Chemical Tools to Explore Ubiquitin Signaling Our research program is focused on the development of chemical tools to study the role of ubiquitin signaling. Specifically we have developed selective small molecule inhibitors of the ubiquitin activating E1 enzymes that lead to a complete inhibition of the ubiquitin system. These inhibitors contain sulfamate moiety that acts as a nucleophile reacting with the E1~Ub thioester to form ubiquitin adenylate mimic that binds tightly to the E1 enzyme and inhibits its function. During these studies we have uncovered the potential of N-acylsulfamates as useful pH-cleavable linkers for a variety of applications. We used those to design a novel class of pH-cleavable photocrosslinkers to study E2/E3 interactions, and discovered the second E2 enzyme binding site on E6-AP ligase. ! Our other research directions included the development of irreversible tethering technology, a useful technology to discover covalent enzyme and protein-protein interaction inhibitors. Using this technology we discovered first-in-class inhibitors of HECT E3 Nedd4-1 enzyme processivity. This types of inhibitors are novel and offer opportunities to decouple physiological functions of HECT E3 ligases related to their ability to mono- and polyubiquitinate protein substrates. To visualize the binding mode of these inhibitors, we obtained the first crystal structure of Nedd4-1 enzyme bound to its covalent small molecule inhibitor, and subsequently developed more potent inhibitors of Nedd4-1 enzyme. ! Finally, we will discuss the development of novel fluorescent assays to screen for inhibitors or activators of HECT E3 ubiquitin ligases. Typical assays for E3 ligases are very complex and require ATP, Ubiquitin, E1, E2, and E3s which increases the cost of the assay, and leads to false positive results due to the inhibition of E1 or E2 enzymes. To simplify the assay, we have developed a novel reaction, in which protein ubiquitination and polyubiquitin chain synthesis are achieved in the absence of ATP, E1 and E2 enzymes, and in the presence of the chemically activated ubiquitin. Finally we will discuss future directions. Host:  Liming Zhang