Development of optochemical strategies to study proteome-wide ubiquitination dynamics in a linkage-specific manner and target protein-specific ubiquitination events
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Date
2025
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Abstract
Proteins are the primary drivers of cellular processes and normal cellular functioning depends on their precise balance of homeostasis and degradation. Protein degradation is largely brought about by ubiquitination, a post-translational modification that adds multiple molecules of the protein Ubiquitin (Ub) to substrates and targets them for proteasome-mediated degradation. Ubiquitination is a highly conserved, complex phenomenon involving multiple enzymatic reactions that covalently adds Ub to substrates via an isopeptide linkage between the ɛ-amino group of a substrate lysine (Lys, K) and the carboxyl group of the terminal glycine (Gly, G) of Ub. Ub itself has seven Lys on which more Ub molecules can be added giving rise to polyUb chains. Initially discovered as an important mediator of protein degradation and thus turn over, subsequent studies have revealed that ubiquitination also plays important roles in cellular signal transduction. Linkage-specificity of Ub chains is derived from the specific Lys on which chains are built and their specific structure, giving rise to a complex code of Ub chains Consequently, this linkage-specificity decides the biological outcome of the ubiquitination event. Due to the complexity and inter-twined dynamics of ubiquitination, the study of its linkage specific kinetics becomes largely significant. However, due to its highly transient behaviour, studying protein ubiquitination in vivo has proved to be challenging and is often met with background noise from endogenous Ub and the various possibilities of Ub chain structure formation. In this study, a light-activated Ub has been developed by incorporating a photocaged lysine at specific sites, through amber codon suppression, for the monitoring of proteome-wide linkage-specific polyubiquitination and to gain insights into the kinetics of the same. This study reveals rapid, minute-scale ubiquitination kinetics for Lys11 (K11), Lys48 (K48) and Lys63 (K63) linkages. Also, the roles of individual components of the ubiquitin-proteasome system have been studied in K48-initiated chain synthesis by small molecule inhibition. This approach expands the repertoire of current cellular ubiquitination perturbation strategies with the ability to control linkage-specific ubiquitination at high temporal resolution and is a promising tool for studying ubiquitinome dynamics. Furthermore, in this study, advances were made towards looking into the kinetics of a monoubiquitination event in Ten-Eleven translocation (TET) proteins.
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Keywords
Ubiquitination, Light-activated non-canonical amino acids, Genetic codes expansion
Subjects based on RSWK
Ubiquitin, Genetischer Code