Theses - Cambridge Institute for Medical Research
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Item Open Access Organelle remodeling and V-ATPase dynamics in the lysosome regeneration cycleSava, IoanaLysosomes play an essential role in intracellular degradation and cellular homeostasis, contributing to crucial functions within cellular metabolism. Ongoing research continues to expand our knowledge of the molecular biology and morphology of these organelles, revealing their diverse range of functions. The heterogeneity in lysosomal pH, catalytic activity, and localisation, within the lysosome regeneration cycle has been investigated in this work. This thesis provides evidence that during organelle remodelling in the lysosome regeneration cycle, lysosomal pH is regulated in fed cells by the reversible assembly/disassembly of the proton pumping V-ATPase and that the enzyme lysosomal phospholipase A2 (LPLA2/PLA2G15), a previously proposed target of cationic amphiphilic drugs (CADs), plays a role in bis(monoacylglycerol) phosphate (BMP) synthesis. Chapter 1 of this thesis provides an introduction to lysosomes and the background to the experimental studies undertaken. It includes a description of endolysosome formation (ELF) as a result of the kissing and fusion of late endosomes with lysosomes as well as of lysosome reformation from endolysosomes (ELR), which together make up the regeneration cycle. It also includes a description of the sucrosome system used to study ELR. Chapter 2 describes the materials and experimental methods used throughout my research. In Chapter 3, I describe experiments utilising cathepsin activity probes, MRB and BMV109, to study the intracellular distribution of catalytically active endolysosomes and inactive terminal lysosomes. Using BMV109 in normal rat kidney (NRK) cells expressing EGFP-tagged lgp120 revealed how organelle size and function during ELF and ELR from sucrosomes is regulated. In cells expressing fluorescently tagged subunits of the V-ATPase and other proteins, I found that during the lysosome regeneration cycle, there was an increase in apparent recruitment (by colocalisation with the late endosomal/lysosomal marker lgp120/LAMP1) of the V1G1 subunit of V-ATPase to the lysosomal membrane during ELF (sucrosomes formation). However, colocalisation decreased after 4 hours of invertase-triggered reformation of terminal lysosomes from sucrosomes. In my investigation, I also found that the changes in Rab7a and RILP colocalisation with lgp120/LAMP1 during sucrosome formation (ELF) and ELR follow a similar time course to that observed for the V1-ATPase subunit V1G1. However, there was no change in the colocalisation of the Vo subcomplex subunit Voa3 or the small GTPase Arl8b with lgp120/LAMP1. This suggested the reversible assembly/disassembly of V-ATPase during ELF and ELR. I obtained quantitative evidence that peripheral terminal lysosomes were negative for V1G1 but positive for Voa3, whereas perinuclear endolysosomes were positive for both V1G1 and Voa3. Using pHLARE, a genetically-encoded pH probe, I demonstrated that increased colocalisation of V1G1 with lgp120 during ELF corresponds to decreased lysosomal pH, while the opposite was observed during ELR. In experiments with cells not treated with sucrose, in collaboration with Dr N. A. Bright, I was able to show that the recruitment of V1G1-EGFP to endolysosomes occurs shortly after kissing/fusion of late endosomes with lysosomes. Using fluorescence recovery after photobleaching, in collaboration with Dr N. A. Bright, I observed the dynamic equilibrium and rapid exchange between the cytosolic and membrane-bound pools of this subunit. In Chapter 4, I used Western Blotting using pelleted membranes from NRK cells expressing Lgp120-mCherry/V1G1-EGFP to confirm that the changes in colocalisation of V1G1-EGFP and lgp120-mCherry during ELF (sucrosome formation) and ELR translated into changes in protein abundance at the lysosomal membrane. However, this process was time-consuming, labour-intensive, and yielded variable results. Therefore, lysosomes and sucrosomes were isolated from NRK cells, employing a magnetic isolation protocol. The distribution of V1 and Vo subunits during ELF (sucrosome formation) and ELR was assessed by analysing the magnetically enriched fractions Western blotting and aligned with the confocal microscopy data presented in the previous chapter. These findings support the reversible assembly and disassembly of V-ATPase during ELF and ELR from sucrosomes. In Chapter 5, I describe experiments that showed that mammalian target of rapamycin complex 1 (mTORC1) activation and RILP were likely not necessary for V-ATPase disassembly during ELR in continuously fed cells. The colocalisation of lgp120-mCherry and V1G1-EGFP in NRK cells showed similar changes during