|M.Sc Student||Inbar Duek|
|Subject||A Shifting Ubiquitin Landscape at Mitochondria: The Effect|
of Protein Degradation Pathways
|Department||Department of Biology||Supervisor||Full Professor Glickman Michael|
|Full Thesis text|
Proteins are the building blocks of the cell and therefore they are under tight regulation throughout their synthesis and function. The two main processes for protein
degradation in the cell are proteasomal degradation and lysosomal degradation.
Ubiquitin is a 76 amino acid protein that serves as a signal molecule for both pathways. Abnormal or misfolded proteins, as well as proteins that have fulfilled their
purpose, are being ubiquitinated and degraded by the ubiquitin proteasome system (UPS). Autophagy is one of the lysosomal pathways, by which bulk cytoplasm is engulfed by an isolated membrane. Originally, autophagy was discovered as a non-specific starvation coping mechanism. Nowadays it is clear that this mechanism can also degrade cellular components in a specific way. Ubiquitin signals proteins to localize near the elongating membrane of the autophagosomes, which enables their degradation by the lysosome. The autophagy process can lead to degradation not only of proteins but of whole organelles as well. Mitophagy is a pathway by which mitochondria are being degraded by the lysosome. The mitochondria are a unique organelle in the cell. Mitochondria takes part in many
biological pathways, but its main role is to produce energy through oxidative phosphorylation. One of the by-products of oxidative phosphorylation are ROS (reactive oxygen species). ROS can oxidize proteins and cellular component thus damaging their structure and function. Being the main source for ROS formation, mitochondria are highly sensitive to oxidative stress and ROS. Mitochondria dysfunction was found in early stages of many neurodegenerative diseases such as Alzheimer disease, Parkinson disease and Huntington disease. In addition, recent studies claim involvement of ubiquitin and the UPS in aging and neurodegeneration as well. Several studies on these subjects have identified elevation in ubiquitination or proteasomal dysfunction. Moreover, in the last two decades it was established that mitochondrial proteins are being ubiquitinated for regulatory purposes. In this study, we investigated the change in the ubiquitination pattern of the mitochondria, upon inhibition of the major proteolytic pathways. We were able to extract highly enriched mitochondria from cells treated with autophagy inhibitor Bafilomycin A1 or proteasome inhibitor MG132. We discovered that autophagy inhibition with Bafilomycin A1 did not cause any changes in the ubiquitin levels at the mitochondria. Moreover, it had a mild effect on the mitochondria morphology. On the other hand, proteasome inhibition causes mitochondria fragmentation and an increase of almost 25 fold in the ubiquitin levels at the mitochondria. We were able to detect an increase in numerus linkage types: K11, K27, K29, and K48. Based on our results we were able to build a model to describe the mitochondria reaction to the proteasome inhibitor MG132. According to our model, the mitochondrial sphere is expanded due to accumulation of ubiquitin, UPS components including proteasome, and regulatory proteins such as chaperons in the mitochondrial fraction. Lastly, we identified induction of autophagy upon proteasome inhibition by MG132. This suggests a link between the two major proteolytic pathways in the cell, where autophagy is induced in order to remove excess or damaged mitochondria.