Kinetic and structural influences of acetylation on ubiquitin processing

Rachel E. Lacoursiere, The University of Western Ontario

Abstract

Ubiquitin (Ub) is a small modifying protein abundant in cells where it serves numerous regulatory roles including immune signaling, transcriptional regulation, and proteostasis. To exert its function, Ub covalently interacts with a series of E1, E2, and E3 enzymes before final substrate modification. Dysregulation of Ub signaling has implications in human maladies such as cancer, autoimmune disorders, and neurodegenerative diseases. In these diseases and associated in cellulo models, modifications to Ub serve an additional role in Ub regulation. Post-translational modifications like acetylation or phosphorylation modulate protein-protein interactions and Ub signaling. To understand how acetylation of Ub alters the central E2 step in the Ub cascade, we used orthogonal translation to synthesize seven unique versions (K6, K11, K27, K29, K33, K48, or K63) of acetylated Ub (acUb) and showed them to be competent in RING and HECT-type ubiquitination experiments. We then created a library of acUb proteins labeled with the CyPet fluorophore, and in conjunction with the YPet-labeled E2 proteins UBE2D1 and UBE2L3, we optimized a Förster Resonance Energy Transfer (FRET) experiment that highlighted how Ub acetylation modulates the catalytic activity of the E1 protein UBA1. We found that Ub acetylation at most sites significantly altered the formation of both the UBE2D1~Ub and UBE2L3~Ub conjugates. The unloading of the UBE2D1~Ub and UBE2L3~Ub conjugates was then evaluated with the acUb proteins in the presence of small nucleophiles. Our findings that both E2 proteins were sensitive to nucleophilic cysteine prompted the use of cysteine-dependent E3 proteins IPAH3 and HUWE1 to study how acUb alters transthiolation from the E2 protein to an E3 ligase. We used Nuclear Magnetic Resonance (NMR) spectroscopy to study conformation of the UBE2L3~Ub and acUb conjugates. Together, these sets of experiments demonstrated that a subset of the acUb proteins were ineffective in supporting E3 function likely through E2~Ub conformational changes and E3-chain building preference. Overall, we provide the first analysis of the complete set of acUb proteins and how they modulate E2-dependent steps in ubiquitination. We expect our data will guide future experiments to understand the full involvement of Ub acetylation in regulating cellular processes.