Decoding the Mechanisms and Regulatory Networks of Plant U-Box/RING Ubiquitin Ligase Complexes under biotic stress - PROJECT SUMMARY/ABSTRACT The ubiquitin-proteasome system is a cornerstone of eukaryotic cellular regulation, orchestrating critical processes such as protein homeostasis, signal transduction, cell cycle progression, and stress responses. Central to this system are E3 ubiquitin ligases, which determine substrate specificity and mediate the transfer of ubiquitin from E2 enzymes to target proteins. This research focuses on unraveling the molecular mechanisms, structural dynamics, and regulatory networks of two highly conserved and essential classes of E3 ligases: Plant U-box (PUB) ligases and a specific family of RING ligases, with a particular emphasis on their roles under biotic stress conditions. U-box and RING ligases are conserved across eukaryotes, including humans, where their dysregulation is associated with various diseases. In plants and human U-box and specific RING ligases are integral to immunity, development, and stress adaptation, yet their precise biochemical mechanisms, including substrate recognition and regulation, remain poorly understood. Our preliminary studies have provided significant insights into these ligases. We determined the crystal structure of the U-box domain of a PUB E3 ligase in complex with E2 enzyme that represents a large and highly conserved class of E2s in eukaryotes, revealing a novel dimeric interface. We discovered a redox- sensitive disulfide bond switch in the E2 enzyme that dynamically modulates ubiquitin transfer and chain formation, both in vitro and particularly under biotic oxidative stress conditions. Using proximity labeling coupled with mass spectrometry, we performed distinct experiments including mapping the interactome of a PUB E3 ligase, and investigating the RING complex module under both normal and pathogen-exposed conditions. The experiment informed dynamic proteome changes in response to pathogen exposure, uncovering new relationships and previously unknown target substrates. Additionally, our CryoEM studies of the RING complex module provided critical insights into its dynamic substrate recognition mechanisms, proposing how structural adaptations can drive ubiquitination activity. Together, these findings provide a strong foundation to scale up and expand our studies, enabling deeper exploration of structure-function relationships as well as interactome networks to reveal novel regulatory pathways and mechanisms. This project seeks to decode how U-box and RING ligases integrate into and regulate broader cellular processes. It aims to uncover the molecular mechanisms of substrate specificity, ubiquitin transfer dynamics, and regulatory networks of these ligases. Leveraging structural and functional approaches in vitro and in planta, this research will elucidate plant stress responses and reveal conserved ubiquitination pathways with broad implications for eukaryotic biology and therapeutic innovation.
