Theses - Cambridge Institute for Medical Research
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Item Embargo Identification and characterisation of novel interactors of surfactant protein CYing, HaoyangInherited pathogenic variants of surfactant protein C (SFTPC) can cause familial pulmonary fibrosis. The most common, SFTPC-I73T, aberrantly localises to the plasma membrane and causes alveolar type 2 (AT2) dysfunction via a poorly understood toxic gain-of-function. Understanding the mechanisms of SFTPC trafficking and their interactors in health and disease is crucial to defining how pathogenic variants induce cellular dysfunction. To investigate trafficking components required for SFTPC trafficking that are perturbed by the I73T variant, I undertook SFTPC-WT and -I73T proximity labelling using TurboID and identified labelled proteins by mass spectrometry. I discovered >100 proteins preferentially labelled by SFTPC-WT and therefore likely to be required for normal SFTPC trafficking. I undertook validation of biologically relevant hits by CRISPR/Cas9 deletion of candidates involved in membrane trafficking, ubiquitination and lysosomal acidification. I found that many induced misprocessing and relocalisation of SFTPC-WT to the plasma membrane, phenocopying the I73T mutation. I undertook further validation of top hits in a novel CRISPR interference AT2 organoid model. I also undertook an exploration of the biology of MPZL1, a poorly characterised Ig-superfamily member which differentially interacts with, and is relocalised by, SFTPC-WT and I73T. This suggests that the normal function of MPZL1 may require interaction with SFTPC-WT. I defined novel aspects of its expression, cleavage and activation and showed from RNA sequencing that MPZL1 activation upregulates genes involved in cell migration; this may be crucial for AT2 function in alveolar repair. Together, this work has identified novel interactors involved in SFTPC trafficking and explored the biology of MPZL1, offering further insights into how SFTPC-I73T contributes to the development of pulmonary fibrosis.Item Embargo Serpin polymers enforce molecular filtration in the endoplasmic reticulumZubkov, Nikita; Zubkov, Nikita [0000-0001-8912-0073]Newly synthesised secretory proteins fold in the endoplasmic reticulum, failure of which can be toxic and cause disease. Alpha1-antitrypsin is the serine protease inhibitor (serpin) that is secreted mainly by hepatocytes and acts in lungs where its major function is to inhibit neutrophil elastase. Neuroserpin is a serine protease inhibitor expressed in the nervous system. Mutations in these two proteins can cause them to polymerise and accumulate within endoplasmic reticulum (ER) causing its fragmentation into ER inclusions. Accumulation of these serpins leads to cirrhosis and early-onset dementia respectively. Molecular mechanisms of these diverse pathologies remain incompletely understood. Utilising a range of advanced live-cell imaging techniques I showed that serpin polymers undergo a liquid:solid phase transition, filling the lumen of the ER with a protein matrix that imposes molecular filtration, retarding the mobility of ER proteins in a size-dependent manner. I demonstrated that serpins’ phase transition is promoted by the ATF6 branch of the unfolded protein response during ER stress, and overexpression of ER chaperones calreticulin and BiP promotes this solidification and increases the stiffness of the resultant protein matrix. Single particle tracking of ER proteins revealed that this process initiates in cells with normal reticular ER morphology. This novel mechanism of ER dysfunction, involving phase transition of a protein in the ER lumen, provides a template for understanding related proteinopathies as diverse as an autosomal dominant form of dementia and diabetes insipidus, and identifies ER quality control components as potential therapeutic targets.Item Embargo Disease-modifying effects of human small heat shock proteins in zebrafish models of neurodegenerationLager Gotaas, IngridNeurodegenerative diseases are frequently characterised by the build-up of misfolded proteins and degeneration of brain structures. Tauopathies are a collective of >20 such diseases featuring abnormally aggregating tau, a microtubule-associated protein normally acting to stabilise these protein cargo tracks. There is currently no cure. Small heat shock proteins (sHSPs) are highly conserved molecular chaperones known to act as holding partners for substrates. Studies have reported that some sHSPs can act as chaperones for disease-related proteins to prevent their aggregation and may be beneficial in such diseases. However, there is a lack of a thorough screen of the sHSPs and their disease-modulating effects in an in vivo model. The aim of this thesis research was to screen the sHSP family to determine which, and how, any of the human sHSPs could ameliorate tau toxicity in zebrafish tauopathy models. Of all eleven sHSPs, HSPB1, B4 and B5 ameliorated morphological disease phenotypes induced by pan-neuronal expression of mutant A152T tau. HSPB4 and B5 have never been demonstrated to be beneficial in a tauopathy model in vivo, and peptides created from the central domain of these similarly ameliorated the morphological phenotype. To further investigate the underlying mechanism of action, I created and characterised a transgenic zebrafish line ubiquitously expressing human HSPB5. I demonstrate that overexpression of HSPB5 in the pan-neuronal A152T tau line results in reduced levels of hyperphosphorylated and insoluble tau. Additionally, a synthetic HSPB5 peptide similarly ameliorated