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dc.contributor.authorPardo Hernández, Carlos
dc.date.accessioned2022-04-28T12:19:57Z
dc.date.available2022-04-28T12:19:57Z
dc.identifier.isbn978-91-8009-671-3 (print)
dc.identifier.isbn978-91-8009-672-0 (pdf)
dc.identifier.urihttps://hdl.handle.net/2077/70924
dc.description.abstractMitochondria are membrane-bound organelles that produce the majority of ATP used to drive metabolic processes in eukaryotic cells. A unique feature of mitochondria is the existence of a separate mitochondrial DNA (mtDNA), which codes for thirteen proteins needed for oxidative phosphorylation (OXPHOS). The double-stranded mtDNA is copied by a dedicated enzymatic machinery, which includes mitochondrial DNA polymerase γ (POLγ) and a set of accessory proteins. Mitochondria also harbor transcription and translation machineries, which are distinct from those in the nucleus. Mutations that affect factors required for mtDNA maintenance or expression can cause mitochondrial disease. In the present thesis, we have characterised a subset of proteins which play an important role in replication and transcription of mtDNA, both from a structural and functional point of view. We have studied mitochondrial helicase TWINKLE and analysed the molecular consequences of disease-causing mutations in this protein. By means of electron microscopy, we have correlated structural changes caused by these mutations, with functional consequences on the protein activity. Our findings demonstrate that mutations impair TWINKLE’s ability to undergo large-scale conformational changes required for proper function. We have also studied one of the most prevalent mitochondrial disease-causing mutations affecting POLγ, A467T. Using a combination of biochemical characterisation and in vivo studies using a murine model, we demonstrate that the mutation impairs interactions between the catalytic and accessory subunits of POLγ. As a result, POLγ dissociates and the catalytic subunit is degraded by LONP1, a protein involved in maintaining mitochondrial matrix protein homeostasis. Details of LONP1 function is further characterised in the two other studies. In these works, we use cryo electron microscopy (cryo-EM) to determine the structure of full-length, human mitochondrial LONP1. In combination with a range of biochemical experiments we use this structural information to establish the mechanisms by which LONP1 can degrade mitochondrial proteins. We also analyse the functional consequences of two LONP1 mutations causing human disease. In vitro analysis demonstrated that the mutations impaired LONP1’s ability to bind and degrade substrates in an ATP-dependent manner. In summary, our work has provided new structural and functional information about POLγ, TWINKLE, and LONP1 – three key factors required for proper mitochondrial function. Our work has also characterised disease-causing mutations affecting these proteins and established the molecular consequences of these alterations.en_US
dc.language.isoengen_US
dc.relation.haspartI. Structural basis for adPEO-causing mutations in the mitochondrial TWINKLE helicase. Bradley Peter, Geraldine Farge, Carlos Pardo-Hernández, Stefan Tångefjord and Maria Falkenberg. Human Molecular Genetics, 2019, Vol.28, No.7 1090-1099. https://dx.doi.org/10.1093/hmg/ddy415en_US
dc.relation.haspartII. DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion. Pedro Silva-Pinheiro†, Carlos Pardo-Hernández†, Aurelio Reyes, Lisa Tilokani, Anup Mishra, Raffaele Cerutti, Shuaifeng Li, Dieu-Hien Rozsivalova, Sebastian Valenzuela, Sukru A. Dogan, Bradley Peter, Patricio Fernández-Silva, Aleksandra Trifunovic, Julien Prudent, Michal Minczuk, Laurence Bindoff, Bertil Macao, Massimo Zeviani, Maria Falkenberg† and Carlo Viscomi†. Nucleic Acids Research, 2021, Vol.49, No.9 5230-5248. https://doi.org/10.1093/nar/gkab282en_US
dc.relation.haspartIII. A dual allosteric pathway drives human mitochondrial Lon. Genís Valentín Gesé†, Saba Shahzad†, Carlos Pardo-Hernández†, Anna Wramstedt, Maria Falkenberg and B. Martin Hällberg. Manuscript submitted to eLife, 2022.en_US
dc.relation.haspartIV. Novel mutations in the substrate binding domain of the mitochondrial matrix protease LONP1 are a cause of mitochondrial disease. Carlos Pardo-Hernández, Mariella T. Simon, Shaya S. Eftekharian, Alexander Stover, Daniel D. Nguyen, Bradley Peter, Wei-Lin Huang, Lauren O’Grady, Inderneel Sahai, Raymond Wang, Susanne M. Rafelski, Claes Gustafsson, Maria Falkenberg† and Jose E. Abdenur†. Manuscript, 2022.en_US
dc.subjectmtDNAen_US
dc.subjectLONP1en_US
dc.subjectPOLγen_US
dc.subjectmtDNA maintenanceen_US
dc.subjectmitochondrial diseaseen_US
dc.subjectTWINKLEen_US
dc.subjectmitochondriaen_US
dc.titleMolecular and structural studies of proteins required for mitochondrial DNA maintenanceen_US
dc.typetexteng
dc.type.svepDoctoral thesiseng
dc.gup.mailcarlos.pardo.hernandez@gu.seen_US
dc.gup.mailcapae24@gmail.comen_US
dc.type.degreeDoctor of Philosophy (Medicine)en_US
dc.gup.originUniversity of Gothenburg. Sahlgrenska Academyen_US
dc.gup.departmentInstitute of Biomedicine. Department of Medical Biochemistry and Cell Biologyen_US
dc.gup.defenceplaceFredagen den 20 Mai 2022, kl 13.00, Hörsal Gösta Sandels, Medicinaregatan 9, Göteborg. https://gu-se.zoom.us/j/61610510869?pwd=ajJiWnlOSkFJL2RjQXNPNThVTXFBZz09en_US
dc.gup.defencedate2022-05-20
dc.gup.dissdb-fakultetSA


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