Programmed cell death is essential for animal development and homeostasis, and its disruption is associated with many disorders including neurodegeneration and cancer. Apoptosis is a prominent cell death form, however mutations in key apoptotic regulators only cause minor developmental defects in vertebrates. Non-apoptotic programs also exist, but their molecular basis remains poorly understood. Linker Cell-type Death (LCD) is a non-apoptotic and caspase-independent cell death program operating in C. elegans development. Remarkably, the morphological hallmarks of LCD are observed throughout vertebrate development, suggesting LCD is conserved. The Ubiquitin Proteasome System (UPS) is a key effector of LCD. The heat shock factor HSF-1 stimulates transcription of the E2 ligase
let-70/Ube2D2. LET-70/UBE2D2 functions with Cullin Ring E3 ubiquitin ligases comprising the substrate recognition subunit BTBD-2 and other proteins to stimulate proteasome activity and effect LCD. The proteolytic targets of the UPS during LCD, however, and how their degradation trigger linker cell demise are unknown. To identify candidate targets of the UPS, we performed a yeast 2-hybrid screen against the substrate recognition subunit BTBD-2. Our preliminary evidence suggests that BTBD-2 interacts with an enzyme required for cellular methylation. Importantly, loss of this enzyme restores LCD to animals lacking the E2 ligase
let-70, suggesting that this gene is a bona fide UPS target during LCD. To determine how loss of this enzyme precipitates LCD, we will confirm that the gene is degraded by the UPS, determine the role of its enzymatic activity in LCD, and assess similar genes and known interacting partners for a role in LCD. In parallel to these studies, we have found that EBAX-1, a substrate recognition subunit of Cul2-based E3 ligases, is also required for LCD. Two independent
ebax-1 loss of function mutations display inappropriate linker cell survival, and these defects are rescued by expression of wild-type
ebax-1 genomic clones. Furthermore, an endogenous translational reporter for
ebax-1 is expressed throughout the lifetime of the linker cell. To determine how
ebax-1 promotes LCD, we are performing epistasis studies with other LCD genes and are identifying its target substrates. Together these studies will reveal mechanisms that execute non-apoptotic cell death that can elucidate the etiology of associated disorders and eventually identify novel therapeutic targets.