morphological phenotypes by treatment via embryo immersion and reduced levels of insoluble tau. These results indicate that HSPB5 and its peptide may be of therapeutic interest for use against tauopathies as it can improve various disease phenotypes in an in vivo model.Item Embargo Scalable methods for the discovery of autophagy and disease genesDjajadikerta, AlvinMacroautophagy (henceforth referred to as autophagy) is a major and conserved cellular process in which cells deliver cytoplasmic contents to lysosomes for degradation. As autophagy has been linked to various human diseases, the discovery and characterisation of autophagy genes is of interest for both basic science and medical research. This thesis develops scalable experimental and computational methods for the discovery of autophagy genes and elucidation of their relationships to human disease. To enable scalable genetic screens for autophagy, we develop two high-throughput autophagy assays: (1) an autophagic flux assay, SRAI-LC3B; and (2) an assay for levels of the ATG12-ATG5 conjugate, an important early autophagy marker. SRAI-LC3B can be used to assess autophagic flux via flow cytometry or microscopy and responds robustly to established pharmacological and genetic controls. Additionally, we exploit the optical properties of SRAI-LC3B to develop a novel high-throughput autophagy assay in a human neuronal model. By conducting a targeted screen with the ATG12-ATG5 assay, we identify two chaperone proteins as novel autophagy regulators. These chaperones act together to regulate autophagy at multiple stages, by mechanisms likely to involve stabilising the VPS34 complex and enabling DNM2-mediated autophagosome scission. Computational techniques such as network propagation and machine learning can help to condense large, complex data into testable predictions. We exploit these tools to develop a model that predicts new autophagy genes by analysing systematic datasets. Top predictions were screened using the SRAI-LC3B assay, resulting in eighteen new candidate autophagy genes. Predicted genes were enriched >5-fold in significant screen hits compared to a randomly selected control set. Subsequently, we develop a systematic procedure for process-to-phenotype prediction, which analyses interaction networks to predict diseases that may be associated with autophagy. A targeted screen of top predictions identifies twenty-eight diseases in which disruption to one or more associated genes caused significant changes in autophagy, raising the prospect that dysfunctional autophagy may play a role in some of these diseases.Item Open Access Epigenetic repression of intronless mobile elements by the HUSH complexSeczynska, MartaThe mammalian genome is under constant threat from invasion by mobile genetic elements including transposons and viruses. To defend the genome, cells recognize incoming DNA and limit its transcription through repressive chromatin modifications. The human silencing hub (HUSH) complex transcriptionally represses long interspersed element-1 retrotransposons (L1s) and retroviruses through histone H3 Lys9 trimethylation (H3K9me3). How HUSH recognizes and initiates silencing of these invading genetic elements is unknown. By monitoring transcription from L1 transgenes, I found that incoming L1s are recognized by HUSH independently of their integration sites, a critical difference with previously studied viral silencing. By studying sequence determinants of L1 repression by HUSH, I discovered that HUSH is able to recognize and transcriptionally repress a broad range of sequence-diverse, intronless invading DNAs, despite no prior exposure to such DNAs. Sequence length and high adenine (A) content in the sense strand are important additional determinants of susceptibility to HUSH-targeting. My further work demonstrated that Periphilin binds transcripts from the target locus, prior to and independent of, H3K9me3 deposition, which explains why target transcription is essential for both initiation and propagation of HUSH-mediated H3K9me3. Whilst diverse intronless transgenes are susceptible to HUSH-repression, I found that the presence of an intron counteracts repression, even in the absence of intron splicing. A subset of endogenous intronless loci are silenced by HUSH, and Periphilin specifically binds transcripts from endogenous intronless loci. Therefore, intronless DNA, the product of reverse transcription, provides a versatile means to distinguish invading RNA-derived retroelements from intron-containing host genes and provides a mechanism by which HUSH protects the genome from ‘non-self’ DNA. I propose that HUSH is a component of the innate immune system and intronless DNA the molecular pattern recognized by HUSH. By silencing cDNA, the product of reverse transcription, HUSH controls the reverse flow of genetic information (i.e. from RNA to DNA) in the human genome.Item Open Access Modelling neurodegenerative diseases in human iPSC-derived neuronsPrestil, RyanNeurodegeneration is a pathology shared by a varied class of diseases, and many of the mutations that are known to cause such diseases have been linked to protein aggregation and autophagy dysfunction. Improvements to gene editing and neuronal differentiation strategies have enabled the derivation of in vitro disease models using human iPSC-derived neurons to provide a more accurate understanding of how disease mutations affect neuronal health. I first sought to model the polyglutamine disease spinal and bulbar muscular atrophy (SBMA), detailed in Chapter 4. Using iPSCs derived from a healthy donor and an SBMA patient, the CAG repeat of the endogenous