder: Wellcome Trust

Abstract: My research is focused on uncovering the molecular mechanisms of DNA interstrand crosslink (ICL) repair in humans. Disruption of the underlying DNA-repair pathway causes Fanconi Anemia (FA), with serious cancer susceptibility. Also, ICL-forming drugs are used therapeutically in non-FA cancer patients, however resistance is a major problem. Targeting the FA-pathway could allow clinicians to treat these patients. A key and fundamental event in the FA-pathway is the recruitment of repair proteins to ICLs. Specific and timely recruitment is essential for accurate repair. We have recently discovered proteins specifically detecting ICLs and we have obtained the cryo-EM structure of other ICL-repair proteins. My aim over the next five years is to advance the field further by uncovering mechanistically how repair factors are activated and recruited to ICLs at the single-molecule level. We will: 1) Dissect the mechanism of initial recruitment of repair factors to ICLs. 2) Uncover functions of identified proteins in FANCD2-complexes in ICL-repair. 3) Determine roles of novel phosphorylation sites on FANCD2/FANCI. 4) Elucidate mechanism of FANCD2/FANCI activation. Addressing these central questions will not only greatly advance our understanding of ICL-repair, but will also likely enhance our understanding of other DNA repair pathways utilizing analogous mechanisms.

Lay abstract: Mutations arising in our genome can cause cancer. To prevent this, humans have so-called DNA repair pathways. One such pathway is the Fanconi Anemia pathway, which when nonfunctional causes the Fanconi Anemia (FA) disease. FA is a disease causing high incidence of cancer. Some patients do not survive beyond childhood. The reason that patients develop cancer is the non-functional FA pathway, which now cannot repair a dangerous type of DNA damage called DNA interstrand crosslinks (ICLs). The aim of my research is to learn more about how our cells repair ICLs. My research applies several approaches designed to address this important question from different angles. I will study several proteins that are involved in this biological process, some of which we have discovered recently, and others, the importance of which have been known longer. My group will uncover how these proteins work together in the complex DNA repair process.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Genetic and functional interactions in the mammalian DNA-damage response
Funder: Wellcome Trust

Abstract: DNA in our cells is frequently subject to a wide array of molecularly-distinct forms of damage. To cope with this, life has evolved multiple DNA repair and associated processes, collectively termed the DNA-damage response (DDR). While considerable progress has been made in identifying DDR proteins and their regulators, much remains to be learned about how they operate and are controlled. Building on our successful proof-of-concept studies, we will carry out genome-wide and focused genetic screens via state-of-the-art CRISPR-Cas9 approaches and haploid-cell based chemical mutagenesis. Together with ensuing validation and mechanistic studies, the proposed research has the following interconnected goals: • To identify novel genetic and functional relationships between DDR genes/proteins, and between these and other cellular components, thereby providing fundamental insights into how mammalian cells respond to DNA damage and defining how such responses are controlled and coordinated. • To establish how defects and deregulation of certain DDR processes affect cellular sensitivity and resistance to established and emerging cancer therapies, and to explore the potential clinical relevance of these affects. • To expand our knowledge of how DDR processes are affected by protein post-translational modifications, particularly ubiquitylation and phosphorylation.

Lay abstract: The genetic information of our cells, DNA, is continually being damaged by factors that arise through normal cell metabolism and from environmental agents. To cope with this damage, our cells have evolved a large number of sophisticated proteins that detect this damage and, in most instances, mediate its effective repair. Defective repair of such damage, such as occurs occasionally in normal cells and more often in cells with DNA-damage response (DDR) defects, leads to mutations that disrupt cell function and can lead to cancer and other diseases. Our proposed research builds on our past experience in identifying and characterising DDR proteins, and couples this with our recent successful implementation of novel “CRISPR-Cas9” and other technologies to systematically screen for new DDR proteins and regulators and then characterise how they function. Our planned research should thereby provide new mechanistic insights into fundamental DDR processes. Furthermore, it will help explain how certain DNA-damaging chemotherapeutic agents and novel DDR-enzyme targeted drugs kill cancer cells, and how cancers can evolve resistance to such therapeutic agents. This knowledge may also suggest ways to more effectively treat cancers, as well as various other genetic diseases that are caused by DDR defects.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Molecular mechanisms of telomere protection and poly(ADP-ribosyl)ation-regulated telomere length homeostasis
Funder: Wellcome Trust

