Intracellular communication via ubiquitin (Ub) signaling impacts all aspects of eukaryotic cell biology and regulates pathways critical to human development and viability. Aberrations or defects in Ub-signaling can result in numerous debilitating diseases including neurodegenerative diseases, infections, and cancer. Despite remarkable progress over the decades, we still have only a rudimentary understanding of the molecular factors and networks of interactions that govern the assembly of the proteins required for regulated Ub transfer and signaling. The ability to intervene in diseases related to Ub-signaling requires a thorough understanding of these pathways at the molecular level. The basic scheme for Ub modification involves the concerted activities and interactions of several different proteins. This proposal focuses on expanding our understanding of a central player in Ub-transfer reactions, the class of enzymes known as Ub-Conjugating Enzymes or E2s. Once thought simply to shuttle Ub to the site of modification, emerging evidence suggests that E2s can play a pivotal role in substrate recognition, determining the nature of Ub modification of a target protein, and even interacting with and influencing the activities of proteins outside the traditional Ub-transfer pathway. There are ~40 E2s in the human genome and most are thought to be directly involved in Ub transfer. Very little functional information is available for the majority of these E2s. The Research Plan outlined in this proposal seeks to substantially expand our understanding of E2 function. Using various biochemical approaches, NMR spectroscopy, crystallography, and mass spectrometry, this project seeks to develop a molecular understanding of the factors that govern 1) the intrinsic activity of E2~Ub conjugates toward particular residues, 2) E2~Ub recognition of substrates, and 3) and regulatory interactions of E2~Ub conjugates that regulate proteins outside the main Ub-transfer pathway. We have adapted and developed new tools with which to identify proteins selectively modified by particular E2~Ub conjugates. We make use of a new "in coli" expression system that can reconstruct Ub-transfer pathways in E. coli. This system allows us to generate particular E2~Ub conjugates and investigate their activity in cells without complications from competing Ub- transfer reactions. The new knowledge we seek to obtain will have a significant impact our understanding of human E2 structure and function and greatly accelerate biological research involving ubiquitylation and ubiquitin signaling.
Ubiquitin (Ub) signaling regulates pathways critical to human viability and defects or aberrations in Ub- signaling can result in numerous debilitating diseases including neurodegenerative diseases, bacterial and viral infections, and cancer. As a consequence, Ub signaling pathways are currently the focus of intense investigations as potential targets for intervention and treatment of disease. By focusing on the enzymes known as Ub-conjugating enzymes, or E2s, this study seeks to understand the networks of protein interactions that govern the assembly of the proteins required for Ub transfer and signaling. Despite their importance, very little information is available for most human E2s, the proteins they target, or the pathways in which they function. This project takes a unique perspective to understand at the molecular level how E2s function to transfer Ub and to identify human proteins targeted by select E2s. The new knowledge will greatly facilitate the ability of researchers to identify, test, manipulate, and target specific ubiquitylation events in a cell, provide insight into how bacterial pathogens can hijack Ub pathways, and will have a significant impact our understanding of human E2 structure and function to further accelerate biological research involving Ub signaling.
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