Structural and Functional Characterization of Non-Canonical Ubiquitin Conjugating Enzymes
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Thirty-five years ago, during its initial discovery, the field of ubiquitination could not have been more aptly named. In fact, as the study of ubiquitin (Ub) expands, it is becoming clear that this small post-translational modification is far more ubiquitous than the founders of this field could have originally imagined. Initially identified as a protein degradation signal, Ub is now known to regulate vastly disparate cellular functions, including the DNA-damage response and immune signaling. This small (8.5kDa) modification achieves its diverse signaling capabilities partly through the enzymes that catalyze its attachment to cellular substrates. These enzymes (E1, E2, and E3) work in succession to identify and modify substrates with varying Ub signals. The manner in which Ub is attached (monoubiquition or 8 types of polyubiquitin chains) and the site of attachment dictate a substrate's cellular fate. Furthermore, much of the diversity in Ub signaling can be attributed to the structural characteristics of the enzymes in the ubiquitin cascade. This thesis examines the structural and biochemical features of two E2 ubiquitin-conjugating enzymes, Ube2w and Ube2h. First, we show that Ube2w specifically attaches monoubiquitin to the N-terimni of disordered protein substrates. To attach this unique Ub modification, Ube2w has evolved a non-canonical domain architecture, solved by our group using nuclear magnetic resonance (NMR) techniques. We also show, that another E2, Ube2h, contains a disordered C-terminus and that it can ubiquitinate a substrate, histone H2A/H2B, in vitro, in the absence of an E3. Studies presented here build the foundations to understand the cellular impact of Ube2w and Ube2h.
- Biological chemistry