Abstract: Telomeres, the ends of linear chromosomes, face two unique challenges. The first, the end protection problem, arises from the resemblance of chromosome ends to DNA double-strand breaks, which elsewhere in the genome trigger a rapid DNA damage response that needs to be averted at telomeres. The second, the end replication problem, refers to the gradual loss of telomeric DNA with every round of DNA replication. Telomere end protection is achieved by the six-membered telomeric Shelterin complex. Shelterin mutations in cancer and telomere disorders illustrate the importance of the complex in telomere maintenance. Homeostatic telomere extension (in stem and most cancer cells) is achieved by telomerase and a Shelterin-dependent telomere length-sensing mechanism. However, end protection collaterally impedes the access of telomerase to telomeres, a dilemma that in human cells is solved by the poly(ADP-ribose)polymerase (PARP) tankyrase, which remodels the Shelterin complex. The mechanism of telomere protection and length maintenance remains incompletely understood. Moreover, how tankyrase remodels the Shelterin complex to enable telomere extension without triggering a DNA damage response is unknown. We aim to unravel the mechanisms of telomere protection and length homeostasis by combining single-particle cryo-electron microscopy with biochemical and biophysical assays as well as functional studies in mammalian cells.

Lay abstract: An elaborate machinery contantly detects and repairs damage to our DNA. While a break of both strands of the DNA double helix is dangerous and needs to be repaired rapidly, how come normal chromosome ends, which resemble DNA breaks, do not trigger the same response? The answer lies in special sequences at chromosome ends, the telomeres, which are packaged up by proteins that collectively avert the damage response. At the same time, telomeres serve as templates for the telomerase enzyme, which maintains telomeres at a constant length in long-lived cells. Telomere packaging, however, impedes the access and function of telomerase. This dilemma is solved by a protein known as tankyrase, which orchestrates both telomere packaging and extension such that they can occur simultaneously. We aim to use electron microscopy and biochemistry to study how exactly this occurs and why defects in telomere packaging can lead to cancer and heritable diseases.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: The cellular regulation of oxygen and metabolite sensing
Funder: Wellcome Trust

Abstract: HIFs are central to the adaptive oxygen response in multicellular organisms, but these transcription factors are also activated by changes in mitochondrial metabolites, even in the presence of oxygen. Exploring this link between oxygen and metabolite sensing is key if we are to understand the hypoxic and metabolic changes intrinsic to inflammation or tumorigenesis. Our identification of the 2-oxoglutarate dehydrogenase complex (OGDHc) and mitochondrial lipoylation as regulators of HIF activation in aerobic conditions provides a novel entry point to explore the relationship between mitochondrial function and HIFs. My goals are to: 1) Characterise metabolic pathways that control 2-OG metabolism and HIF activation. 2) Determine how HIF binding to chromatin activates a transcriptional response following hypoxic or metabolic stimuli. To address these aims, we have established an innovative discovery platform based on our forward genetic approaches, and have engineered fluorescent reporters to respond dynamically to changes in different HIF isoforms. Together with our biochemical and cell biological expertise, these studies will provide important insights into the cellular mechanisms involved, with the ultimate goal of identifying new therapeutic targets for inflammatory respiratory disease and tumorigenesis.

Lay abstract: The ability of cells to sense and respond to changes in oxygen and nutrients is essential; when disrupted, this leads to diseases such as cancer, impaired immunity or metabolic disorders. Using genome-editing approaches we have discovered new genes that influence the cell’s ability to sense both oxygen and nutrients. Our goal is to now determine how they work, understand how oxygen and nutrient sensing are linked, and how this influences human health. We are particularly interested in how mitochondria, the powerhouses of the cell, respond to changes in nutrient availability and relay this information to the DNA genome in the nucleus (where all genes are encoded). We envisage that understanding these basic aspects of cell function will lead to the identification of new therapeutic targets that can combat diseases where oxygen sensing or energy metabolism is impaired.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Structures, Recruitment and Regulation of Key Components in DNA Damage Response
Funder: Wellcome Trust

Abstract: Genome stability and integrity are extremely important for proper cellular functions. However, each cell suffers from tens of thousands of DNA damage daily. Cells have thus evolved a myriad of ways to deal with these damages. A double-strand break (DSB) is the most severe form of damage and cells have developed multiple pathways to ensure efficient repairs are carried out. Homologous recombination is a faithful repair process and a large number of proteins are involved in this pathway. We aim to characterize some of the key proteins in homologous recombination including the master signaling kinases ATR/ATM (Mec1/Tel1 in yeast), the chromatin remodeler INO80 and the tumour suppressors BRCA2, PALB2 and BRCA1. We have obtained significant breakthrough in some of the areas in the last five years funded by a Wellcome Trust Investigator award. In this current proposal, we will continue to investigate their structures and core mechanism of actions. More importantly, we now want to investigate how these key players are recruited and how they are regulated. The knowledge obtained will help us understand the causes of cancer and aging and can also provide new avenues for therapeutic development.