androgen receptor (AR) gene was CRISPR-edited to encode a series of lengths or an early stop codon. However, AR expression was silenced upon transcription factor-mediated differentiation to a lower motor neuron-like state, and chemical differentiation prevented ligand-induced AR nuclear translocation. Deriving the cell lines in this work highlighted that purification of transgenic cells is a key bottleneck to gene editing. I therefore adapted a synthetic marker gene that presents a streptavidin binding peptide (SBP) tag on the extracellular membrane, detailed in Chapter 3. Expression of this tag in iPSCs enabled transient fluorophore staining and effective sorting of mixed populations with magnetic streptavidin beads. Finally, Chapter 5 establishes L1CAM as a novel autophagy modulator; iPSC-derived neurons showed that reduction of the L1CAM transcript with shRNAs, but not genetic knockout of the L1CAM protein, is sufficient to reduce transcription of the ATG8 gene family, which are core components of macroautophagy. This work exemplified both the strengths and weaknesses of iPSC-derived neurons; namely, they are tractable and able to recapitulate neural phenotypes, but deriving new model lines requires a high initial investment, so adequate proof-of-concept is crucial.Item Open Access Lysosomes and Ribonucleoprotein Dynamics in NeuronsFernandopulle, MichaelThe lysosome is canonically known as a major organelle for protein degradation and nutrient sensing in the cell. Mutations in lysosomal enzymes and membrane proteins often result in neurological disease, demonstrating the critical role for this organelle in neuronal function and homeostasis. However, little is known about why diseases that impair this ubiquitous organelle often give rise to neuron-specific deficits. Given the selective vulnerability of neurons to lysosomal dysfunction, a better understanding of the unique functions of lysosomes in neurons is necessary. To gain deeper insight into the biochemistry of neuronal lysosomes, we developed methods to engineer and culture human iPSC-derived neurons. Within this system, we used proximity labeling proteomics to profile the lysosomal surface proteome. We discovered that annexin A11 (ANXA11), a protein linked to familial amyotrophic lateral sclerosis (ALS), is a novel component of the lysosomal surface. ANXA11 is also a component of RNA granules, and its bipartite domain organization allows it to act as a tether between RNA granules (N-terminal phase separation) and the lysosomal surface (C-terminal phospholipid binding). We find that ANXA11 enables RNA granule hitchhiking on motile lysosomes, promoting the long-distance transport of RNA within neuronal processes. Critically, disease-linked mutations in ANXA11 disrupt this transport, providing support for failed RNA transport as a pathogenic mechanism in neurodegenerative disease. We identified ALG-2 and calcyclin (CACY) as two important modifiers of ANXA11 phase separation properties, and find that ALG-2 in particular restricts ANXA11-RNA granule contact and RNA granule-lysosome contact. Finally, we identify an unexpected interaction between a ribosome biogenesis protein (NOP14) and endosomal SNARE proteins (STX7, STX8, STX12, and VTI1B) on the surface of lysosomes, and provide some evidence that this interaction mediates ribosome-lysosome tethering. Disrupting these interactions is linked with a depletion of stalled ribosomes in neurites, potentially providing a mechanism for ribosome hitchhiking on lysosomes for neuritic transport. Together, this work illuminates a previously unknown dimension of lysosome biology in neurons, and positions the lysosome as a broadly important organelle for multiple aspects of protein and RNA trafficking and metabolism.Item Open Access Functional and Mechanistic Analysis of Protein Degradation by Human Cytomegalovirus to Uncover Viral Immune Evasion MechanismsFletcher-Etherington, Alice; Fletcher-Etherington, Alice [0000-0001-7313-9247]Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that represents a significant global health burden. In immunocompetent individuals, HCMV establishes a lifelong persistent and typically asymptomatic infection that is controlled by a multifaceted host immune response. However, immunocompromised patients, including transplant recipients and those with acquired immunodeficiency syndrome, are at a high risk of HCMV-associated morbidity and mortality. HCMV is also the most prevalent infectious cause of congenital disease, with the ability to cause neurodevelopmental complications. Cell-intrinsic immune responses are the first line of defence against viruses, mediated by constitutively expressed host proteins and processes that respond directly to virus infection. As a result of virus-host coevolution, viruses often antagonise antiviral host proteins by driving their downregulation, mislocalisation or inactivation. A quantitative proteomic analysis of HCMV infection published by our group found that at least 133 proteins are likely targeted for degradation by HCMV during early infection. For the project presented in this thesis, seven of these candidate antiviral factors were screened for antiviral activity using a novel restriction assay system and plaque assays. Two proteins, mixed lineage kinase domain-like pseudokinase (MLKL) and DmX-like protein 1 (DMXL1), were then selected for further mechanistic and functional characterisation. MLKL is the terminal effector of a form of cell death called necroptosis. Many herpesviruses suppress necroptotic signalling to evade cell death. However, the mechanism of HCMV-mediated inhibition of necroptosis