UC San Diego

Eukaryotic organisms have evolved to recycle proteins in process called Ubiquitylation. Attachment of ubiquitin molecules to a substrate protein act as a signal for the substrate protein to be degraded. The last step of ubiquitylation typically requires an enzyme called the E3 ubiquitin ligase. This work investigates the interactions between the CUL5-ASB9 E3 ligase and a novel substrate, histone octamers, through Hydrogen-Deuterium exchange and MALDI-TOF mass spectrometry.

Chapter II explains the in vitro expression and purification of the CUL5-ASB9 E3 ligase (AECR). There are many components to AECR which are not very soluble if purified separately. The purification strategy of co-expression in E. coli and co-purification of AECR showed to be successful. Additionally, we show AECR is active a capable of ubiquitylating histone octamers through in vitro activity assays and SDS-PAGE analysis.

Chapter III quantitatively confirms and characterizes the activity of AECR through liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further confirm through mutagenesis of AECR and in vitro assays that AECR’s histone ubiquitylation activity is not dependent on the modification called neddylation.

Chapter IV investigates the interactions between histones and ASB9, which is known to be the substrate receptor for AECR. Here we use Hydrogen-Deuterium exchange to identify changes in ASB9 upon histone binding. We observe that ASB9 increases in exchange when histones are present, but there is a small region of protection that may indicate the position where histones bind.