ELF (sucrosome formation) and ELR, regardless of treatment with the mTORC1 inhibitor torin1. My conclusions were also supported when comparing the relative abundance of V1G1 and V1A1 subunits of the V1 subcomplex to the amount of the Vod1 subunit of the Vo subcomplex in magnetically isolated lysosomes before and after sucrosomes formation, as well as after a 4-hour invertase addition, both in presence or absence of torin1. Additionally, the siRNA knockdown of RILP did not affect the colocalisation pattern between lgp120-mCherry and V1G1-EGFP described in chapter 3. The role of Rab7a and its interactors’ associating with the V-ATPase may still play a significant role in regulating assembly during ELR. Chapter 6 describes experiments focused on the relationship between lysosomal phospholipase A2 (LPLA2/PLA2G15) inhibition and drug induced phospholipidosis by cationic amphiphilic drugs (CADs). I was able to show, in human embryonic fibroblast (HEK) cells, that the increase in TFEB-GFP translocation after treatments with CADs corresponded to an increase in the fluorescent output of a commercially-available Phospholipidosis detection reagent, in a dose-dependent manner. I investigated whether the properties of LPLA2/PLA2G15 could be studied by expression in yeast cells. However, expressed human LPLA2/PLA2G15 was unable to rescue yeast growth in high oleate conditions and following starvation after depletion of its yeast ortholog, Lro1. Depletion of LPLA2/PLA2G15 in HEK cells, using two different siRNA oligonucleotides, led to an increase in the fluorescent output of the Phospholipidosis detection reagent and a decrease in the number of bis(monoacylglycerol) phosphate (BMP) puncta. This could be rescued after expressing LPLA2-mCherry in LPLA2-depleted HEK cells. This suggests that LPLA2/PLA2G15 is involved in BMP synthesis and its inhibition is linked to phospholipidosis. Lysosomal pH and catalytic activity remained unaffected following LPLA2/PLA2G15 depletion. Chapter 7 is a final discussion including suggestions for future experiments.Item Open Access Stress-signal recognition in the unfolded protein responseNeidhardt, Lisa; Neidhardt, Lisa [0000-0003-0256-5040]The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER) by adjusting its folding capacity to the load of unfolded proteins in the compartment. This critical feedback mechanism governs the functioning of the secretory pathway, impacting various proteins such as receptors, extracellular matrix components, and signalling molecules (Kelly, 1985). Malfunctions in this pathway are linked to diseases like cancer, diabetes, and neurodegeneration (Sherwood, L., 2015; Uhlen *et al*, 2015; Pohlschroder *et al*, 2005; Wang & Kaufman, 2016). Whilst effector mechanisms in the UPR are well characterised, sensing of the unfolded protein load in the ER is incompletely understood. It is widely accepted that the key UPR transducer – IRE1 – responds to the unfolded protein burden by dimerisation/oligomerisation-dependent activation, however, the molecular basis of this upstream event in the UPR remains elusive. This thesis provides insight into regulatory mechanisms acting on the two IRE1 paralogues IRE1α and IRE1β to understand their unique physiological function and infer general principles of UPR regulation. For IRE1α, it has been proposed that the Hsp70 chaperone BiP, a major component of the ER protein folding machinery, couples IRE1α signalling to the folding state of the compartment. This is supported by data showing that the luminal J-domain co-chaperone ERdj4 promotes the formation of a complex between BiP and IRE1's stress-sensing luminal domain (LD) *in vitro* using recombinantly expressed proteins. This observation suggests that the interaction with BiP favours IRE1α LD’s monomeric, inactive state. In line with this concept, loss of ERdj4 derepresses IRE1α activity in cells (Amin-Wetzel *et al*, 2017). However, evidence linking these *in vitro* and cellular observations is sparse. Data reported here show that enforced loading of endogenous BiP onto endogenous IRE1α represses UPR signalling in cells. Furthermore, deletions in the IRE1α-encoding ERN1 locus that de-repress the UPR in cells, encode flexible regions in IRE1α LD that are required for BiP binding and BiP-induced monomerisation *in vitro*. In contrast to IRE1α, little is known about the regulation of IRE1β, a tissue-restricted IRE1 paralogue expressed in mucin producing cells. The evidence presented here identifies the mucin chaperone AGR2 as a repressor for UPR signalling mediated by IRE1β. In cells, AGR2 is a selective repressor of IRE1β signalling without affecting IRE1α activity. *In vitro*, AGR2 binds IRE1β’s LD and promotes monomerisation. In summary, these findings support the concept that the physiological regulation of IRE1 paralogues is