has so far remained elusive. HCMV protein pUL36 was found to be necessary and sufficient for the downregulation of MLKL and the inhibition of necroptosis. pUL36 has previously been shown to inhibit another mode of cell death, apoptosis, making it a multifunctional cell death inhibitor. DMXL1 interacts with the vacuolar-type H+-ATPase to regulate endosomal acidification, with implications for endosomal trafficking, autophagy, immune signalling and many other cellular processes. The viral gene responsible for downregulation of DMXL1 was identified as pUS33A, which may recruit the E3 ligase Kip1 ubiquitination-promoting complex (KPC) to target DMXL1 for proteasomal degradation. Therapeutics currently available for treating HCMV are associated with significant toxicity and drug resistance. Characterisation of the protein-protein interactions underlying the viral evasion of cell-intrinsic immune responses may permit the development of small molecule therapeutics that disrupt these interactions and facilitate endogenous inhibition of viral replication. As well as contributing to our understanding of how HCMV regulates cell death and endosomal acidification, this thesis also presents proteomic data that will enable the identification of additional antiviral host proteins and the characterisation of virus protein function.Item Restricted Exploring Pain Neurobiology: Molecular Investigation of Genetic Sensory DisordersSarveswaran, Nivedita; Sarveswaran, Nivedita [0000-0002-2341-2142][Restricted]Item Open Access A study on non-canonical autophagy signalling(2021-05-22) Karabiyik, Cansu; Karabiyik, Cansu [0000-0001-7993-1825]An essential requirement for cell viability is the ability to restore energy supplies to avoid exhaustion of all resources upon nutrient depletion. Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the engulfment of intracellular content in double-membrane vesicles known as autophagosomes, which fuse with lysosomes for the degradation and recycling of the autophagic cargo. Nutrient starvation leads to the induction of autophagy by activating the master regulator AMP-activated protein kinase (AMPK). AMPK activates multiple downstream regulators such as ULK1, which in the canonical pathway is known to activate the VPS34 complex, resulting in the formation of PI(3)P-containing autophagosomes. A failure to induce functional autophagy has been implicated in a range of neurodegenerative diseases, in which the aggregation of toxic proteins and organelles cause neuronal loss. Since studies suggest that canonical PI(3)P-dependent autophagy is impaired in many neurodegenerative diseases, the potential of upregulating non-canonical autophagy holds great therapeutic value. As earlier research showed that autophagy can be upregulated in a VPS34-independent, PI(5)P-dependent manner upon glucose starvation, in this thesis I elucidated the mechanism leading to upregulation of PI(5)P-dependent autophagy. Here, a new role has been revealed for ULK1. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on amino acid S1548, thereby increasing its kinase activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels such as the neurodegeneration-associated mutant polyQ-huntingtin. This study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve, revealing a novel mechanism by which autophagy is induced.Item Open Access Identification and Characterisation of Human Cytomegalovirus-Mediated Degradation of Helicase-Like Transcription FactorLin, Kai-Min; Lin, Kai-Min [0000-0003-2109-1530]Viruses are known to degrade host factors that are important in innate antiviral immunity in order to infect successfully. To systematically identify host proteins targeted for early degradation by human cytomegalovirus (HCMV), the lab developed orthogonal screens using high resolution multiplexed mass spectrometry. Taking advantage of broad and selective proteasome and lysosome inhibitors, proteasomal degradation was found to be heavily exploited by HCMV. Several known antiviral restriction factors, including components of cellular promyelocytic leukemia (PML) were enriched in a shortlist of proteasomally degraded proteins during infection. A particularly robust novel ‘hit’ was helicase-like transcription factor (HLTF), a DNA repair protein that participates in error-free repair of stalled replication forks. HLTF was found degraded very early during infection, and its expression remained low throughout the course of HCMV lytic cycle. De novo expression of UL145, a previously uncharacterized viral protein, was found necessary and sufficient to degrade HLTF via recruitment of the cullin 4/DDB1 E3 ligase complex. HLTF degradation was reported in human immunodeficiency virus (HIV) infection, however the interaction between HLTF and viruses remain largely elusive. The roles of UL145 were explored in hopes of understanding functions of HLTF in HCMV infection. UL145 was identified as a non-essential immediate early protein, however had a possible role in type I interferon (IFN) induction regulation in later stages of HCMV lytic progression. As the key host protein to be rescued by UL145 deletion, depletion of HLTF was found to transiently impair IFNβ transcription and HCMV infection. I hypothesise that HLTF is an undiscovered nuclear viral DNA sensor that triggers an antiviral interferon response during viral DNA replication. Additionally, work presented here expands the range of powerful screening technologies to identify HCMV restriction factor candidates by identifying virally degraded host proteins. Further investigation of these candidates will contribute to our understanding of how HCMV modulates