Abstract
Proteostasis – the balance of protein synthesis, folding, and degradation – integrates with stress signaling, metabolism, and cell death. Central to this network is the Arg/N-degron pathway and its defining enzyme, arginyltransferase 1 (ATE1). This dissertation explores ATE1 and the N-degron pathway as both evolutionarily conserved, mechanistic regulators in eukaryotic biology and as possible translational tools.
First, I show that ATE1 regulates HIF1α stability in a hydroxylation-dependent manner, contributing to our lab’s work in establishing arginylation as a parallel axis to canonical VHL-mediated hypoxia sensing. Second, I identify a redox-sensitive [Fe-S] cluster in ATE1 that is required for its activity and demonstrate that both [Fe-S] cluster and heme biosynthetic pathways are necessary for ATE1-induced cell death in yeast. Third, I reveal that this cell death occurs independently of ATE1’s canonical role in ubiquitin-mediated degradation, instead pointing to a mitochondria-dependent mechanism that may involve ATP synthase subunit c and the mitochondria permeability transition pore. Finally, I extend N-degron logic into applied contexts, developing proof-of-principle strategies for targeted protein degradation: a type II N-degron PROTAC and a cleavage-resistant ubiquitin fusion, each capable of reducing levels of the oncogenic protein MCL1 in preliminary studies.
Together, these findings elaborate on the N-degron pathway, and more specifically ATE1, as a redox-sensitive mediator situated at the intersection of proteostasis, mitochondrial biology, and cell fate, while also demonstrating how its underlying principles can inspire next-generation protein degradation technologies.