PROJECT SUMMARY The objective of this proposal is to illuminate the fundamental mechanisms by which ubiquitin ligases recognize and ubiquitinate substrates within protein complexes. Our interest in this topic arose after our discovery that the multifunctional ubiquitin ligase Anaphase-Promoting Complex (APC) is mutated in inherited neurodevelopmental disorders. Studies from the Ferguson and Brown labs in APC mutant in vivo and in vitro systems demonstrated a previously unknown role for APC-mediated ubiquitin signaling in the regulation of the composition of neuronal heterochromatin through clearance of specific protein substrates. In post-mitotic neurons of the developing APC mutant brain, we found that the most significantly dysregulated target of the APC in neurons was the Chromosome-Passenger Complex (CPC), which includes the kinase Aurora B and the scaffold INCENP. Imaging analysis showed that Aurora B and its product phosphorylated Histone 3 (p-H3, H3S10ph) accumulate within heterochromatin in APC mutant neurons during post-mitotic terminal differentiation. Through the proposed aims, we will examine the interaction between the APC, its target the CPC, and the CPC product H3S10ph, to generate critical structural insight into the molecular pathogenesis of neurodevelopmental disorders. In Aim 1, we will build upon past successes in similar experiments by producing the CPC and the APC as purified recombinant protein complexes and dissect their interaction using in vitro enzyme assays and mass spectrometry. In Aim 2, we will perform cryo-EM to produce a structural map of the APC interacting with the CPC. We predict that the completion of these aims will shed light on the molecular interactions required for the recognition and ubiquitination of the CPC by the APC, providing key mechanistic insight into chromatin regulation by ubiquitin signaling and the pathogenesis of APC-related neurodevelopmental disorders. Through the proposed combination of hypothesis-driven and unbiased experiments, we will build upon our discovery of the neurodevelopmentally essential APC-CPC-H3S10ph axis. Insight gained from these aims will likely serve as the foundation for us to expand in a multitude of long-term experimental directions in vivo and in vitro. The molecular understanding gained from these lines of inquiry would provide critical insight that will be necessary for the development of rational interventions for a class of debilitating diseases that represents a major unmet medical need.