Theses - MRC Mitochondrial Biology Unit

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Open Access
Investigating the rate-limiting step of mitochondrial complex I catalysis
Choy, Man Nok
Respiratory complex I (NADH:ubiquinone oxidoreductase) is a key enzyme in metabolism and is the least understood protein in the mitochondrial electron transfer chain (ETC). It couples the energy released from NADH oxidation and ubiquinone (Q) reduction to the translocation of four protons across the inner mitochondrial membrane, contributing to the proton motive force (PMF) used to synthesise ATP. Although structural knowledge of complex I is now extensive, the mechanism by which it couples the redox energy for proton translocation remains unclear. First, the rate-limiting step of catalysis in complex I was investigated from the point of view of the proton. Using the proteoliposomes system, the pH and solvent isotope dependence of kinetic parameters of purified bovine and *Yarrowia lipolytica* complex I mutants were measured under a range of conditions. I find complex I robustly displays a solvent kinetic isotope effect (KIE), signifying that the rate-limiting step involves a proton transfer. This isotopic sensitive step is dependent on Q-chain length, Q binding-site mutations, and Qconcentration, but not dependent on Δp, suggesting that this step is Q-reduction. Proton inventory experiments suggest that a single proton is transferred in the rate-limiting step, and that complex I is rate-limited by a combination of a proton transfer step and an isotopically insensitive step, which was assigned as the product release of ubiquinol. Next, the role of conserved charged residues in the central axis were investigated using the *Paracoccus denitrificans* model system. Mutants of residues involved in the energy propagation pathway and subunit hydration channel gating were evaluated using solvent isotope effect and proton pumping experiments. These mutations all exhibited a greater isotope dependence than WT, showing that proton pumping steps have become robustly rate-limiting. Then, experimental results were evaluated against computational and mechanistic proposals, to identify the role of these residues. Finally, the role of a putative re-protonation channel in NUCM was investigated using the *Yarrowia lipolytica* model system. Point mutations, made up of conserved buried charged residues connecting the Q-binding site to the matrix were generated. Characterisation of mutants revealed that residues with strong ionic interactions (arginine and glutamate) did not express complex I, and that mutation effects were inconsistent with the abrogation of a protonation channel. I conclude that these residues likely play no role in re-protonation, but instead may be important for structural stabilisation.
Embargo
Applications of mitochondrial gene therapy
Nash, Pavel
Genetic aberrations in the mitochondrial genome (mtDNA) can often manifest as clinical pathologies. Along with genetic mutations in nuclear encoded mitochondrial genes, these pathologies form a group of genetic disorders referred to as mitochondrial diseases. Engineered mitochondrially targeted zinc finger nucleases (mtZFNs) have been successfully used to selectively degrade mutation bearing mtDNA both *in vivo* and *in vitro*, resulting in a shift in the genetic makeup of affected mitochondria and subsequently to phenotypic rescue. Due to an uneven distribution in the mtDNA mutation load across tissues in patients, as well as a great diversity in pathogenic mutations, it is of interest to develop selective gene therapy techniques that could be delivered to a particular affected tissue and curated for specific mutations. Specifically, many mitochondrial diseases are characterized by dysfunctions of the muscular and/or central nervous systems (CNS). This thesis demonstrated the effectiveness of *in vivo* mitochondrial gene therapy using mtZFNs in targeting clinically relevant tissues delivered using an adeno-associated viral (AAV) platform to a murine model harboring a pathogenic mtDNA mutation. The work demonstrated effective reduction in mutation load in skeletal muscle, which was accompanied by molecular phenotypic rescue. The gene therapy treatment was shown to be safe when markers of immunity and inflammation were assessed. This work subsequently explored refinements in design methods for mtZFNs using both rational design and directed evolution assays for a novel murine model, with the later method being novel. Lastly, the work demonstrated the phenotypic rescue of a homoplasmic cell line derived from this novel murine model, which was previously an incorrigible genetic defect prior to the advent of mitochondrial base editing. In particular, the combined use of base editing and nuclease treatments showed greater reduction in mutation burden as well as a reduction in the off-target effects associated with base editing. In summary, this work expanded the potential clinical scope of mitochondrial gene therapy, demonstrating effectiveness and safety *in vivo*, improving the design capabilities for therapeutic nucleases and enabling therapy on homoplasmic cells for the first time.
Embargo
Genetic Engineering as a Tool to Investigate Mitochondrial Gene Expression
Mutti, Christian
Mitochondria are complex and dynamic organelles found in most eukaryotic cells, and involved in many cellular functions, most notably the production of cellular energy through the oxidative phosphorylation (OXPHOS) machinery. In mammals, mitochondria contain their own genome that encodes thirteen essential polypeptides for the OXPHOS system, alongside mitochondrial rRNAs and tRNAs required for their expression. Gene expression in mitochondria is distinct from the bacterial and eukaryotic cytosolic counterparts, and many aspects of the processes of transcription and translation are not fully understood. Given the recent advancements in genome engineering in mitochondria, in particular the development of base editing, it is now possible to investigate these processes in more detail than ever before. Using these reverse genetics approaches, this work aims to utilise this new toolkit to probe different aspects of mitochondrial gene expression and broaden the understanding of these processes. The work covers mitochondrial transcription, ribosome biogenesis, translation initiation and protein synthesis chronologically. In the first section of this work, following the discovery of a second light strand promoter (LSP2) in mitochondria in vitro by collaborators, cytosine base editing was used to probe its activity in living cells. By the precise mutation of two individual sites in LSP2, one which increased promoter activity and another which decreased it in vitro, it was possible to demonstrate the same effect in living cells on transcription in living cells and confirm its activity as a bona fide mitochondrial promoter. This promoter likely allows mitochondrial genome replication and gene expression to occur from two sites, and its discovery changes the understanding of these processes. In the next chapter, the role of m4C methyltransferase METTL15 in ribosome biogenesis and rRNA stability was studied. The METTl15 gene was first inactivated in cells using CRISPR/Cas9, and the lack of m4C modification on the mitochondrial 12S rRNA shown. METTL15 knock-out cells exhibited a decrease in mitochondrial translation rates, reduced OXPHOS steady-state levels and perturbed mitoribosomal biogenesis. The levels of the mitoribosomal small subunit were severely depleted in these cells, with proteins close to the modification site showing the most significant reduction. Through the complementation of catalytically inactive METTL15 into knockout cells, the role of METTL15 as a chaperone in ribosome biogenesis, rather than its enzymatic activity, was shown. Base editing of the 12S rRNA was then performed to demonstrate the importance of the integrity of the rRNA decoding centre in ribosome activity. The following chapter explores the roles of enzymes NSUN3 and ALKBH1 in modifying tRNAMet, allowing it to decode non-conventional AUA methionine codons. Ribosome profiling data showed that, in the absence of NSUN3 and ALKBH1, the mitochondrial ribosome stalls at AUA codons, confirming a long-held hypothesis on the role of these enzymes in aiding AUA recognition by tRNAMet. The mutation of the conventional AUG start codons of CYTB and ATP6 was performed to corroborate these findings, yielding unexpected results on mRNA processing. In the final chapter, a library of cytosine base editors was designed and optimised for the precise ablation of each mitochondrially-encoded protein. The library was used to generate near-homoplasmic knockout cells of each protein, with minimal off-targets, allowing for the systemic investigation of the role of each mitochondrially-encoded protein. The library was further used to test the emerging adenine base editors. Taken together, this work provides a comprehensive use of genetic engineering to study different aspects of mitochondrial gene regulation, making important discoveries and contributions to the field.
