ABSTRACT
Many harmful environmental exposures humans experience, such as UV radiation, air pollutants, and exposure
to arsenic compounds, lead to oxidative stress from the accumulation of reactive oxygen species (ROS) in the
cell. A vast array of disease progression results from oxidative stress, such as cancer, neurodegeneration, and
atherosclerosis, and chronic kidney disease. In response to oxidative stress, humans rewire cellular processes
at the transcriptional, translational, and post-translational level. A central aspect of this response includes the
ubiquitin modification, as its global accumulation during oxidative stress is essential for cell survival. These roles
that promote cellular defense include proteasome degradation, autophagy, translational control, and antioxidant
protein production. However, many roles of the ubiquitination in the context of oxidative stress remain
unexplored. My sponsor’s laboratory has begun to uncover new roles for ubiquitination under oxidative stress,
such as how mutations preventing specific K63 ubiquitin linkages increase sensitivity to oxidative stress in yeast.
Additionally, recent unpublished findings highlight an accumulation of K63 ubiquitin at the endoplasmic reticulum
(ER) under oxidative stress in mammalian cells, which is likely to modify ribosomes. This proposal aims to
characterize vital roles of the ubiquitin modification within the oxidative stress response in humans, in my first
aim by defining the outcome of this localized K63 accumulation, and in my second aim by defining vital roles for
ubiquitin enzymes in cell viability through use of a pooled CRISPR loss-of-function screen. For aim one, I will
first test the impact of DUB activity, autophagy and proteasome degradation on removing ER K63 ubiquitin during
stress recovery by western blot and immunofluorescence. If related to DUB activity, which is my leading
hypothesis, I will then use siRNAs to identify a specific ER-resident DUB from a candidate list whose depletion
prevents recovery, then determine how redox levels regulates the DUBs through in vitro and in vivo studies. For
aim two, I will use sgRNA libraries to target 40/40 E2s, 512/~600 E3s, and 90/102 DUBs to identify candidate
enzymes whose sgRNAs become underrepresented under higher ROS levels. Once identified, I will then define
the roles of the most underrepresented enzyme by identifying its stress-associated targets of ubiquitination, as
well as how oxidation regulates its function. Deeper understanding of ubiquitin’s role in the basic mechanisms of
oxidative stress response pathways will inform clinical strategies to mitigate disease progression under ROS
accumulation. This proposal will ultimately support my training to become an independent physician-scientist.
My training plan includes presenting my findings of this work at multiple conferences, and structured interactions
with my mentoring team. With the support of this F30, I will develop the required skill set to successfully transition
to my post-doctoral and residency training.