Advancing Proteomics Technologies to Decipher the Ubiquitin-Proteasome System

Protein-protein interactions (PPIs) are key to the formation of protein complexes, the active components responsible for a multitude of cellular functions. Aberrant PPIs can have detrimental effects on essential biological processes and lead to various human diseases. Elucidating PPI networks and their structural features within cells is central to understanding fundamental biology and molecular alterations associated with human pathologies, and ultimately facilitating therapeutic development. However, delineation of proteome networks to define the cell’s functional states at the systems-level is challenging due to limitations in existing approaches. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful technology for PPI studies, owing to its unique capability of preserving and capturing protein interactions in their native environments, as well as uncovering PPI identities with structural details. Although current XL-MS technologies are successful in global PPI analysis, unmet challenges still remain especially for complete illustration and quantitative assessment of cellular networks, and spatial PPI mapping. This necessitates new developments to enhance technical capabilities for advancing systems structural biology. The ubiquitin-proteasome system (UPS) is the major degradation pathway in eukaryotes, whose network is remarkably dynamic and made of a large number of compositionally and structurally dynamic machines that orchestrate protein ubiquitination and degradation. Dysregulation of the UPS has been linked to many human diseases including cancer and neurodegenerative disorders. Given the clinical success of proteasome inhibitors, the UPS has become an effective platform for drug discovery. Although basic functions of the UPS are understood, molecular details underlying its multi-layer regulation and mechanistic action remain elusive. Therefore, elucidating the interaction and structural dynamics of the UPS network in its physiological context is essential not only for advancing the understanding of UPS biology, but also for augmenting their therapeutic potential in human health and medicine. In the next five years, we plan to address several outstanding technological challenges in PPI studies to better decipher the UPS pathways, by pursuing the following two research directions: 1) Advancing XL-MS technologies for interactomics and structural proteomics at the systems-level; 2) Mapping the UPS network to uncover molecular details underlying protein ubiquitination and degradation. Specifically, we will center our efforts on developing novel XL- MS technologies to enable in-depth and quantitative analysis of proteome networks with structural details and enhanced spatial resolution to define cellular functional states. In addition, we will employ the newly established technologies to delineate action mechanisms of proteasome regulators in protein degradation, investigate Cullin- RING Ligase mediated protein ubiquitination and dissect the organization of the UPS network. Together, these studies will result in an exciting technological advancement in proteomics research, and facilitate answering important but unresolved biological questions associated with UPS biology.