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Activation of Parkin Ching Activation of Parkin Ching (Jean) Luo Department of Biochemistry McGill University, Montréal April 2019 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science. © Jean Luo 2019 Table of Contents Table of Contents Abstract ........................................................................................................................................... 4 Résumé ............................................................................................................................................ 5 Acknowledgements ......................................................................................................................... 7 List of Abbreviations ...................................................................................................................... 8 List of Figures .............................................................................................................................. 10 List of Tables ................................................................................................................................ 11 1. Introduction .......................................................................................................................... 12 1.1 Parkinson’s Disease .................................................................................................................. 12 1.2 Genetics of Parkinson’s Disease .............................................................................................. 14 1.3 Ubiquitin-Proteasome Pathway .............................................................................................. 15 1.4 Parkin ........................................................................................................................................ 18 1.5 Activation of parkin by PINK1 ............................................................................................... 21 1.6 Activating Element ................................................................................................................... 23 1.7 Links between parkin/PINK1 mutations and Parkinson’s disease ..................................... 27 1.8 Parkin substrates and PICK1 ................................................................................................. 28 1.9 Research objectives .................................................................................................................. 31 2. Materials and Methods ......................................................................................................... 33 2.1 Parkin construct ....................................................................................................................... 33 2 2.2 Mutant constructs of parkin ................................................................................................... 33 2.3 Transformation of parkin constructs into Escherichia coli BL21 strain. ........................... 35 2.4 Expression and purification of parkin ................................................................................... 35 2.5 Expression and purification of GST ....................................................................................... 36 2.6 Expression and purification of GST-PICK1 ......................................................................... 36 2.7 SDS-PAGE ................................................................................................................................ 37 2.8 Phosphorylation of human parkin by PINK1 ....................................................................... 37 2.9 Autoubiquitination assay ......................................................................................................... 38 2.10 In vitro binding assay ............................................................................................................... 38 3. Results ................................................................................................................................... 39 3.1 The ACT element is conserved in invertebrates .................................................................... 39 3.2 Purification of ACT mutants of parkin .................................................................................. 39 3.3 Autoubiquitination assay ......................................................................................................... 41 3.4 In vitro binding assay for PICK1 and parkin ........................................................................ 49 4. Discussion/Conclusion ......................................................................................................... 56 References ..................................................................................................................................... 60 3 Abstract Parkinson’s disease is the second most common neurodegenerative disease, and 10% of the cases can be genetically inherited. Mutations in PARK2 gene, which encodes for parkin, are responsible for most cases of autosomal-recessive juvenile parkinsonism (AR-JP). Parkin is an E3 ubiquitin ligase, which promotes the ubiquitination of specific substrate proteins, and it is activated by PINK1 in order to participate in mitophagy. Parkin is basally autoinhibited, where its E2 binding site and catalytic cysteine are occluded; however, when parkin is bound to phosphorylated ubiquitin (pUb), the released Ubl domain becomes phosphorylated and binds to RING0 domain, releasing the catalytic cysteine on RING2. Recently, an activating element (ACT) has been suggested to participate in the activation of parkin by competing with RING2 for its binding site on RING0, promoting the release of RING2. The first topic of this thesis investigates the effect of each conserved ACT residue on parkin activation through auto- ubiquitination assays. The autoubiquitination assays suggest that the ACT element may enhance the activity of parkin, but it is not essential for its activity. The second topic examines the interaction between parkin and protein-interacting with C-kinase I (PICK1). PICK1 is a substrate of parkin, but unlike most polyubiquitinated substrates, PICK1 becomes monoubiquitinated by parkin, and monoubiquitinated PICK1 inhibits parkin. Its unique inhibitory effect on parkin makes it a potential target as the reduction of PICK1 may enhance parkin’s protective function. PICK1 was previously reported to bind to the C-terminus of parkin through its PDZ domain, but it was recently reported to bind to RING1 through its BAR domain. The latest findings in the domain rearrangement of parkin at different stages of activation suggested that phosphorylated parkin may result in stronger binding between PICK1 and parkin. However, the GST-pull down assays showed no binding between phosphorylated parkin and PICK1, suggesting that phosphorylated parkin does not enhance the interaction between parkin and PICK1. 4 Résumé La maladie de Parkinson est la deuxième maladie neurodégénérative la plus courante dont 10 % des cas peuvent être héréditaires. Les mutations du gène PARK2, qui code pour l’enzyme parkine, sont responsables de la plupart des cas de parkinsonisme juvénile récessif autosomique (AR-JP). La parkine est une ubiquitine ligase E3, qui favorise l'ubiquitination de protéines de substrat spécifiques, et elle est activée par PINK1 afin de participer à la mitophagie. La parkine est autoinhibée basalement, où son site de liaison E2 et la cystéine catalytique sont occlus; cependant, lorsque la parkine est liée à l'ubiquitine phosphorylée (pUb), le domaine Ubl libéré devient phosphorylé et se lie au domaine RING0, libérant la cystéine catalytique sur RING2. Récemment, l'élément activateur (ACT) a été identifié comme participant à l'activation de la parkine en rivalisant avec RING2 pour son site de fixation sur RING0, ce qui favorise la libération de RING2. Le premier sujet de cette thèse porte sur l'effet de chaque résidu d'ACT conservé sur l'activation des parkines par des tests d'auto-ubiquitination. Les essais d'autoubiquitination suggèrent que l'élément ACT peut améliorer l'activité du parkin, mais qu'il n'est pas essentiel pour son activité. Le deuxième sujet présenté dans cette thèse examine l'interaction entre la parkine et l'interaction des protéines avec la C-kinase I (PICK1). Le PICK1 est un substrat de parkine, mais contrairement à la plupart des substrats qui sont polyubiquitinés, le PICK1 devient monoubiquitiné par parkine et le PICK1 monoubiquitiné inhibe la parkine. Son effet inhibiteur unique sur la parkine est en fait une cible potentielle car la réduction du PICK1 peut renforcer la fonction protectrice de la parkine. Le PICK1 se fixait auparavant au terminal C du parkin par l'intermédiaire de son domaine PDZ, mais on a récemment signalé qu'il se fixait à RING1 par l'intermédiaire de son domaine BAR. Cette recherche utilise des essais in vitro pour étudier la liaison entre le parkin et le PICK1 afin d'étudier plus en profondeur les sites de liaison exacts. Cependant, aucune liaison n'a été observée 5 entre parkin et PICK1, ce qui suggère que différentes approches devraient être adoptées pour étudier leurs interactions. 6 Acknowledgements I would like to express my deep gratitude to Dr. Kalle Gehring, my principal investigator, for accepting me into this laboratory and for his patient guidance and constructive critiques of this research work. I would also like to thank Dr. Guennadi Kozlov for his continuous encouragement,
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