UC San Diego

Ubiquitin (Ub) is an 8,500 Da, 76 amino acid protein that is among a family of similar Ubiquitin-like proteins. E3 Ligases facilitate the attachment of Ub onto lysine residues of substrate proteins. Ub can also be attached to another Ub molecule, forming polyubiquitin chains which signal for proteasomal degradation and the immune response. Small ubiquitin-like modifier (SUMO) is involved in cell cycle control and the DNA damage response. Another Ubiquitin-like protein, Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8), can modify some E3 Ligases to regulate their activation.

Chapter II presents a novel method for the side-by-side identification and quantification of Ubiquitin and SUMO modifications. Trypsin digestion of a Ubiquitin-modified protein leaves a diglycyl-lysine at the site of modification, which can be detected by mass spectrometry. Unlike Ubiquitin, detection of SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. This chapter describes detection of endogenous SUMOylation using α-lytic protease, WaLP.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) probes the solvent accessibility and conformational dynamics of proteins by measuring deuterium uptake on backbone amides over time. Chapter III describes an open-source software package designed to automate the correction, analysis and visualization of HDX-MS data.

Chapter IV investigates the structure, dynamics and regulation of an ASB9-CUL-RING E3 Ubiquitin Ligase. The structure of the substrate-receptor complex was determined for the first time by Cryo-EM, identifying changes in both the receptor and the substrate that were not predicted by previous models.

Chapter V further investigates the dynamics, regulation and activity of the ASB9-CUL-RING E3 Ubiquitin Ligase. In vitro assays, western blots and mass spectrometry data describe the locations and intensity of ubiquitin modifications. These data show that ARIH2, another E3 ligase, is necessary for initial substrate modification, and that the addition of other E2 enzymes can modulate the poly-ubiquitin chains formed. HDX-MS studies show that modification of the ASB9-CUL-RING ligase causes conformational restructuring that facilitates the binding and activation of ARIH2.