I’m happy to say in the past few weeks I’ve made some progress with my project. While the beginning was a pit rocky, after a few rounds of optimization and discovering we were using a faulty antibody results have started to come in and I couldn’t be more excited. What I’ve been told is that’s kind of how science goes and that a lot of results come in very quickly towards the end of a given amount of time. I’m excited to see what comes out of these last two weeks!
Ubiquitination is a cellular response to damaged proteins caused by oxidative stress. But the mechanisms and targets of different ubiquitin structures have not been well characterized. This research investigated the ubiquitinating enzymes and relative targets related to K48 ubiquitination, a signal for protein degradation, and how the efficiency of relevant molecular machineries, the proteasome and deubiquitinating enzymes, is impacted by different levels of oxidative stress. This was done by using mutant yeast strains with ubiquitinating enzyme knockouts, exposing them to oxidative stress, and analyzing their K48 ubiquitin content with a western blot. Ribosomal isolation and K48 ubiquitin antibody tagging was used to examine ubiquitin targets. Proteasome and deubiquitinating enzyme activity was analyzed using a substrate that fluoresces when processed and tracking light levels emitted by stressed cell lysates. Our data suggests the K48 ubiquitination system involves multiple ubiquitinating enzymes and genes and the greater impact of oxidative stress on the deubiquitinating enzymes than previously anticipated. Improving our understanding of K48 ubiquitination in stressed cells could allow us to better understand diseases such as Parkinson’s and Alzheimer’s that are caused by protein aggregates in stressed cells, and potentially provide new targets for treatment to make cells more resistant to stress.
