Investigating the Functional Significance of BRAP-2 in Response to Oxidative Stress in Caenorhabditis Elegans

In order to prevent cellular damage caused by reactive oxygen species (ROS), mammals have developed sophisticated defense mechanisms such as the Keap1-Nrf2 oxidative stress response pathway to maintain homeostasis. Deregulation of Nrf2 could result in detrimental neurodegenerative diseases and cancer. The mammalian BRAP2/IMP is a Ras effector protein with a known role in negatively regulating the ERK/MAPK pathway through the KSR scaffold protein. We are interested in studying the homolog of BRAP2 in C. elegans known as BRAP-2. Our lab has previously shown that a mutation in brap-2 causes an enhanced expression of the SKN-1/Nrf2 target gene gst-4 in the intestine and hypodermis. In this study we utilized genetic and biochemistry approaches to show that BRAP-2 is a negative regulator of SKN-1. Our results demonstrate that brap-2 mutants have enhanced SKN-1 expression within intestinal nuclei. Western blot analysis indicates BRAP-2 physically interacts with activated LET-60/Ras and KSR-2/KSR. Endogenous expression of pMAPK is also higher in brap-2(ok1492), revealing that BRAP-2 is required in activating Ras and the MAPK cascade for SKN-1 regulation. To better define the BRAP-2/SKN-1 signaling cascade, an RNAi screen was performed and 20 novel transcription factors or co-activators of SKN-1 were identified to elevate gst-4 expression in brap-2 mutants. The screen revealed elt-3 and nhr-49 as viable candidates for participation in the SKN-1/Nrf2 signaling pathway to promote this biological effect. Analysis through various experiments show that ELT-3/GATA or NHR-49/PPAR are required for enhanced gst-4 expression in brap-2(ok1492) animals. Our data also indicates that lifespan extension conferred by overexpressing SKN-1 is dependent on functional ELT-3 and NHR-49. Together, this work provides further understanding of how BRAP-2 can regulate SKN-1 in coordination with other transcription factors in response to oxidative stress through the ERK/MAPK pathway. The stress gene regulatory network is a complex model and many of its signaling pathways are evolutionary conserved. My research using the C. elegans model may provide a mechanism on how BRAP2 and Nrf2 are regulated in higher organisms, including humans to facilitate research and development of therapies for the treatment of diseases caused by signaling dysregulation such as cancer.