governed by ER chaperones with a dual function: Firstly, as UPR repressors, and secondly, as effectors directly involved in maintaining protein folding homeostasis in the compartment.Item Embargo Elucidating how mutations in EIF2AK4 cause pulmonary vascular diseaseSchwiening, Max Henry; Schwiening, Max [0000-0001-8134-534X]Pulmonary veno-occlusive disease (PVOD) is an incurable condition characterised by the progressive remodelling of pulmonary veins, venules and capillaries. This obstruction of the pulmonary vessels causes increased pulmonary pressures which leads to right ventricular hypertrophy, and death within 1-2 years if untreated. Biallelic mutations in the stress sensing kinase, EIF2AK4 (GCN2), are the main genetic cause of PVOD. I hypothesised that loss of GCN2 may lead to a pro-inflammatory phenotype which could be contributing to the development of PVOD. PVOD was modelled using mice with homozygous null mutations in *gcn2*. *Gcn2-/-* mice spontaneously developed an increase in right ventricular systolic pressure, compared to wild-type controls (28.1 ± 3.4 vs. 24.7 ± 3.7 mmHg, p = 0.04). Both left and right ventricles exhibited hypertrophy in the *gcn2-/-* mice but left ventricular systolic pressures were normal. Inflammatory cytokines, in particular interleukin 6, were raised in *gcn2-/-* mice at baseline in both serum and lung compartments which was exaggerated after stimulation with LPS. Chronic exposure to LPS in *gcn2-/-* mice led to elevated right ventricular systolic pressures which was prevented by genetic deletion of interleukin 6. Since the chemotherapy drug mitomycin-c can also induce PVOD in humans I decided to create an orthogonal model of PVOD by exposing wild-type mice to mitomycin-c. These mice also demonstrated right ventricular remodelling, which was prevented by genetic ablation of interleukin 6. Preliminary single-cell RNA sequencing analysis of lung cells from *gcn2-/-* mice shows potential upregulation of pro-inflammatory pathways in *gcn2-/-* macrophages and fibroblasts. I have shown, for the first time, that *gcn2-/-* mice at baseline reproduce features of PVOD and share a pro-inflammatory phenotype with other forms of pulmonary hypertension and these are prevented by loss interleukin 6.Item Embargo A Single Cell Approach to TCR Signal StrengthMa, Claire; Ma, Claire [0000-0002-4244-7535]Cytotoxic T lymphocytes (CTLs) play a key role in the cell-mediated immune response against virally-infected and tumourigenic cells, killing their targets through the release of cytolytic granules. T cell receptor (TCR) recognition of foreign peptides presented by Class I MHC molecules stimulates naïve CD8+ T cell differentiation to effector cells and triggers the cytolytic activity of effector CTLs. Signal transduction downstream of the TCR is a highly diverse and coordinated network of post-translational protein modifications that ultimately drive transcriptional, translational, metabolic and cytoskeletal changes in the cell. How cytotoxic T cells coordinate their molecular machinery in response to strong versus weak stimuli remains unclear. Utilising single-cell methods including mass cytometry and single-cell RNA-sequencing, this study demonstrates how naïve and restimulated cytotoxic T cells coordinate their responses against peptides of differing stimulation strengths.Item Embargo A study of the causes and consequences of SFTPC mistrafficking in alveolar type 2 cells using novel organoid modelsRutherford, Eimear NiamhIdiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterised by progressive lung parenchymal scarring, relentless lung function decline, and eventual respiratory failure. It is universally fatal with current treatments targeting downstream pathogenic pathways and only able to slow disease progression modestly. IPF is triggered by repeated microinjury to the alveolar epithelium and dysfunction of alveolar type 2 (AT2) cells, which in health produce pulmonary surfactant and act as progenitor cells to enable epithelial repair. AT2 cell dysfunction results in failed alveolar re-epithelisation and fibroblast recruitment, activation, and proliferation but the cellular mechanisms underlying this are poorly understood, precluding the development of therapeutics targeting early pathogenic events. Inherited forms of pulmonary fibrosis, including those caused by pathogenic variants of surfactant protein C (SFTPC), offer an opportunity to study these early events. The commonest pathogenic mutation, SFTPC-I73T, results in aberrant accumulation of immature protein at the plasma membrane and in early endosomes resulting in a toxic gain-of-function phenotype. SFTPC-I73T mislocalisation reflects aberrant proprotein proteolytic cleavage, lack of ubiquitination and failed trafficking to lamellar bodies for packaging with other surfactant