Lay abstract: Our genomic information is stored in the DNA. However DNA is damaged by UV irradiation, drug, smoke, alcohol or metabolic products. Fortunately our cells have developed many strategies to detect and repair these damages. One repair method is to use homologous DNA from a sister chromatin. This process is highly coordinated by the actions of many proteins, working horses of the cell. If this repair process is not working properly, such as when certain proteins are defective, the damaged DNA will not be repaired properly and/or promptly, and this can lead to changes in DNA, causing cancer or aging in the long run. We want to visualize these players, to understand how they work, how they are controlled so that we can understand how they repair our damaged DNA. This knowledge can help us understand the cause of cancer and aging and can also provide new avenues for therapeutic development.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Understanding the mechanisms of Cell Competition and its role in tissue biology
Funder: Wellcome Trust

Abstract: Cell competition is increasingly recognized as playing a key role in selecting the cellular composition of tissues, both in health and diseases such as cancer. My proposed research aims to advance our understanding of the mechanisms of cell competition and how it impacts on adult tissue biology, to advance our fundamental understanding of this biomedically-relevant process and learn how to harness it for therapeutics. Building on my group’s recent discoveries and methodological advances, I will use an integrated strategy combining in vitro and in vivo approaches to: 1) Elucidate how the oxidative stress response, which we found marks cells as ‘losers’ for competitive elimination, triggers cell competition in Drosophila. 2) Elucidate another link we discovered between aberrant autophagy in ‘loser’ cells and cell competition, in Drosophila. 3) Characterize the molecular mechanisms behind our discovery of p53-induced cell competition, using a CRISPR-based genomic screen in mammalian cultured cells. 4) Investigate the impact of cell competition on human adult tissue biology, using human primary keratinocytes and p53 competition-inducing mutations. 5) Carry out targeted proof-of-principle experiments to investigate for the first time whether cell competition can be exploited to improve tissue engraftment and repopulation in vivo, using the mouse airway.

Lay abstract: The health of an organism relies on the health of its constituent cells. To help maintain a healthy tissue composition, a process of competition between cells has developed, which selects the best cells within tissues based on their survival characteristics ('fitness'). When cells of differing fitness levels come into contact, weaker cells (‘losers’), which would be perfectly viable on their own, are eliminated by fitter cells (‘winners’) through induction of cell death. Cell competition likely impacts on numerous aspects of tissue biology, possibly acting as a quality control mechanism to eliminate unfit or harmful cells as they arise. Correspondingly, if properly harnessed, cell competition could open the door to novel therapeutic strategies, allowing for example to improve the efficiency of stem cell therapies. In this project we will combine work in the fruitfly as a model organism with work using mammalian cultured cells to significantly deepen our knowledge of how cells compete in tissues, identify and validate new molecules that control cell competition, identify key health and disease contexts where cell competition plays a role, and investigate for the first time how cell competition could be exploited therapeutically to replenish unhealthy tissues with healthy cells.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;CANCER AND NEOPLASMS
Title: Recognition, activation and targeted degradation of protein kinases clients by the HSP90-molecular chaperone
Funder: Wellcome Trust

Abstract: Many oncogenic protein kinases depend on interaction with the HSP90 molecular chaperone, mediated by the co-chaperone CDC37, for their cellular stability and oncogenic activity. Inhibition of HSP90's conformationally-coupled ATPase mechanism leads to the ubiqtuitylation and degradation of these protein kinase 'clients'. Consequently HSP90 is an important target for therapeutic intervention in cancer. Although there has been substantial progress in this field, important issues remain unresolved. In particular we wish to understand : 1. How protein kinase clients are specifically and selectively recognised by the CDC37 co-chaperone, and recruited to HSP90 ? 2. What structural and biochemical changes are elicited in the client protein by recruitment to HSP90 and by its conformationally-coupled ATPase cycle ? 3. How dephosphorylation of CDC37 by the HSP90-targeted protein phosphatase PP5 regulates client protein release ? 4. How protein kinase clients are targeted for proteasomal degradation when HSP90's ATPase is inhibited ? To address these questions we will use cryoelectron microscopy, X-ray crystallography, NMR spectroscopy, and a range of biochemical and biophysical approaches, to determine structures of key complexes along the pathway from initial client recognition to release or ubiquitylation, and define the structural and biochemical transitions that connect them.

Lay abstract: The normal functioning of cells is controlled by nano-molecular machines made up of protein molecules. Many of these require help from specialised nano-machines called chaperones, which look after component proteins until required. Many of the mutated or over-abundant protein molecules that turn a normal well behaved cell into an uncontrolled cancer, are particularly dependent on chaperones, so that drugs that stop chaperones working cause these aberant proteins to be destroyed, thereby selectively killing the cancer cells. One particular class of proteins, kinases, that are often aberant in cancer, depend on a chaperone called HSP90. We wish to understand how these cancer-associated kinase proteins gain access to HSP90 in the cell, how HSP90 binds to them and stabilises them, how they are eventually released from HSP90, and how they become destroyed when HSP90 is stopped from workng by specific drug molecules.

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