host protein expression to evade antiviral factors.Item Open Access Gene expression from unintegrated HIV-1 is restricted by the SMC5/6 complexDupont, Liane; Dupont, Liane [0000-0002-2124-4069]Integration of viral DNA into the host genome is the hallmark of human immunodeficiency virus 1 (HIV-1) infections. However, in early infection, the most abundant viral DNA species are extrachromosomal unintegrated viral genomes, which despite being competent for gene expression, are poorly expressed in the host cell. This suggests the existence of a restriction mechanism acting selectively on extrachromosomal DNA species. Importantly, restriction of gene expression is relieved upon delivery of the HIV-1 accessory protein Vpr, which degrades host target proteins via the host CRL4-DCAF1 cullin-RING ubiquitin ligase complex. To identify the responsible Vpr target, I performed a CRISPR-Cas9 forward genetic screen using a custom designed Vpr target sgRNA library. The screen identified SLF2, a poorly characterised protein which recruits the SMC5/6 complex to sites of DNA damage. I confirmed that SLF2 binds the SMC5/6 complex, and gene knockouts validated a functional role for SLF2 and all core SMC5/6 complex components for restriction of gene expression from unintegrated virus. This was independent of the SLF1-mediated DNA damage recruitment pathway. HIV-1 Vpr therefore selectively degrades SLF2 by exploiting CRL4-DCAF1 activity to antagonise restriction. Unintegrated viral genomes are rapidly chromatinised by the host cell, forming the basis for epigenetic regulation. I showed that the SMC5/6 complex binds unintegrated viral genomes in an SLF2-dependent manner, suggesting that the complex acts directly on viral chromatin. Concomitantly, I showed that SLF2 induces a loss of activating histone marks on unintegrated virus. The SMC5/6 complex belongs to the structural maintenance of chromosomes (SMC) family of proteins that share the ability to topologically entrap and translocate DNA, which can lead to chromatin compaction. Using ATAC-seq, I showed that delivery of Vpr protein or loss of SLF2 increased chromatin accessibility suggesting a loss of compaction. I propose a model in which the SMC5/6 complex is recruited via SLF2 to unintegrated viral genomes, causing chromatin compaction with ensuing silencing of gene expression. Expression of hepatitis B virus, another extrachromosomal DNA virus, is also restricted by the SMC5/6 complex. This suggests that the SMC5/6-mediated pathway of extrachromosomal DNA silencing may have wide-ranging importance for understanding HIV-1 as well as other viral and cellular extrachromosomal DNAs.Item Open Access The role of ABHD11 in the regulation of the hypoxia inducible transcription factorsBailey, Peter Stephen John; Bailey, Peter Stephen John [0000-0001-7707-3521]Hypoxia inducible transcription factors (HIFs) mediate a highly conserved cellular oxygen sensing system, driving a diverse set of transcriptional changes that allow adaptation to hypoxic conditions. However, HIFs can also be activated independently of oxygen by changes in levels of mitochondrial metabolites, a mechanism which is important in the development of certain HIF-driven cancers. Critical to understanding this metabolic axis is the recognition that the prolyl hydroxylase enzymes (PHDs), the oxygen sensors within the HIF pathway, are members of a diverse group of enzymes termed 2-oxoglutarate-dependent dioxygenases. These enzymes all require oxygen, iron and the Tricarboxylic Acid (TCA) cycle metabolite, 2-oxoglutarate (2-OG/a-ketoglutarate) for catalytic activity. Therefore, understanding how 2-OG levels are regulated is essential to dissect the relative contribution of metabolism to activation of HIFs. A key determinant of 2-OG metabolism is the 2-oxoglutarate dehydrogenase complex (OGDHc) – a rate limiting step for conversion of 2-OG to succinyl-CoA within the TCA cycle. Genetic disruption of the OGDHc leads to HIF stabilisation through the accumulation of 2-OG and formation of L-2-hydroxyglutarate, an inhibitor of PHDs. Patients with hereditary mutations in the OGDHc develop tumour syndromes, typical of HIF activation. However, how the OGDHc is regulated is not known. In this thesis, I optimise CRISPR/Cas9 forward genetic screens in oxygen replete conditions to identify genes involved in HIF metabolic activation, focusing on the OGDHc. These screens successfully identify known determinants of 2-OG metabolism, including core components of the OGDHc. In addition, they identified ABHD11 as an uncharacterised mitochondrial protein that, on depletion, leads to metabolic stabilisation of HIF-1⍺. Using a combination of cell biology, LC-MS metabolomics and in vitro enzymatic assays, I demonstrate that ABHD11 localises to the mitochondrial matrix and is required for normal TCA cycle function. ABHD11 depletion decreases the activity of the OGDHc, leading to 2-OG accumulation and L-2-hydroxyglutarate formation, consistent with a central role for OGDHc function in HIF metabolic activation. ABHD11 associates with the OGDHc, but ABHD11 depletion does not alter protein levels of the subunits. Instead, ABHD11 is required to maintain an essential post-translational fatty acid modification on the OGDHc E2 subunit (DLST), lipoylation, which is sensitive to damage from reactive oxygen species and lipid peroxidation products. Together, these studies identify ABHD11 as a new component maintaining TCA cycle function through regulating the lipoylation of the OGDHc.Item Open Access Studies on the autophagy gene WIPI4 and its interactor UBR5(2020-01-15) Zhu, YeAutophagy is a tightly regulated process that