Embargo
Development of pseudosymmetry analysis to identify key residues in transport protein mechanisms
King, Alannah
Transport proteins, despite making up approximately 10% of the human genome, are understudied. Multiple computational tools exist to analyse them, but no tool exists to predict which residues are likely to be involved in substrate binding or in the mechanism of the transporter. In this thesis, I present GAPS-Pro, a tool to analyse transport proteins based on their pseudosymmetrical properties and to predict the function of individual amino acid residues based on sequence information alone. The mechanisms and structures of transport proteins are symmetrical, however their substrates and coupling ions are not. Therefore, asymmetry has had to evolve within the substrate binding site of the transporter for it to adapt to different functions. However, the symmetric mechanism and structure needs to be maintained such that the transporter remains functional. By identifying strongly symmetric or strongly asymmetric residues, it is possible to predict the function of a residue from sequence information alone. Parameters for GAPS-Pro are developed for three major superfamilies of transporter: the mitochondrial carrier family, the major facilitator superfamily, and the amino acid polyamine organocation superfamily. The software is then tested on transporters for which there is a large amount of experimental data to test the effectiveness of the procedure. GAPS-Pro is then used to select residues for analysis of the human phosphate carrier and the human citrate carrier by mutagenesis, and its versatility is shown by using it to predict the sequence of the ancestral mitochondrial transporter. Work is then presented which uses other computational techniques, such as phylogenetic analysis, to study transporters in a variety of protists. Finally, a discussion about the future directions of GAPS-Pro and the current limitations is presented.
Embargo
The role of mitochondrial transporters in human physiology and adverse drug effects
Jaiquel Baron, Stephany
Commonly prescribed medications often cause off-target mitochondrial dysfunction, but the underlying molecular mechanisms are largely unknown. Mitochondrial transport proteins form a significant class of potential off-targets that remain largely unexplored. The main aim of this thesis is to evaluate the role of human mitochondrial transporters in adverse drug effects, to overcome the technical challenges in their functional characterisation and to identify compounds that can inhibit substrate transport. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), therefore it was validated first as a model for studying drug-carrier interactions. For the first time, pure human ADP/ATP carrier 1 (hAAC1∆1-10), heterologously expressed in yeast, was obtained in a functional form, and two types of studies were carried out. Firstly, thermostability shift assays were used to investigate the binding of drugs, previously reported to inhibit AAC1. Secondly, their effect on transport was assessed in proteoliposomes with reconstituted hAAC1∆1-10, enabling characterization of their inhibition kinetics. This approach confirmed that chebulinic acid, CD-437 and suramin are potent hAAC1∆1-10 inhibitors with IC₅₀-values in the low micromolar range. Next, GSK compiled a library of 33 medications that failed clinical trials or were discontinued due primarily to mitochondrial toxicity. To investigate whether mitochondrial carrier inhibition could cause mitochondrial dysfunction, hAAC1∆1-10 and the human citrate carrier (hCIC), another abundant carrier, were purified and assayed using the strategies described above. Remarkably, from this limited subset of mitotoxic compounds, benzbromarone was shown to inhibit hAAC1∆1-10 and hCIC with an IC50 of 8.4 μM and 12.5 μM respectively. Moreover, tolcapone inhibits hCIC with an IC50 of 5 μM, three times more potent than the canonical inhibitor benzene tricarboxylic acid. Moreover, detailed understanding of the transport processes underpinning the translocation of substrates of other carriers may also provide opportunities to study potential drug off-targets. Therefore, an investigation was carried out into the broad substrate specificity and proton coupling properties of hCIC, which are poorly understood. As inhibitors above have features in common with endogenous substrates, it is also important to identify orphan transporters as new potential off-targets. Hence, human SLC25A44, a transporter that was recently proposed to transport branched-chain amino acids, was also purified, and identified as a potential candidate for structural work, as it is remarkably stable in some short-chain detergents, therefore, crystallisation trials were set up. Consequently, this study has demonstrated that mitochondrial transport proteins can be drug off-targets, since prescription drugs inhibit multiple mitochondrial transport proteins with low micromolar affinity. Thus, these results highlight the importance of exploring drug-transporter interactions further, identifying orphan transporters, and understanding the transport processes. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will ultimately contribute to the development of drugs with an improved safety profile.
Open Access
Mechanisms Controlling the Segregation of Mitochondrial DNA Heteroplasmy
Glynos, Angelos
Mutations of the mitochondrial DNA (mtDNA) are often the cause behind primary mitochondrial disorders affecting 1:5000 individuals. However, the full extent of the impact that mtDNA mutations have is yet to be comprehensively understood. One of the main reasons behind our slow progress in the field is the multi-copied nature of mtDNA, which suggests that even healthy individuals will carry a small percentage of mutated mtDNA molecules alongside healthy ones, in a state termed heteroplasmy. In cases where the proportion of mutant to healthy mtDNA molecules reaches a critical threshold, diverse and multisystem pathological phenotypes begin to appear. While an individual’s mtDNA heteroplasmy level is largely dependent on that of his maternal germline, studies have shown that there are diverse forces, both intra and extracellular in nature that drive segregation. Further complicating this phenomenon, the observed driving forces appear to be mutation- and cell type-specific in their effect. In this dissertation I first describe my work on optimising and validating a protocol that allows us to measure single cell heteroplasmy. Developing this in-house technique, enabled us to perform high-throughput analyses of cell populations of interest while revealing for the first time the intricacies governing single mtDNA heteroplasmy variability at the single cell level. With this protocol in place, I set out to study the heteroplasmy of mouse brain- and spleen-derived populations. In this endeavour, I made use of two novel mouse models that carry a mutation on mitochondrial-tRNA Alanine (mt-Ta), m.5019A>G and m.5024C>T. Recording single cell heteroplasmy values at different timepoints throughout development, we observed that both mutations followed the principles of random genetic drift. The rate of drift exhibited mutation-specific patterns. Moreover, I present a collaborative project geared towards uncovering the impact the two mt-Ta mutations have at the level of the transcriptome on difference cell lineages belonging to E8.5 mouse embryos. I describe the identification of 17 distinct cell lineages and their inherent variability in mtDNA transcript abundance. While no developmental disparities were observed in mutant embryos compared to controls, we did detect an upregulation of mtDNA transcripts in response to the mutation. At the same time, genes that were previously defined as epistatic suppressors/buffers were found to be downregulated. Pseudobulk analysis revealed differential expression of genes both at the level of the organism and that of the cell-lineage. Overall, mice carrying the m.5024C>T mutation seem to mount a greater compensatory transcriptional response compared to their m.5019A>G counterparts. Finally, I explore the relationship between mtDNA heteroplasmy, copy number and the cell cycle. More specifically, making use of a fluorescent cell cycle reporter, I examine mtDNA changes along the cell cycle. Having established a consistent pattern, I assess the impact of genetic manipulation of mtDNA copy number and restriction of glycolysis on cell cycle progression. Finally, I delve into the consequences of large scale mtDNA deletions on the cell’s respiratory capacity and examine whether that defect impacts their ability to complete the cell cycle.
