E1 enzymes catalyze a multi-step reaction, in which ubiquitin or a ubiquitin-like (Ubl) protein is initially activated by C-terminal adenylation, then thioester-linked to the catalytic cysteine residue of the E1 enzyme, and finally transferred to the catalytic cysteine of an E2 enzyme. Two ubiquitin-specific E1 enzymes, Uba1 and Uba6, have been identified in mammals. Interestingly, Uba6 and its cognate E2, Use1, can recognize both ubiquitin and the Ubl FAT10 (human leukocyte antigen F-associated transcript 10). Target protein modification with FAT10 (“FATylation”) is cytokine-inducible and serves as a signal for proteasomal degradation. FAT10 has been implicated in antigen presentation, mitotic regulation, and xenophagy, and its expression is upregulated in various types of cancer. Project A1 will elucidate the structural framework of ubiquitin/FAT10 activation by Uba6 and will compare the selectivities of Uba1 and Uba6 for particular E2 enzymes.