Studies of Ubiquitin Activation and Chain Synthesis

Abstract

The Ubiquitin Proteasome System (UPS) is among the most extensive cellular pathways, with over 1200 components, and it has profound implications for cellular homeostasis and disease. In this dissertation, we will discuss in vitro studies of its mechanism. We will begin in Chapter Two by characterizing a computationally derived small molecule library to target the ubiquitin fold domain of UBE1 for enzymatic inhibition. This work will describe a screen conducted to test a library generated in silico and discuss alternative methods for targeting E1. Next, we will transition to investigating free ubiquitin chain formation by the E3 ligase Hul5 in Chapter Three. We will describe rapid di-ubiquitin formation by Hul5 and the suppression of this reaction by calcium, which is mediated by calcium binding to calmodulin. Characterization of di-ubiquitin identified a preference for K48-linkages and formation by an acceptor site on Hul5. Lastly, we will conclude by discussing the role of Hul5 as the only identified processivity factor of the proteasome. In Chapter Four, we will establish a framework and method for the in vitro characterization of Hul5 as a processivity factor to uncover its mechanism of action. Together, this body of work elucidates therapeutic and mechanistic features of the UPS to contribute to our biological understanding of the pathway.