The regulatory process known as protein ubiquitination modifies cellular proteins with far-reaching impacts on human health and disease. It is involved in every known biological process and is implicated in a growing range of diseases that includes cancers, neurodegenerative diseases, muscle wasting, etc. Studies conducted worldwide over the past twenty years have defined the mechanisms by which protein ubiquitination is carried out, with one obvious exception. Almost little is known about how substrates (i.e., the cellular proteins that are modified by this system) are recognized and how they fit into the ultimate mechanism. In the coming grant period, we propose to rectify this situation for two important ubiquitin E3 ligases, BRCA1/BARD1 and HHARI. Our goals are made possible by several important advances, including definition of bona fide substrates for each of these E3s and the ability to chemically synthesize biochemically homogeneous version of the products. We will also investigate two uncharacterized human E2 Ubiquitin-conjugating enzymes that each have unusual/unique activities in that they attach Ub to protein groups other than lysines, thereby expanding the universe of possible attachment sites. We will use a combination of biochemical, structural, and cellular approaches to address questions of fundamental importance and general relevance.