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Ubiquitination and Proteasomal Regulation of Pannexin 1 and Pannexin 3

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Ubiquitination and Proteasomal Regulation of Pannexin 1 and Pannexin 3 Ubiquitination and proteasomal regulation of Pannexin 1 and Pannexin 3 Anna Blinder A thesis submitted in partial fulfillment of the requirements for the Master’s degree in Cellular and Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Anna Blinder, Ottawa, Canada, 2020 ABSTRACT Pannexin 1 (PANX1) and Pannexin 3 (PANX3) are single-membrane channel glycoproteins that allow for communication between the cell and its environment to regulate cellular differentiation, proliferation, and apoptosis. Their expression is regulated through post-translational modifications, however, their regulation by ubiquitination and the ubiquitin proteasome pathway (UPP) has not been examined. Here, I show that PANX1 is monoubiquitinated and K48- and K63-polyubiquitinated, and PANX3 is polyubiquitinated. While treatment with MG132 altered the banding profile and subcellular distribution of both pannexins, data suggested that only PANX3 is degraded by the UPP. To study the purpose of PANX1 ubiquitination, a PANX1 mutant bearing nine lysine mutations was engineered. Results revealed increased cell surface expression of the mutant, suggesting that ubiquitination may regulate trafficking. I thus demonstrated for the first time that PANX1 and PANX3 are polyubiquitinated and differentially regulated by ubiquitin and by the proteasome, indicating distinct mechanisms that stringently regulate pannexin expression. II TABLE OF CONTENTS ABSTRACT ................................................................................................................................................ II TABLE OF CONTENTS ........................................................................................................................ III LIST OF TABLES ..................................................................................................................................... V LIST OF FIGURES .................................................................................................................................. VI LIST OF ABBREVIATIONS ................................................................................................................ VII ACKNOWLEDGEMENTS .................................................................................................................. XIV 1.0 INTRODUCTION ................................................................................................................................. 1 1.1 An overview of pannexins ................................................................................................................ 1 1.1.1 Pannexin post-translational modifications and life cycle .............................................................. 2 1.2 Ubiquitination ................................................................................................................................... 5 1.2.1 Substrate recognition for ubiquitination ........................................................................................ 6 1.2.2 Ubiquitin chain diversity ............................................................................................................... 7 1.2.3 Deubiquitinating enzymes ............................................................................................................. 8 1.2.4 Ubiquitin-like modifiers and signalling ....................................................................................... 10 1.3 Degradation pathways .................................................................................................................... 10 1.3.1 Ubiquitin Proteasomal Degradation Pathway (UPP) ................................................................... 11 1.3.2 Endoplasmic Reticulum Associated Degradation (ERAD) ......................................................... 13 1.3.3 Endocytosis and endolysosomal degradation .............................................................................. 16 1.3.4 Autophagosomal degradation ...................................................................................................... 19 1.4 ER stress and the unfolded protein response (UPR) ....................................................................... 21 1.5 Prototypical ubiquitinated substrates .............................................................................................. 22 1.5.1 Connexin 43 ................................................................................................................................ 22 1.5.2 Tropomyosin-related kinase A .................................................................................................... 24 1.6 Hypothesis and objectives .............................................................................................................. 25 2.0 MATERIALS AND METHODS ....................................................................................................... 26 2.1 Cell lines and cell culture ............................................................................................................... 26 2.2 Plasmids and transfections ............................................................................................................. 26 2.3 Engineering of the PANX1 and PANX3 ubiquitination-resistant mutants .................................... 26 2.4 Pharmacological treatments............................................................................................................ 28 2.5 Cell lysis and western blot analysis ................................................................................................ 28 2.6 In vivo ubiquitination assays .......................................................................................................... 29 2.7 Protein degradation assays ............................................................................................................. 31 2.8 Cell surface biotinylation assay ...................................................................................................... 32 III 2.9 Immunofluorescence staining ......................................................................................................... 32 2.10 Statistical analysis ........................................................................................................................ 33 2.11 Cross-referencing PANX1 protein interactors ............................................................................. 33 3.0 RESULTS ............................................................................................................................................ 36 3.1 PANX1 is post-translationally modified by ubiquitination ............................................................ 36 3.2 PANX1 interacts with numerous components of the ubiquitin-conjugating system and the proteasomal degradation pathway ........................................................................................................ 39 3.3 Proteasomal inhibition regulates PANX1 protein levels ................................................................ 40 3.4 PANX1 is not primarily degraded by the 26S proteasome............................................................. 46 3.5 Lysine residues 307, 381 and 409 do not mediate PANX1 regulation by ubiquitination and by the proteasome............................................................................................................................................ 49 3.6 The lysine residues targeted in the KR9 PANX1 mutant regulate PANX1 protein expression ..... 51 3.7 PANX3 is polyubiquitinated .......................................................................................................... 55 3.8 Proteasomal inhibition regulates PANX3 protein levels ................................................................ 56 3.9 PANX3 is targeted for ubiquitin-dependent proteasomal degradation ........................................... 58 4.0 DISCUSSION ...................................................................................................................................... 63 5.0 CONCLUSION ................................................................................................................................... 77 REFERENCES .......................................................................................................................................... 78 IV LIST OF TABLES Table 1. Sequences of the primers used to introduce lysine to arginine mutations by multisite-directed mutagenesis ......................................................................................................................................... 27 Table 2. Primary and secondary antibodies used for immunoblotting. ...................................................... 29 V LIST OF FIGURES Figure 1. myc-PANX1 is both mono- and polyubiquitinated .................................................................... 37 Figure 2. myc-PANX1 is modified by both K48- and K63-linked polyubiquitin chains in Rh30 cells .... 38 Figure 3. The ubiquitin pathway-related proteins, ubiquitin, UBE2L3, UBR4, PSMC2, PSMD2, and UBXN4 were identified as PANX1 interactors by two proteomic screening methods ...................... 40 Figure 4. Proteasomal inhibition by MG132 changes the PANX1 banding pattern and induces the expression of lower molecular weight bands in Rh30 cells ................................................................ 43 Figure 5. MG132 treatment increases myc-PANX1 localization
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