Proteostasis signaling in cell cycle control - SUMMARY-ABSTRACT Dynamic oscillations in protein abundance represents the most salient molecular feature of cell cycle progression. This is typified by the cyclins and cyclin kinase inhibitors (CKIs), that oscillate during cell cycle progression and determine the activation kinetics of Cyclin Dependent Kinases, which propel the cell cycle forward. Protein dynamics are not confined to kinase regulators, but rather are exhibited by hundreds of proteins, including regulators of transcription, chromatin organization, cytoskeleton, and metabolism. Defining the pathways, networks and mechanisms underlying these dynamics is essential to understanding proliferative control. Cell cycle protein dynamics are controlled, in part, by the ubiquitin proteasome system. Ubiquitin is the major regulator of protein degradation in eukaryotes and plays an essential and highly conserved role in cell cycle. A cascade of enzymes coordinates the conjugation of ubiquitin onto substrates. However, E3 ligases are the enzymes that ultimately determine when, where and who is ubiquitinated. Like other post-translational modifications, ubiquitin is reversible, and can be removed from substrates by deubiquitinases (DUBs). Thus, the countervailing activities of E3s and DUBs sculpts the proteome to regulate cell cycle progression and the maintenance of genome integrity. The goal of my research is to define enzymes in the ubiquitin pathway that control cell cycle, their cognate substrates, and how specific ubiquitination events contribute to cell cycle progression. To address this goal, the proposed project seeks to address two fundamental questions, which remain understudied. The first relates to key molecular events of G2-phase. Cells arrest in G2 in response to stress and damage and utilize this time to make key decisions about whether to proliferate. However, G2 remains the most poorly studied phase in the cell cycle. We uncovered a widespread program of protein degradation that occurs in G2 and G2/M, and which is regulated by the activity of the multi-functional kinase PLK1. The identity and timing of substrate degradation, the E3 ligases involved, and the consequences of this regulation are almost entirely unstudied. The second question relates to the role of DUBs in cell cycle, which relative to the E3s they antagonize, remain vastly understudied. Using computational and proteomic methods we identified DUBs with understudied roles in cell cycle. We are determining their enzyme kinetics and preferences, substrates, mechanisms of action, and structures when bound to E3 counterparts. We pursue these questions using a combination of cell, molecular and biochemical assays, combined with proteomics and cryo-electron microscopy. Collectively, addressing these questions will identify new regulatory pathways that control cell cycle, proliferative decision making, and the maintenance of genome integrity.