Open Access
Investigating the role of mitochondrial dysfunction in a Drosophila model of C9orf72 ALS/FTD
Au, Wing Hei
Mitochondrial dysfunction is a prevalent feature in many neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). ALS is a debilitating and incurable disease characterised by the loss of upper and lower motor neurons leading to symptoms such as muscle weakness and paralysis. Most patients die from respiratory failure after 2–5 years however, only one globally licensed treatment (Riluzole) is available which only prolongs survival by a modest 2–3 months. A hexanucleotide repeat expansion consisting of GGGGCC (G4C2) in the first intron of C9orf72 is the most common pathogenic mutation in ALS/FTD. Three disease mechanisms have been proposed including haploinsufficiency and the sequestration of RNA binding proteins at accumulations (foci) of the transcribed RNA. Although intronic, the repeats are translated to produce 5 dipeptide repeat proteins (DPRs) through a mechanism known as repeat associated non-AUG (RAN) translation. Various pathogenic mechanisms have been proposed and it is generally accepted that mitochondrial dysfunction is an early alteration in ALS. Mitochondria are vital organelles important for cellular processes regulating energy metabolism and cell survival. The role of mitochondria specifically in C9orf72 ALS/FTD has been relatively understudied, especially in an in vivo system. Excess production of reactive oxygen species (ROS) and defective mitochondrial dynamics are common features of ALS, but it is not clear whether these phenomena are causative or a consequence of the pathogenic process. In this thesis, I have utilised 3 different Drosophila models of C9orf72, including a (i) 36 repeat GGGGCC (G4C2x36), (ii) poly GR36 DPR-only model and (iii) GR1000-eGFP DPR-only model. Firstly, I recapitulated established phenotypic characterisations that have been previously published. Briefly, pan-neuronal expression of the various transgenes exhibit locomotor deficits which I used as a readout for testing different genetic manipulations to modulate and ultimately rescue these behavioural phenotypes. Next, I performed a thorough characterisation of mitochondrial dysfunction in all the models, analysing impacts on ROS, morphology and mitochondrial turnover (mitophagy). I found alterations in mitochondrial morphology, specifically hyperfusion, a reduction in mitophagy, increased ROS production and impaired respiration in these models. Unexpectedly, genetic manipulation to restore mitochondrial fission/fusion dynamics or boosting mitophagy were unable to rescue the locomotor deficits in larvae. However, genetic upregulation of antioxidants such as mitochondrial superoxide dismutase 2 (SOD2) and catalase were able to rescue impaired larval locomotion. Surprisingly, overexpression of cytosolic superoxide dismutase 1 (SOD1) exacerbated larval crawling phenotypes. Together, these data suggest a causative link between mitochondrial dysfunction, ROS and behavioural phenotypes. To elaborate on this connection, I investigated whether the nuclear factor erythroid 2–related factor 2 (NRF2)/Keap1 signalling pathway might play a role. I found that NRF2 was translocated to the nucleus suggesting an activation of the pathway. However, there were minimal changes to NRF2 targeting transcript genes although changes were observed using a glutathione S-transferase D1 (gstD1-GFP) reporter for NRF2 activity. Despite these variable effects, both genetic reduction in Keap1 and pharmacological treatment with an NRF2 activator, dimethyl fumarate (DMF), showed a behavioural rescue in climbing activity of G4C2x36 and GR36 flies. While more research is needed, these results provide compelling evidence that mitochondrial oxidative stress is a major upstream pathogenic mechanism leading to downstream mitochondrial dysfunction such as alterations in mitochondrial function and turnover. Consequently, targeting one of the main intracellular defence mechanisms to counteract oxidative stress – the NRF2/Keap1 signalling pathway – could be a viable therapeutic strategy for ALS/FTD.
Open Access
Cryo-EM studies of substrate and inhibitor binding to mammalian respiratory complex I
Chung, Injae
Mammalian respiratory complex I (NADH:ubiquinone oxidoreductase) is an intricate multi-subunit, energy-transducing membrane protein that is essential for aerobic energy metabolism and NADH/NAD⁺ homeostasis. It couples the energy released from NADH oxidation and ubiquinone (Q) reduction to pump four protons across the inner mitochondrial membrane, contributing to the proton motive force used to synthesise ATP. Despite recent advances in structural knowledge and decades of biochemical investigations, the mechanism of redox-coupled proton translocation by complex I is still unknown. In the work presented in this thesis, electron cryomicroscopy (cryo-EM) was used as a primary tool to provide fundamental insight on this mechanism by generating three-dimensional reconstructions of complex I bound to substrates, ligands or inhibitors. Analyses of the structural data and further interrogation by complementary biochemical, biophysical, and computational approaches were used to develop an integrated understanding of substrate and inhibitor binding to complex I. First, to investigate the mechanism of action of a drug in phase 1 clinical trials against cancers reliant on oxidative phosphorylation (IACS-010759), the structure of mouse complex I inhibited by IACS-2858 – a tighter binding derivative – was resolved to a global resolution of 3.0 Å. The inhibitor, which bears little resemblance to ubiquinone-10 (Q₁₀), occupies the entrance to the Q-binding channel in a ‘cork-in-bottle’ binding mode not previously observed for complex I. Key inhibitor-enzyme interactions were identified, providing a molecular basis for understanding cross-species differences in binding affinities. Modelling of kinetic data showed that IACS-2858 is a simple one-site competitive inhibitor, and the structural motif of a ‘chain’ of aromatic rings was proposed as a characteristic that promotes complex I inhibition. Next, a strategy for reconstituting bovine complex I into lipid nanodiscs supplemented with exogenous Q₁₀ was devised to probe how the native substrate Q₁₀ binds to the ‘reactive site’ of the Q-binding channel. Five structurally and biochemically distinct conformational classes were identified at global resolutions up to 2.3 Å. These structures fall into three major states: an ‘active’ ready-to-catalyse state, a ‘deactive’ pronounced resting state, and a ‘slack’ state that appears partially disrupted and is of uncertain physiological and biochemical relevance. Comparisons of the deactive structures suggested how substrate/ligand binding restructures the Q-binding site and why both Q and NADH are required for reactivation. Importantly, a Q₁₀ molecule spanning the entirety of the Q-binding site was observed with the Q-headgroup close to its proposed ligating partners NDUFS2-His59 and NDUFS2-Tyr108. Combined with results from molecular dynamics simulations, these structures reveal how the charge states of key active-site residues influence the Q₁₀ binding pose. The bound Q₁₀ species is attributed to a quinone paused in a ‘pre-reactive’ conformation.
