Year: 2024
Type: conference-abstract
Abstract: In all eukaryotes, the protein ubiquitin serves as a key post-translational modification of cellular proteins in multiple essential processes. Although understanding the dynamics of protein ubiquitination as a critical point of regulation is of great interest in many biomedical contexts, few tools are available to directly monitor substrate ubiquitination in mammalian cells. In cells, particularly abundant forms of ubiquitin-protein conjugates are found on histones, where these modifications serve diverse roles that include transcriptional activation, gene silencing, and facilitation of DNA double strand break repair. We are designing protein biosensors to investigate the dynamics of site-specific histone ubiquitination in live cells. The genetically-encoded protein sensors utilizes a “split” fluorogenic reporter protein called splitFAST. The splitFAST N- and C-terminal fragments are expressed as fusion products with a ubiquitin binding domain (UBD) and histone H2A, respectively. This yields an avidity-based sensor capable of recognizing ubiquitination of histone H2A. Importantly, the splitFAST fluorescence is reversible; thus, the sensor can report histone ubiquitination/deubiquitination dynamics. Additionally, we will discuss how the avidity-based strategy using splitFAST can be extended to detect ubiquitination of other proteins. This approach will make possible the development of new tools to monitor dynamics of substrate post-translational modifications in live cells.
Source: Journal of Biological Chemistry
Institutions: Colorado State University, Tufts University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,800
OpenAlex ID: W4393139028