UBC Theses and Dissertations
Gp78 E3 ubiquitin ligase mediates both basal and damage-induced mitophagy Alan, Parsa
Mitochondrial degradation through the lysosomal delivery pathway, otherwise known as mitophagy, is a strongly conserved evolutionary process that cells and tissues use to adapt to various cellular environments. Defects in mitophagy have been linked to neurodegenerative pathologies, aging phenotypes, as well as cancer progression. Here, we show that CRISPR/Cas9 knockout (KO) of Gp78 in the HT-1080 cell line results in an approximately two-fold increase in mitochondrial volume. Knockdown of the essential autophagy gene ATG5 in the wildtype HT-1080 cells showed similarly increased levels of mitochondrial mass as present in the Gp78 KO clones. Therefore, the increased mitochondrial mass exhibited by the Gp78 KO clones is because of its reduced capacity for autophagy upon loss of Gp78. The increase of mitochondria phenotype upon loss of Gp78 was recapitulated in vivo, where xenograft tumors in Rag2 immunodeficient mice showed increased mitochondrial staining in tumor sections for all Gp78 knockout clones relative to the parental HT-1080 cells. To directly assess Gp78-mediation of basal mitophagy, we developed a sensitive assay using the HT-1080 and Gp78 KO cells stably transfected with the GFP-mRFP-LC3 (tfLC3) autophagy reporter and assessed mitophagic flux upon the chemical inhibition of autophagy with bafilomycin A1. We applied SPECHT, a novel Laplacian-based image analysis algorithm, to quantify tfLC3 puncta association with mitochondria, and found that loss of Gp78 results in impaired autophagic and mitophagic flux. We also show that loss of Gp78 results in the accumulation of defective mitochondria and increased levels of reactive oxygen species (ROS) that are closely associated with mitochondria. Collectively, the data presented provide evidence that Gp78 mediates basal mitophagy, and that impaired basal mitophagy due to loss of Gp78 results in increased ROS and the accumulation of defective mitochondria.
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