Embargo
Electron cryomicroscopy structures of respiratory supercomplexes from alphaproteobacteria suggest mechanisms to enhance catalysis and to prevent deactivation of respiratory complex I
Yaikhomba, Mutum
In mitochondria, the electron transport chain complexes that generate the proton motive force to drive ATP synthesis form oligomeric membrane assemblies known as supercomplexes. Even though they are found throughout nature and several physiological consequences are associated with their depletion, it is not clear why they have evolved and what their selective advantage is. While the role of mitochondrial CIII-CIV-type supercomplexes in the context of enhancing catalysis is being delineated, the function of respiratory supercomplexes, particularly at the level of complexes I and III, is unresolved. Moreover, the structures of respiratory supercomplexes comprising the entire electron transport chain in bacteria and those with a transmembrane cytochrome c are not known. Here, the structures of five distinct supercomplex assemblies are presented from Paracoccus denitrificans, a close relative of the mitochondrial progenitor. The initial part of the thesis describes the procedure to purify the respiratory supercomplexes from P. denitrificans membranes, the biochemical and mass-spectrometric characterisation of the purified samples, the single-particle data processing scheme to obtain their structures and the subsequent atomic model building for these five different supercomplexes. These supercomplexes – CI2CIII2CIV2, CI1CIII2CIV2(cbb3)1, CI1CIII2CIV2, CI1CIII2CIV1 and CIII2CIV2 were resolved at resolutions of ~3 - 7 Å. The structures demonstrate that bacterial supercomplexes are modular and assembled from minimal components essential for its catalytic function. Intriguingly, the ~200 Å interface between the bacterial complexes in the membrane is held together by predominantly lipid-mediated interactions, reminiscent of 2D crystals of bacteriorhodopsin. Despite this minimal protein-interaction interface, the bacterial structures bear a striking resemblance with the mammalian counterparts, shedding light on the evolutionary origins of eukaryotic supercomplexes. The alphaproteobacterial supercomplex may also suggest a mechanism to enhance catalysis between CI and CIII by providing structural scaffolds to increases the concentration of hydrophobic substrate ubiquinone-10 between the reaction centres. A physical tether anchors the membrane tethered cytochrome c552 not only at the CIII-CIV interface, but also to cytochrome c1, the electron donor site, explaining why cytochrome c552 can function as an efficient endogenous electron relay between them. In our supercomplex structure captured in the oxidised configuration of CIII, the cytochrome domain of cytochrome c552 fails to reach CIV, suggesting a novel ‘switch mechanism’ for its release to allow the cytochrome domain to reach CIV during catalysis. The CI1CIII2CIV2(cbb3)1, supercomplex structure reveals how this is in stark contrast to the mode of electron conduction between CIII and cbb3 oxidase by the water soluble cytochrome c550. The structure of the CI1CIII2CIV2(cbb3)1 supercomplex also reveals how the mechanism employed by the CIII-CIV supercomplex contrasts with the mechanism of electron conduction between CIII and cbb3 oxidase by the water-soluble cytochrome c550. The arrangement of CIII-cbb3 oxidase in the native CI1CIII2CIV2(cbb3)1 supercomplex, also deviates from the genetically engineered CIII-cbb3 counterpart in a related alphaproteobacterium, providing insight into the contrasting mechanism by which the water soluble cytochrome c is utilised between them. In mammals, respiratory complex I is the largest proton pump of the oxidative phosphorylation machinery and is a genetic hotspot for mitochondrial diseases. So far, fundamental mechanistic investigations have been impeded by the lack of a minimal model system which solely exhibits both structural and biochemical characteristics of the catalytically ‘active’ form of the enzyme. Here, the structure of P. denitrificans complex I is solved in a state that resembles the mammalian ‘active’ state at resolutions of 2.9 - 3.1 Å. This structure is characterised by changes in the conformation of several conserved residues along the hydrophilic axis and with a ubiquinone-10 near the active site. Comparison with other structurally resolved homologs reveals that the alphaproteobacterial complex I possesses several evolutionarily unique features that effectively seal the outlets of the quinone reaction chamber, which are prone to solvent exposure and lead to a ‘deactive’ state, an off-catalytic state. This provides a rationale for the long-standing enigma, observed biochemically, why the alphaproteobacterial enzyme does not undergo ‘deactivation’, a process otherwise suggested to be involved in a regulation of mitochondrial physiology and in preventing ischemia-reperfusion injury. Along with other characteristics, such as amenability of the whole complex to genetic manipulation and straightforward growth characteristics, this inability to transition to the ‘deactive’ form establishes the utility of P. denitrificans as a model system for further investigations of the enigmatic catalytic mechanism of complex I.