components. In this thesis, I sought to identify key trafficking factors required for SFTPC maturation in health and to understand the pathogenic consequences of SFTPC-I73T expression. Using targeted forward genetic screens, I identified the E3 ligase ITCH as a novel effector of SFTPC processing, and confirmed its importance in SFTPC maturation by developing a genetically manipulable alveolar organoid model in which to test the consequence of ITCH depletion. Using this novel organoid system, I then generated models of both inducible exogenous SFTPC-I73T expression and its endogenous expression using base editing. These heterozygous models reproduced key phenotypes observed in patient tissue and demonstrated numerous defective cellular pathways including altered apicobasal polarity, perturbed lumenogenesis, global endolysosomal dysfunction, and a markedly altered plasma membrane proteome and lipidome. Together, I have forwarded our understanding of SFTPC handling in health, and the pathogenic consequences of its accumulation in disease. The aberrant cellular pathways I identified are likely to be relevant in sporadic IPF, as is the case with other monogenic inherited causes of pulmonary fibrosis. This could potentially offer novel therapeutic targets for addressing early pathogenic events.Item Embargo An in vitro chemogenomic screening system to identify the molecular targets of antimalarial drug candidatesBower-Lepts, ChristopherThe efficacy of existing antimalarials is threatened by parasite resistance, driving the need for a well-stocked pipeline of potential replacement therapies. A global effort to carry out phenotypic screening in live parasite assays has identified many promising hit compounds, but elucidating the targets of these compounds, often a necessary step in the hit-to-lead development process, has been slow. This is because target identification has rested primarily on one technique, *in vitro* evolution and whole genome sequencing (IVIEWGA), which is time consuming and difficult to scale. Large scale chemical-genetic screening, or chemogenomics, has facilitated rapid and scalable drug target identification in other organisms. To establish a chemogenomic screening system for target identification in asexual *Plasmodium* stages, I leveraged an existing library of *P. berghei* artificial chromosomes (PbACs), of which ~600 containing potential drug targets (transporters, enzymes, metabolic pathways) were engineered to possess a unique DNA barcode. These barcodes can be used to detect and quantify constructs within complex pools using a next generation sequencing (NGS) process termed barcode sequencing (BarSeq), allowing screening of multiple lines in parallel. I transfected PbACs into *P. knowlesi* parasites grown in vitro in human erythrocytes, establishing that PbACs are maintained stably over many generations and the encoded *P. berghei* genes are transcribed in recipient parasites, resulting in systematic target overexpression. *P. knowlesi* was chosen because of its extremely high transfection efficiency, which allowed pooled transfection of up to ~100 constructs at once, generating complex populations of mutant strains. These parasite pools were then exposed to antimalarial drug candidates, and the relative distribution of PbACs within the pool compared before and after < 2 weeks of drug treatment using BarSeq. Pilot screens probing for known gene-compound associations - DSM1-*dhodh*, KDU691-*pi4k* and NITD609-*atp4* - identified those targets with high sensitivity in all cases, and in one case also identified other genes involved in the same mechanism of action pathway (*rab11a*). The remarkable sensitivity of the BarSeq approach also permitted the identification of some of these targets in vastly more complex pools of over 400 strains. I applied the system to a spectrum of approved and developmental antimalarial inhibitors with both known and unknown mechanisms of action, successfully identifying their targets in some cases, particularly those with an underlying copy number variation (CNV) resistance mechanism. In one case the approach elucidated a possible novel target, the parasite signal peptidase enzyme *sec11*. While downstream validation experiments were unable to confirm whether *sec11* was indeed the target for its interacting compound, it could represent a promising druggable target in the parasite warranting further exploration. The PbAC/chemogenomics approach is highly sensitive at identifying targets and is adaptable to high-throughput approaches, thus represents a valuable early-stage tool to explore the mechanisms of action of antimalarial compounds.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 Open Access 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 Open Access 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.