sequesters and delivers proteins and other cellular substances for degradation in the lysosome. Dysfunction of autophagy has been implicated in many diseases including neurodegenerative diseases, cancer, and infectious diseases. Beta-propeller Protein-Associated Neurodegeneration (BPAN), an early onset neurodegenerative disease, is caused by mutations in WIPI4, a member of the WD repeat domain phosphoinositide-interacting family. This thesis identifies WIPI4 as a regulator of the closure of autophagosomes. Although many proteins have been found to indirectly regulate autophagosome closure, the exact mechanism of this process has remained unclear. This thesis explored the regulatory mechanism of GABARAP by WIPI4. GABARAP is a potential closure regulator and is also required for expansion and fusion steps of autophagy. I identified that WIPI4 regulates the stability and trafficking of GABARAP. Altogether, this thesis furthers the understanding of the mechanism of autophagosome closure.Item Open Access CHIC2 and STUB1 regulate interferon-γ receptor cell surface expression(2020-05-22) Rebeyev, NatalieThe regulated expression of cell surface receptors is essential for cellular homeostasis and defective in disease states. Receptor regulation is particularly important as receptor internalization limits the duration and intensity of substrate signaling. Interferon-γ (IFN-γ), a critical antiviral cytokine, transduces its signal through the interferon-γ receptor (IFNGR), a single-pass heterodimeric receptor composed of IFNGR1 and IFNGR2. While the IFN-γ signal transduction pathway is well studied, the cell surface regulation of the IFNGR is less well characterized. To gain a better understanding of the ligand-independent regulation of cell surface IFNGR, I performed a CRISPR/Cas9 genome-wide genetic screen to identify genes critical for IFNGR regulation. Loss of CHIC2 or STUB1, also known as CHIP, increased IFNGR1 cell surface expression and their role in receptor regulation was further characterized. CHIC2 is a poorly studied, cysteine-rich protein, while the cytosolic E3 ligase, STUB1 is well-recognized for its role in the ubiquitination, internalization and lysosome-mediated degradation of cell surface proteins. To determine if CHIC2 also plays a more generalized role in cell surface protein regulation, I performed ‘plasma membrane profiling’, an unbiased, quantitative proteomic approach to compare the cell surface protein abundance in wildtype vs CHIC2 knockout cells. CHIC2 deficiency increased cell surface expression of 61 proteins in THP-1 cells, 43% of which were also STUB1 regulated, suggesting a critical role for CHIC2 in receptor regulation. In the absence of CHIC2, less cell surface IFNGR1 was internalized and the increased cell surface IFNGR1 expression potentiated IFN-γ-mediated signaling, confirming CHIC2’s functional importance. Chromosomal translocation of CHIC2 is associated with hematological malignancies, but its endogenous function is unknown. I showed that palmitoylation of CHIC2 is essential for function as CHIC2’s membrane insertion is required for interacting with STUB1 and regulation of the IFNGR. STUB1 is likely recruited to IFNGR1 through an exposed hydrophobic region that binds Hsp70, an interaction facilitated by CHIC2. Furthermore, CHIC2 is monoubiquitinated by STUB1 and internalized from the plasma membrane in a STUB1-dependent manner. CHIC2’s ubiquitination is essential for its internalization and likely affects IFNGR1 internalization, though the exact details of this interaction remain unclear. I therefore propose a model whereby membrane-associated CHIC2 recruits the soluble STUB1 E3 ligase to the plasma membrane, allowing STUB1 to associate with, ubiquitinate and internalize IFNGR1, and other receptor substrates. CHIC2 therefore plays a novel and essential role in the regulated expression of multiple cell surface receptors.Item Open Access Resolving Cell Fate: Experimental, computational and mathematical methods in single cell transcriptomic analysis(2019-10-08) Jawaid, Wajid; Jawaid, Wajid [0000-0002-4861-6694]Deeper understanding of the embryological origins of tissues and organs is likely to provide insights into novel clinically relevant preventative and therapeutic strategies. To do so effectively and at a large scale so as to have clinical significance requires an exhaustive and meticulously accurate knowledge of normal morphological development and the underlying molecular pathways. A variety of lineage tracing and classical molecular biology techniques have led to key insights but emerging technologies now offer the possibility of more fine grained and precise measurements at the level of the single cell rather than ensembles of heterogeneous cells. In this study the rapidly developing technology of single cell RNA sequencing was combined with the development of state of the art computational methods to study murine gastrulation and early organogenesis at a hitherto unprecedented granularity. A comprehensive analysis of FLK1+ cells harvested from gastrulating murine embryos was performed. In-silico reconstruction of pseudo-temporal and pseudo-spacial relations were demonstrated. Using prior knowledge of known driver genes deeper substructure was revealed in clusters that were initially defined by unsupervised algorithms, illustrating that careful implementation of supervised approaches can outperform naïve unsupervised methods. In this way multiple members of the leukotriene branch of the arachidonic acid pathway were found to be enriched within a subset of the endothelial cluster that has a molecular signature consistent with yolk