Open Access
Mitochondria-Endoplasmic Reticulum Contact Sites: Regulation and Roles in Coordinating Cell Fate Decisions
Morris, Jordan Luke
Mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) form signalling platforms, which are required for cellular processes such as lipid metabolism, mitochondrial dynamics, mitochondrial Ca2+ signalling and apoptosis. MERCs are vital in coordinating cell fate decisions and their deregulation is associated to the aetiology of several diseases including cancer, diabetes and neurodegenerative disorders. This work focuses on the regulation of MERCs by a contact site regulator and the contribution of aberrant ER-to-mitochondria communication in cancer cell death and chemotherapy resistance. The first part investigates a previously uncharacterised role of a fatty aldehyde dehydrogenase (FALDH), which was identified in a proteomic screen performed in the McBride lab (McGill University, Canada), as a candidate regulator of MERCs and mitochondrial dynamics. FALDH has been documented to exist as two differentially localised isoforms, with one localised to the ER membrane (FALDH-ER) and the other localised to the peroxisomal membrane (FALDH-Px). Here, a novel mitochondrial localisation of FALDH-ER was ascribed and was demonstrated to be enriched in mitochondria-associated ER membranes (MAMs). Using mammalian cell culture models and state-of-the-art microscopy, FALDH-ER was shown to be required for MERCs homeostasis, with its loss inducing a reduction in MERCs and a mitochondrial elongation phenotype. Furthermore, FALDH-ER was shown to be required for the induction of MERCs during starvation. The catalytic activity of FALDH-ER was shown to be independent of its capacity to influence both MERCs and mitochondrial dynamics. Super-resolution microscopy and pure mitochondria isolation revealed that FALDH-ER homodimerization was necessary for mitochondrial localisation and both MERCs and mitochondrial network homeostasis. The loss of FALDH-ER-dependent MERCs did not impair the capacity of mitochondria to uptake Ca2+ from the ER upon stimulation. These observations elicit a paradigm shift in our current understanding of MERCs, indicating the existence of discrete classes of MERCs, with both molecular and functional specificities. Interactome analysis of FALDH-ER revealed a potential involvement of FALDH-ER in the regulation of ER homeostasis through the ubiquitin fold modifier (UFM)-ylation pathway. The loss of FALDH impacted steady state UFMylation and resulted in elevated ER stress. Together, ii this work has identified a novel regulator of MERCs, which is required for the coordination of a specific subset of MERCs, which are functionally distinct to previously described MERCs. The second part focuses on the mechanisms of chemotherapy resistance in a subset of aggressive ovarian and lung cancers. These cancers are driven by the loss of SMARCA4/2 expression, which are subunits of the chromatin remodelling complex, Switch/Non- fermentable (SWI/SNF). The loss of SMARCA4/2 induces the epigenetic silencing of ITPR3, which encodes the inositol-1,4,5-trisphosphate receptor-3 (IP3R3). Using live cell spinning disk confocal microscopy, the loss of IP3R3 was demonstrated to impair ER to mitochondria Ca2+ transfer in these cancers, which subsequently inhibits apoptosis. This is proposed to occur through defective cristae remodelling and inefficient cytochrome C release. Finally, the epigenetic reactivation of SMARCA2 by a histone deacetylase inhibitor (HDACi) was able to restore IP3R3 expression, ER-to-mitochondrial Ca2+ uptake and cancer cell death. Together, a SMARCA4/2-dependent mechanism of apoptosis induction was identified, which may be targeted to enhance chemotherapy response in SMARCA4/2-defficient cancers.
Open Access
Paracoccus denitrificans as a model system for studying the mechanism of respiratory complex I
Jarman, Owen
Respiratory complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across an energy-transducing membrane, contributing to the proton motive force used to synthesise ATP. Although structural knowledge of complex I is now extensive, the mechanisms by which it captures the redox energy for proton translocation, and the mechanisms and pathways of the proton pumps, remain elusive. In this thesis, the α- proteobacterium Paracoccus denitrificans is developed and presented as a powerful model system for understanding mitochondrial complex I, combining interrogative biophysical and structural characterisation with the potential for mutagenesis in every subunit. First, aiming to establish conditions for studying the thermodynamic reversibility of P. denitrificans complex I, activation of ATP hydrolysis by the unidirectional F1FO-ATP synthase of P. denitrificans was investigated by the deletion of potential inhibitory subunits and treatment with potential chemical activators. While no conditions were found in which ATP hydrolysis could be sufficiently activated to drive complex I in reverse, insights were gained into the regulatory mechanism of P. denitrificans ATP synthase and the inhibitory role of Mg-ADP. Next, a strain of P. denitrificans containing an alternative NADH dehydrogenase, a bypass enzyme, was generated to facilitate the creation of deleterious complex I variants. In addition, a purification tag was engineered onto complex I, enabling its rapid purification. The isolated complex I was thoroughly characterised and its reconstitution into proteoliposomes was optimised, expanding the toolkit available for the study of complex I variants. The structure of the enzyme was also partially resolved by cryo-EM. The well-resolved map of the hydrophilic domain revealed a novel supernumerary subunit and demonstrated that P. denitrificans complex I exists entirely in the so-called ‘active’ state. However, due to conformational heterogeneity, the cryo-EM map was poorly resolved in the membrane domain, preventing detailed structural modelling of the complete enzyme. Finally, single point variants in the Nqo13 (ND4) subunit of complex I were generated to investigate: (1) key residues in the energy propagation pathway; (2) coordination to the lateral helix of Nqo12 (ND5); and (3) a potential hydration channel controlled by a gating mechanism. Comprehensive characterisation of the variants revealed insights into all three components of the mechanism. Furthermore, no variants were identified that pump fewer than four protons per NADH oxidised, emphasising the tight coupling between ubiquinone reduction and proton translocation, which is conserved even when the rate of catalysis is compromised.
Open Access
Investigating the substrate binding mechanism of the mitochondrial ADP/ATP carrier
Mavridou, Vasiliki
Mitochondrial ADP/ATP carriers catalyse the equimolar exchange of ADP and ATP across the mitochondrial inner membrane, providing metabolic energy for the cell. Crystal structures of the inhibited cytoplasmic-open and matrix-open states support an alternating access mechanism, involving a cytoplasmic and a matrix gate. However, the molecular nature of substrate binding remains unresolved. Substrate binding is concomitant with the structural movements required for translocation, because substrate binding and release provides the free energy changes for the disruption and formation of the gates. The aim of this project was to provide experimental evidence for the residues that are involved in the substrate binding process. For this purpose, all conserved, solvent-accessible residues between the boundaries of the two gates were mutated to alanine, creating a set of 36 variants. A combination of functional complementation, thermostability and transport assays consistently identified five residues (K30, R88, R197, R246 and R287) to be critical for substrate binding. The Ala variants of these residues did not complement growth of an Aac-deficient strain, abolished the concentration-dependent response to substrate, that is observed for the wild-type protein, and had no transport activity. These residues are located roughly in the middle of the central cavity. Around them, another six residues (N85, N96, L135, V138, G192 and Y196) were found to contribute to the binding process. The variants of these residues did not or partially complement growth of the Aac-deficient strain, presented with significantly reduced substrate-induced thermostability shifts compared to the wild-type protein and were either inactive or had altered transport properties. Residues K30, R88, L135, V138, G192, Y196 and R287 cluster together and are accessible with similar conformers in both conformational states, thus forming the main binding site. Residues N96/R197 and N85/R246 form two pairs located on the cytoplasmic and matrix side of the main site and have conformers that change in a state-dependent manner. Hence, they may be involved in the initial binding and guiding of the substrates to the main site and then in their release to the other side of the membrane, inducing conformational changes. The obtained results provide a plausible mechanism for substrate binding, demonstrate that there is a single binding site for ADP and ATP, explain the reversibility of transport and the importance of charge neutralisation in presence of a membrane potential. Results also show that the size and hydrophobicity of the binding pocket are key for the nucleotide base selection.
