UC Berkeley

Metazoan development and homeostasis require the ability of cells to respond to intracellular stresses and stresses from outside sources. Overactive or dysfunctional metabolism, extracellular toxins, invasion by pathogens, or disease can all contribute to increasing the oxidative stress burden on cells. Oxidative stress occurs due to the buildup of reactive oxygen species (ROS) in cells. These toxic ROS species have the ability to damage DNA, proteins and lipids, thereby preventing proper cellular function and eventually leading to cell death. As such, ROS accumulation during neurodegeneration and aging is thought to contribute to neuronal dysfunction and death. Centered on the transcription factor NRF2, the oxidative stress response counteracts ROS to ensure cell and tissue homeostasis. While unstressed cells constantly degrade NRF2, ROS inhibit the E3 ligase, CUL3KEAP1, to stabilize NRF2 and elicit antioxidant gene expression. Because of the cytoprotective function of NRF2 activity, NRF2 and KEAP1 are frequently mutated in cancer in a way that stabilizes NRF2 to constantly protect cancer cells. Using muscle differentiation as a model, I found that depletion of the amyotrophic lateral sclerosis (ALS) gene CCNF prevents NRF2 activation even in conditions of depleted KEAP1. In a follow-up screen I found that other ALS genes, especially p62/SQSTM1, also prevent full NRF2 activation even when KEAP1 is depleted. These data suggest that the inability to respond to intracellular ROS may generally contribute to ALS disease progression.To understand how NRF2 is still degraded even in conditions of low KEAP, I used mass spectrometry and genetic approaches to identify TRIP12, an essential E3 ligase dysregulated in Clark-Baraitser Syndrome and Parkinson’s Disease, as a component of the oxidative stress response. TRIP12 is a ubiquitin chain elongation factor that acts after CUL3KEAP1 to extend K29-linked conjugates for efficient NRF2 degradation. TRIP12 accelerates stress response silencing as CUL3KEAP1 is being reactivated but limits NRF2 activation during moderate stress. Although necessary, dynamic NRF2 regulation comes at the cost of restricted stress signaling, suggesting that TRIP12 inhibition could be used to bolster the oxidative stress response for the treatment of diseases driven by abundant ROS.