Function and regulation of the reductive stress response

PROJECT SUMMARY Mitochondria are essential organelles that supply cells with ATP and metabolic building blocks, but also play key roles as signaling hubs that orchestrate the immune response or cell survival. Mutations in mitochondrial proteins impede development and cause diseases, such as neurodegeneration, and a decline in mitochondrial activity is

considered a hallmark of aging. To prevent such consequences, cells employ conserved signaling pathways that detect and alleviate mitochondrial dysregulation. It is a major goal of this proposal to dissect the regulation of a mitochondrial signaling pathway, the reductive stress response, which safeguards activation of the electron

transport chain (ETC) through sensing an invariant ETC byproduct, reactive oxygen species (ROS). The reductive stress response is built on a Cys redox switch: in healthy cells, Cys residues in FNIP1 are oxidized, which stabilizes this protein and allows it to downregulate ETC activity. When cells run out of ROS,

however, the FNIP1 Cys residues become reduced and FNIP1 is recognized by the E3 ligase CUL2FEM1B. The subsequent ubiquitylation and proteasomal degradation of FNIP1 removes this mitochondrial gatekeeper to re- activate the ETC and re-supply cells with ROS. FNIP1 and CUL2FEM1B are therefore the sensory module of the

reductive stress response. Importantly, mutations in FEM1B that hyperactivate this E3 ligase cause syndromic developmental delay, showing that tissue formation and homeostasis require tight regulation of the reductive stress response. This proposal will dissect three crucial modes of regulation that ensure accurate reductive stress signaling.

We will first investigate spatial control of reductive stress signaling. As with all ubiquitylation reactions, FNIP1 modification by CUL2FEM1B takes places in the cytoplasm, yet how cells can modulate the oxidation state of the critical FNIP1 Cys residues in this already reducing environment is unclear. We found that substrate and enzyme

of the reductive stress response are anchored on the outer mitochondrial membrane via the TOM complex, a channel that connects the oxidative mitochondrial intermembrane space with the reducing cytoplasm. In our first aim, we will dissect how this localization impacts reductive stress signaling. We expect that this work will reveal

a novel function of a membrane channel as an E3 ligase co-adaptor. Moreover, it will likely allow us to pinpoint the source of ROS that mediate reductive stress signaling, thereby revealing a sought-after physiological role for ROS in signaling. We will next focus on the regulation of reductive stress signaling by the cell cycle. ROS have long been

suggested to control cell division, and we had indeed found that hyperactivation of CUL2FEM1B inhibits proliferation. This result implied that ROS control the cell cycle via the reductive stress response. In line with this notion, we identified the cell cycle regulator RNF187, which promotes cell cycle progression downstream of growth factor

signaling, as an inhibitor of CUL2FEM1B. Our preliminary data suggest that RNF187 and CUL2FEM1B collaborate to restrict another E3 ligase, AMBRA1, which drives cyclin D degradation and thereby prevents initiation of DNA replication. In our second aim, we will dissect the mechanistic underpinnings of this E3 ligase crosstalk to reveal

how ROS signaling is integrated into the cell cycle program. We expect this work to explain how redox stress can its exert negative consequences onto development or onto tissue homeostasis during tumorigenesis. While our first aims address physiological modes of regulation, we will finally develop methods to exert

pharmacological control over reductive stress signaling. As the reductive stress response tunes the ETC, activating the reductive stress E3 ligase CUL2FEM1B provides us with a unique opportunity to increase ETC output in pathologies driven by mitochondrial decline or inhibition. Moreover, because CUL2FEM1B acts on mitochondrial

surfaces, compounds that target this E3 ligase could be converted into localized proteolysis-targeting chimera for more efficient and more specific focused degradation of pathological proteins. In our last aim, we will build on our discovery of compounds that displace protein inhibitors from CUL2FEM1B,, thereby activating both FNIP1

degradation and ETC function. This work will lay the foundation for mitochondria-associated protein degradation as a new modality to provide therapeutic benefit during aging or in neurodegenerative disease. Our proposal takes an integrated genetic, biochemical, and pharmacological approach to dissect the

regulation of the reductive stress response as a conserved mitochondrial stress response. This work will reveal fundamental principles of redox signaling and may lead to the development of a new therapeutics that could benefit patients of neurodegenerative diseases that currently have few, if any, treatment options.