Open Access
Pathogenesis and Therapy of Mitochondrial Diseases
da Silva Pinheiro, Pedro
Mitochondria are highly dynamic organelles found in most eukaryotic cells, with a fundamental role in the generation of cellular energy through oxidative phosphorylation (OXPHOS). Critical for their function, mitochondria have retained their own genome the mitochondrial DNA, mtDNA. In mammals, replication of mtDNA is ensured by the DNA polymerase POLγ, which is composed by one catalytic subunit POLγA and two accessory subunits POLγB. Mutations in the nuclear-encoded POLG gene, coding for POLγA, are a common cause of human disease leading to a spectrum of disorders characterised by mtDNA instability, thus compromising mitochondrial function. Despite being relatively frequent, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing, partly due to the lack of relevant in vivo models. Here, I describe the generation of two mouse models: 1) the PolgA449T/A449T mouse, which reproduces the A467T change, the most common human recessive mutation of POLG and 2) the PolgWT/Y933C mouse, which reproduces the Y955C change, the most common human dominant mutation of POLG. I focused on the use of the PolgA449T/A449T mouse and complementary in vitro techniques to provide insights into the molecular pathogenic mechanism of this POLG mutation. I describe the data showing that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interaction with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues, with consequences for the pathogenesis of the disease. In the second part of the dissertation, I explore a gene therapy approach for mitochondrial diseases associated with mutations in nuclear-encoded genes. In particular, I test the use of a novel adeno-associated virus (AAV) capsid (PHP.B) as a gene therapy platform to ameliorate the neurological symptoms of a pre-clinical mouse model of mitochondrial disease, the Ndufs4 knockout (Ndufs4-/-) mouse. A single injection with AAV-PHP.B to express the human NDUFS4 in Ndufs4-/- mice, improved lifespan, body weight gain, motor coordination and several molecular and histological features of the brain. These data provide promising proof-of-concept for the use of AAV-mediated gene therapy as a therapeutic option for the number of patients with, currently incurable, mitochondrial disease.
Open Access
Mitochondrial genome engineering in the murine germline using designer nuclease technology
(2022-07-23) McCann, Beverly
Mitochondria are subcellular organelles with numerous roles in metabolic and cellular pathways. Most notably, mitochondria produce ATP and energetic intermediates through oxidative phosphorylation (OXPHOS). Most of the approximately 1500 proteins needed for a fully functional mitochondrion are encoded in the nuclear genome. However, the mitochondrial genome, which is a circular, multi-copy genome of roughly 17 kb in size, also encodes for 13 polypeptide genes that form key components of the OXPHOS complexes, along with the 22 tRNA genes and 2 rRNA genes required for their expression. As with any other genetic material, mutations in mitochondrial DNA (mtDNA) can have serious consequences and can lead to mitochondrial diseases. Diseases arising from mutations within mtDNA can have effects on any tissue, although tissues with high energy demand, such as the brain, the muscles and the eyes, experience greater deleterious effects. A large proportion of diseases caused by mutations in mtDNA occur in a heteroplasmic manner, whereby wild-type and mutant mtDNA haplotypes co-exist within a cell. While generally a mutation load of greater than 60% of mutated mtDNA is required to manifest in mitochondrial disease, this is dependent on the exact mutation. In this way, the ratio of wild-type to mutant mtDNA represents the penetrance of the mitochondrial disease phenotype. Diseases arising from mtDNA mutations are often fatal and at this point are completely incurable. Over recent years, approaches have been developed to selectively eliminate mutated mtDNA, allowing for the re-population of wild-type mtDNA within a cell. This has been achieved by the application of designer nuclease technologies, where engineered DNA binding domains, consisting of either a dimeric zinc finger protein (ZFP) or a transcription activator-like effector (TALE) domain, is conjugated to a dimeric nuclease domain, producing zinc finger nucleases (ZFN) or TALE-nucleases (TALEN), respectively. By targeting these pairs of engineered nucleases adjacent to a site of interest, a DNA double-strand break (DSB) can be produced, leading to degradation of the mtDNA molecule. Using novel architecture to express and localise these engineered nucleases it has been possible to target these nucleases directly to mitochondria (mtZFN, mitoTALEN) in cell culture. When expressing a mtZFN targeting a nuclear gene, this novel architecture seems to be successful in that the majority of protein localises to mitochondria, however, it has proven insufficient to prevent mutations within the nuclear genome, demonstrating the potential risk of off-target effects caused by designer nuclease technology. The recent development of a pathogenic mtDNA disease mouse model, containing a m.5024C>T mutation on the tRNAAla gene within the mitochondrial genome has provided an in vivo model for the validation of the technique. My research includes in vitro experiments demonstrating the expression, localisation and capacity to selectively degrade mutant mtDNA molecules in m.5024C>T mouse embryonic fibroblasts with both mtZFN and mitoTALEN, leading to a shift in heteroplasmy towards a greater proportion of wild-type mtDNA. Very recently, both, the Minczuk and Moraes laboratories have independently demonstrated, that AAV-targeted delivery of designer nucleases in the tRNAAla mouse, could selectively eliminate mutated mtDNA in vivo by use of either designer nuclease technologies. My research focused on adapting this technique for use in mouse embryos. I was able to demonstrate many of the technical and biological obstacles that have to be eliminated to move closer toward in vivo expression, localisation and selective degradation of mutated mtDNA by use of either mtZFN or mitoTALEN in mouse embryos. Through extensive optimisation steps in designer nuclease synthesis, advancement in embryo culture and embryo handling techniques, revising microinjection setups and making more general experimental improvements I was able to achieve in vivo expression and an implied localisation of designer nucleases to mitochondria in the mouse embryo. Additionally, I was able to showcase a shift in mtDNA heteroplasmy and reduction mtDNA copy number after mitoTALEN injections, while maintaining normal and healthy embryo development. While this procedure remains far from being used as a potential treatment for mitochondrial diseases in humans, this work has elucidated the difficulty, hurdles and labour-intensive state of techniques that must be addressed when using this technology to specifically eliminate mitochondrial disease-causing mutations in human embryos.