sac derived definitive wave haematopoiesis. This was subsequently validated in an in-vitro embryonic stem cell differentiation colony assay. By developing a novel adaptation to tSNE dimensionality reduction that now allows new data points to be mapped backed to previously calculated embeddings, the FLK1+ data set became a reference to which cells from other experiments were mapped. The Tal1-/- knockout mutant was characterised, reaffirming the known phenotype with complete failure of embryonic haematopoiesis. Analysing the Tal1-/- endothelial cluster shows definitively in the in-vivo organism no activation of an alternative cardiac fate programme as was previously postulated from an in-vitro model system. Additionally tSNE mapped Brachyury cells into the void in the FLK1+ dataset and identified a node like population. Loss of spacial context remains an Achilles’ heel of single cell protocols. Generation of the single cell suspensions leads to disruption of cellular contacts and loss of any spacial information. A novel method is described which uses tSNE of bulk spacial data to pre- condition a tSNE map upon which single cells can then be computationally positioned reconstructing spacial context. To model gene interactions and perturbations from single-cell data, a hybrid feed-forward deep neural network was trained on branching pseudo-temporally arranged single cell qPCR data of in-vivo wild-type murine developmental haematopoiesis. Strikingly despite the model never having ‘seen’ a mutant, in-silico gene perturbations in the deep neural network are able to faithfully reproduce the Tal1-/- phenotype. In summary the use of single cell transcriptomics to probe early murine embryology com- bined with development of new methods has uncovered a novel pathway in embryonic haematopoietic development and allowed in-silico reconstruction of a short period of early embryonic haematopoiesis. Critically these methods have broad application within the fields of developmental and stem cell biology.Item Open Access Determinants of clinical phenotype in myeloproliferative neoplasms(2019-10-19) Grinfeld, JacobBackground: Myeloproliferative neoplasms, (MPNs) such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment. Methods: We sequenced coding exons from 69 myeloid cancer genes, common to two separate bait sets, in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes or copy. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort. Cytokine profiles of over 400 patients were also analysed to determine the contribution of the inflammatory microenvironment to phenotype and progression risk. Results: A total of 2035 patients were included in the analysis. 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. In particular a set of mutations that included ASXL1, SRSF2, U2AF1 and EZH2 was enriched in myelofibrosis and associated with poor outcomes. The JAK2 46/1 haplotype strongly correlated with the presence of 9pUPD and independently with a PV phenotype, demonstrating that the underlying germline background can play a role in determining somatic events and can affect the patient’s phenotype in its own right. We defined eight subgroups based solely on clustering of genomic data that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and overall survival. These included a sub-group defined by mutations the same set of chromatin and splicesome component genes described above, and a subgroup enriched for TP53 mutations and chromosomal changes, which carried a significant risk of AML transformation. Patients with no detectable mutations had very low rates of progression or death. By integrating 63 clinical, demographic and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. The prognostic model performed as well as or better than a number of existing risk scores including the high molecular risk genetic score and international prognostic scoring systems and even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy. Cytokine profiles varied significantly across MPN subtypes, with high levels of TNFalpha and IP-10 seen in myelofibrosis, and to a lesser extent in polycythemia vera. Patients with essential thrombocytosis however, were found to have high levels of GROalpha and EGF, and levels of these at single time points or when measured longitudinally were predictive for the risk of progression to myelofibrosis. Conclusions: Comprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients’ outcomes and may support the treatment of patients with myeloproliferative neoplasms.Item Open Access Investigating the function of the hereditary spastic paraplegia protein spastin in the endomembrane system(2019-07-26) Pearson, Guy James; Pearson, Guy James [0000-0001-8623-9239]Hereditary spastic paraplegias (HSPs) are genetically inherited neurological diseases characterised by the distal axonal degeneration of corticospinal neurons. Of the 80 genes currently associated with HSP, mutations in SPAST, encoding the protein spastin, are by far the most common cause of pathology. Spastin functions as a microtubule remodelling enzyme by using energy derived from ATP hydrolysis by its ATPase domain. The location of this activity is governed by spastin’s localisation domains which mediate recruitment to membrane sites including endosomes and the ER. In this thesis I aimed to elucidate the function of spastin at these sites, as well as to analyse the resulting effects on the cell surface proteome. Through this work, I have shown that spastin functions to mediate the fission of endosomal recycling tubule and have confirmed spastin’s localisation to ER exit sites, but show using synchronised secretion