Open Access
Biogenesis and Function of the Mitochondrial Ribosome
Palenikova, Petra
In this thesis, I present results of my PhD research into the biogenesis and function of the human mitochondrial ribosome. The mammalian mitochondrial ribosome (mitoribosome) is one of the largest ribonucleoprotein complexes in the cell, with the overall molecular mass of the fully assembled 55S monosome being ~2.7 MDa. It is indispensable for cell survival, as it translates 13 polypeptides encoded in mitochondrial DNA, which are essential for oxidative phosphorylation and therefore for supplying the cell with energy in the form of ATP. The mammalian mitoribosome consists of small 28S and large 39S subunits. It is of dual genetic origin, with all protein components encoded in the nucleus and all RNA components encoded in the mitochondrial DNA. A total of 82 proteins, 2 rRNAs and a structural tRNA comprise one mitoribosomal particle. Proper function and assembly of this molecular machine is reliant on numerous associated factors, such as RNA modifying enzymes and assembly factors. Although recent significant progress has been made in our understanding of how the mitoribosome assembles and its functions in translation, we still lack the knowledge about many factors that are necessary for these processes. This work aims to provide insight into the function of selected proteins that were predicted to be involved in biogenesis and/or function of mitochondrial ribosome, namely mitochondrial rRNA methyltransferase 1 (MRM1) and GTP binding protein 8 (GTPBP8). The work also presents genomics and proteomics methods for the study of mitochondrial gene expression machinery and of mitoribosome integrity and assembly, respectively. The first two chapters provide background information for the research performed in the remaining part of the thesis. In the first chapter, I summarise current knowledge of mitochondrial gene expression, with a focus on post-transcriptional RNA modifications and mitochondrial translation. The second chapter describes the materials and methods used. In the third chapter, I present a computational method for analysis of complexome profiling data from experiments that employ stable isotope labelling by amino acids in cell culture (SILAC). This method is implemented in R and is freely available as the Bioconductor package ComPrAn. It provides tools for analysis of peptide-level data as well as normalisation and clustering tools for protein-level data, dedicated visualisation functions and is accompanied by a graphical user interface. Throughout this thesis ComPrAn has been used for quantitative and qualitative analysis of mitoribosomes in studied cell lines. In the fourth chapter, I introduce a CRISPR/Cas9-based screening approach designed to target genes with known or predicted function in mitochondrial gene maintenance and expression. I apply this method to identify genes that show genetic interaction with MRM1. The screen identifies MRM2 as a top candidate for genetic interaction with MRM1. This finding is followed up by the generation of a double knockout cell line which shows severe mitochondrial deficiency, with uridine auxotrophy and disruption of assembly of small mitoribosomal subunit being the most striking effects observed. These findings provide further insight into the role of MRM1 in mitochondria and highlights the complexity of regulation of mitochondrial translation. The fifth chapter focuses on establishing the role of uncharacterised GTPBP8 protein in the cell. I localised GTPBP8 to mitochondria and studied its function by production of a knockout cell line. GTPBP8 knockout presents a strong oxidative phosphorylation defect due to impaired mitochondrial translation. Quantitative analysis of mitoribosome reveals accumulation of both small and large subunits in the knockout, suggesting that GTPBP8 might play a role in very late assembly of either of the subunits, subunit joining or translation initiation. Overall, this work improves our understanding of the regulation of mitochondrial translation by characterising two mitochondrial proteins and their role in mitoribosome biogenesis and function. The CRIPSR/Cas9 screening methodology and ComPrAn R package presented here have potential to be used in the study of other proteins, extending the portfolio of methods available for research of mitochondrial function.
Open Access
Disruption of mitochondrial redox homeostasis as a cellular signal.
Cvetko, Filip
Mitochondria are crucial components of eukaryotic cells and exchange signalling molecules, metabolites, proteins and lipids with the rest of the cell. The organelle is key for energy metabolism as they provide most of the cellular ATP through oxidative phosphorylation and regulate intermediate metabolism. Mitochondria are also a major source of reactive oxygen species (ROS), which are by-products of aerobic respiration and recently recognised as important signalling molecules that control various cellular functions. To avoid the potential damaging effects of ROS, mitochondria contain protein antioxidant systems to help maintain thiol homeostasis. Mitochondria are emerging as an important redox signalling node and are involved in a myriad of signalling pathways, which have a redox component, either through a response to a particular ROS or the shift of the redox state of a responsive group. It is not surprising that mitochondria are therefore heavily regulated by retrograde signalling of the master regulator of cellular antioxidant defence, nuclear factor erythroid-derived 2-related factor 2 (Nrf2). Until now it has not been possible to disentangle the overlapping effects of mitochondrial ROS signalling compared to a redox signal stemming from disruption of mitochondrial thiol homeostasis. Furthermore, it is important to distinguish between disturbing the cytosolic and mitochondrial protein antioxidant systems. I characterised the effects of mitochondrial thiol homeostasis disruption on mitochondrial physiology with MitoCDNB, showing mitochondrial fission. I found that selective disruption of the mitochondrial glutathione pool and inhibition of its thioredoxin system led to Nrf2 activation, while using MitoPQ to enhance production of mitochondrial superoxide and hydrogen peroxide alone did not. To further our understanding of how mitochondrial redox homeostasis is sensed in the cytoplasm and signalled to the nucleus I used an RNAseq approach to investigate the intricacies of early mitochondrial retrograde signalling.
Open Access
Using molecular approaches to understand Complex I deficiency in mouse models
Yin, Zhan; Yin, Zhan [0000-0002-3846-0147]
Complex I (NADH:ubiquinone oxidoreductase), a major electron entry point to the mitochondrial respiratory chain, couples electron transfer from NADH to ubiquinone to proton pumping across the mitochondrial inner membrane, and generates the proton motive force that drives ATP synthesis and transport processes. The ~1 MDa mammalian complex contains 45 subunits, and pathological mutations in both its mitochondrial and nuclear encoded subunits result in diverse neuro-muscular disorders. Recent high-resolution mammalian complex I structures have been solved by single-particle cryo-electron microscopy (cryo-EM) in characterised biological states and provide mechanistic insights. However, the molecular bases of genetically-determined complex I dysfunctions remain unclear. Here, two mouse models of complex I-linked mitochondrial disease were analysed structurally by cryo-EM to understand the mechanisms of their pathogenesis. The first part of this thesis explores complex I from the ND6-P25 mouse model, which contains a mitochondrial-DNA point mutation leading to a proline to leucine substitution at position 25 in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I showed a subtle local structural change resulting in rapid global conversion to a deactive state of the enzyme. Furthermore, the mutant enzyme was unable to catalyse reactive oxygen species production by reverse electron transfer, and the mutant mouse heart is protected against ischemia-reperfusion injury, substantiating a direct link between the two effects. The second part of this thesis describes a structural study of complex I from the ndufs4 knockout mouse model. Although the variant complex I is highly unstable, following sample optimisation its structure was obtained at 2.9 . resolution by cryo-EM. The variant complex I lacking the NDUFS4 subunit is in the active state and, unusually, contains a density resembling ubiquinone in its active site. Absence of NDUFS4 allows motion of the NADH dehydrogenase domain and loss of the NDUFA12 subunit, explaining the instability of the variant complex. Finally, investigations aimed at improving cryo-EM grid preparations for complex I and tackling the problems of limited sample concentration and preferred orientation are described. Grids were modified with graphene, graphene-oxide, polylysine and thiol-PEG; improved numbers of particles could be observed using very low protein concentrations, although with preferred orientation and partial loss of enzyme integrity.
Controlled Access
Expression, Purification and Molecular Characterisation of Sideroflexin Proteins
(2021-05-01) Jones, Daniel
Members of the sideroflexin family are poorly characterised membrane proteins of the mitochondrial inner membrane. Understanding their function is now a high priority, as patients have been identified who carry mutations in human sideroflexin 4, causing a severe mitochondrial, neurological and haematological disease phenotype. The work in this thesis aims to describe the molecular properties of the sideroflexin proteins in order to elucidate their function. In the first part, yeast and the five human sideroflexin proteins were successfully expressed in mitochondria of Saccharomyces cerevisiae and purified to homogeneity. In the absence of a functional assay, thermostability assays and size exclusion chromatography were used to show that the purified proteins were folded, and therefore amenable to further biophysical and functional analyses. Next, the effect of lipids on the stability of human sideroflexin 5 was investigated, demonstrating that cardiolipin is an important stabilising factor. Using these stabilising conditions, it was subsequently established that the protein is dimeric in detergent solution. Based on the principle that interactions with compounds increase the stability of proteins, a compound library was screened in thermostability assays. The results show that human sideroflexin 5 binds zinc ions, which was confirmed subsequently by native mass spectrometry and elemental mass spectrometry. Following this result, the groundwork was laid to find the zinc binding site by combining scanning alanine mutagenesis with thermostability assays. Furthermore, all five human paralogues and yeast sideroflexin were successfully expressed in Lactococcus lactis for zinc binding or transport studies to establish the functional role of the sideroflexins. Finally, in an unexpected way, haem was found to be associated with concentrated human sideroflexin 5, opening up the possibility that sideroflexins could be involved in haem metabolism, which would explain the haematological disease phenotype well. This notion was explored further by Förster resonance energy transfer experiments and binding equilibrium assays using hemin, but hemin intercalates into detergent micelle and it was not possible to separate specific binding from non-specific hemin-detergent interactions, but this lead needs to be explored further.
Open Access
Analysis of PINK1/Parkin-related mitochondrial quality control in Drosophila
Lee, Juliette; Lee, Juliette [0000-0002-4281-2491]
Mitochondria are essential organelles that perform many critical metabolic functions but are also a major source of damaging reactive oxygen species (ROS) and harbour pro-apoptotic factors. Multiple homeostatic processes operate to maintain mitochondrial integrity; however, terminally damaged organelles are degraded through the process of targeted mitochondrial autophagy (mitophagy) to prevent potentially catastrophic consequences. Such homeostatic mechanisms are particularly important for post-mitotic, energetically demanding tissues such as nerves and muscles. There is increasing evidence that failure of this mechanism is linked to normal ageing and some neurodegenerative disorders. Interestingly, two proteins linked to Parkinson’s Disease (PD), Parkin, a cytosolic ubiquitin ligase, and PINK1, a mitochondrially targeted kinase, have been shown to play key roles in this mitophagy. However, little is known about their impact on basal mitophagy in vivo. Moreover, while the consequences of mitophagy defects and the mechanisms that lead to neuronal cell death are currently unclear, aberrant induction of inflammatory signalling is becoming recognised as a key pathogenic mechanism in PD. The work conducted for this thesis aimed to explore the activation of the innate immune system, in the context of PD, using in Drosophila as an in vivo model. First, to analyse mitophagy events in vivo, I developed and characterised transgenic Drosophila expressing the fluorescent mitophagy reporters, the mt-Keima and the mito-QC, were generated to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. My results show that mitophagy is readily detectable and abundant in many tissues including the PD-relevant dopaminergic neurons. However, mitolysosomes were almost completely absent in flight muscles. mechanism associated with the PINK1/Parkin pathway. My work provides evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure. Recently, aberrant activation of immune signalling triggered by the DNA-sensing receptor cyclic GMP–AMP synthase (cGAS) and its downstream signalling effector stimulator of interferon genes (STING) has been implicated in PINK1/Parkin pathology. In order to determine whether the role of Sting in the Pink1/parkin pathology is conserved in Drosophila, I analysed loss of Sting coupled with Pink1/parkin mutants. My work demonstrates that loss of Sting, or the downstream effector Relish, is not sufficient to rescue the behavioural defects or the disruption of the mitochondrial integrity of Pink1/parkin mutant flight muscles, indicating that these phenotypes are not due to aberrant activation of the cGAS-STING pathway in Drosophila. In a broader effort to understand the involvement of the immune system in the Pink1/parkin pathology, I knocked down key components involved in various other immune pathways, in combination with Pink1 and parkin mutants. Like the Sting axis, most of the immune pathways investigated did not seem to modify Pink1 and parkin mutant phenotypes; however, my data revealed that knockdown of key players of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway significantly improved Pink1 mutant phenotypes. Interestingly, this genetic interaction seems to be restricted to Pink1 as loss of JAK/STAT components failed to modify the parkin mutant phenotypes. Although further work needs to be carried out to in order to understand the mechanism behind the interaction between Pink1 and the JAK/STAT pathway, these findings suggest that dowregulation of the particular pathway could be considered as a new therapeutic intervention for PD.
Open Access
Investigations of the mechanism of mitochondrial complex I by electron cryomicroscopy
Grba, Daniel; Grba, Daniel [0000-0003-2915-951X]
Mitochondria are double membrane-bound organelles found in the cytosol of eukaryotic cells. They generate energy for the cell by oxidising the breakdown products of metabolism and relaying the liberated electrons through a series of membrane-embedded oxidoreductase proteins known as the electron transport chain (ETC). The first enzyme in the ETC is complex I (NADH:ubiquinone oxidoreductase). Complex I oxidises NADH in the matrix, reduces ubiquinone in the inner membrane, and couples the energy released to the translocation of four protons across the inner membrane, generating the proton motive force that powers vital cellular processes. The major question of how the energy liberated by NADH:ubiquinone oxidoreduction is captured and efficiently exploited to drive proton translocation is still unanswered. Addressing this question is important for understanding clinically relevant complex I dysfunctions and for pharmacological manipulation of the enzyme. The projects presented here aimed to investigate the mechanism of complex I by using single-particle electron cryomicroscopy (cryo-EM) to generate three-dimensional reconstructions of its structure.