assays that spastin is dispensable for generalised cargo secretion of at least 2 classes of secretory cargo. Finally, through quantitative cell surface proteomics, I show that mutation of spastin’s ATPase domain induces substantial remodelling of the cell surface proteome, and through this have generated a list of pathological candidates whose change in surface abundance could drive the pathogenicity of spastin-HSP.Item Controlled Access Dissecting the early steps of MLL induced leukaemogenic transformation using a new mouse model of AML(2019-07-19) Basilico, SilviaTo maintain a balanced production of all mature haematopoietic lineages and at the same time secure a stem cell reservoir, intricate regulatory programs have evolved to control multi-lineage differentiation and self-renewal in haematopoietic stem and progenitor cells. Leukaemogenic mutations commonly disrupt these regulatory programs causing a block in differentiation with simultaneous enhancement of proliferation. AML is a genetically heterogeneous disease characterized by chromosomal rearrangements that often produce fusion proteins with aberrant transcriptional regulatory activities. Among those, t(11;19) generates the MLL-ENL fusion protein, an aberrant transcriptional factor with an unusual mechanism of transcriptional activation targeting the elongation step of transcription. MLL rearrangements develop a unique category of AML and ALL, mainly affecting infants and paediatric patients, whose transcriptional profile differentiates them from other types of leukaemias. While multiple mechanisms driving this disease have been reported, early cellular and molecular consequences of MLL-ENL expression are still poorly understood. The first aim of this thesis was the generation of a new in vitro mouse model of MLL-ENL driven AML. Since even highly purified haematopoietic stem/progenitor cell populations are recognised to be heterogeneous, it is impossible to define a precise wild-type parental control in conventional retroviral transduction leukaemia models. To circumvent this problem, the strategy devised here was based on the mouse haematopoietic progenitor cell line Hoxb8-FL. Hoxb8-FL cells are able to proliferate indefinitely in vitro in the presence of Flt3 ligand (Flt3L) and an estrogen (β-estradiol)-regulated form of Hoxb8, and show lympho-primed multipotent progenitor (LMPP)-like differentiation capacity both in vitro and in vivo. Transduction experiments generated MLL-ENL expressing Hoxb8-FL cells which, following cytokine enriched culture conditions, generated a preleukaemic cell line able to cause AML development in vivo. The second aim of the thesis was to investigate the transcriptional consequences of MLL-ENL expression at early stages of preleukaemic evolution. The main approach applied was scRNA-seq since cell fate choices, including the stepwise acquisition of a malignant phenotype, are made by single cells. These studies were carried out culturing cells either in a self-renewal condition or in differentiating condition since the previously reported requirement of myeloid differentiation for AML development. The third aim of the thesis was to explore whether any of the early leukaemogenic events identified, could represent genetic vulnerabilities specifically associated with MLL-ENL expression. To this end, genome-wide CRISPR-Cas9 screening was performed on the new AML model developed. The data obtained were integrated with scRNA-seq results in order to identify and validate new potential therapeutic targets associated with early MLL-ENL driven transcriptional changes. In summary, the approaches developed and validated within this thesis have generated a new in vitro murine AML model which has been exploited to gain novel insights into the early steps leading to MLL-ENL driven leukaemogenesis and to validate new candidate therapeutic strategies.Item Open Access The study of two transmembrane autophagy proteins and the autophagy receptor, p62(2019-03-28) Runwal, GautamAutophagy is an evolutionarily conserved process across eukaryotes that is responsible for degradation of cargo such as aggregate-prone proteins, pathogens, damaged organelles, macromolecules etc. via its delivery to lysosomes. The process is known to involve the formation of a double-membraned structure, called autophagosome, that engulfs the cargo destined for degradation and delivers its contents by fusing with lysosomes. This process involves several proteins at its core which include two transmembrane proteins, ATG9 and VMP1. While ATG9 and VMP1 has been discovered for about a decade and half, the trafficking and function of these proteins remain relatively unclear. My work in this thesis identifies and characterises a novel trafficking route for ATG9 and VMP1 and shows that both these proteins traffic via the dynamin-independent ARF6-associated pathway. Moreover, I also show that these proteins physically interact with each other. In addition, the tools developed during these studies helped me identify a new role for the most common autophagy receptor protein, p62. I show that p62 can specifically associate with and sequester LC3-I in autophagy-impaired cells (ATG9 and ATG16 null cells) leading to formation of LC3-positive structures that can be misinterpreted as mature autophagosomes. Perturbations in the levels of p62 were seen to affect the formation of these LC3-positive structures in cells. This observation, therefore, questions the reliability of LC3-immunofluorescence assays in autophagy-impaired cells as method of assessing autophagy and points towards the homeostatic function played by p62 in autophagy-impaired cells.