Design and Synthesis of an E3 ligase Activity-Based Probe and its Application for the Discovery of a New Class of E3 Ligase

The ubiquitylation cascade regulates multiple cellular functions and is involved in numerous diseases. The distinct transfer cascade, involving E1-E2-E3 enzymes, serves as a promising target for drug development. However, E3 ligases (E3s) represent an important class of enzymes yet there are currently no effective tools for profiling their activity. Herein, a new class of E3 activity-based probe (ABP) is presented which is built by re-engineering ubiquitin (Ub)-charged E2 conjugating enzymes. The utility of these probes has been demonstrated by the rapid dissection of the activation determinants of the RING-Between-RING E3 (RBR) E3, Parkin. Furthermore, biotin-E3 ABPs allow us to systematically discover and dissect the E3 activities of a broad spectrum of E3s that are associated with different diseases. By interfacing the ABPs with mass spectrometry, we establish an activity based protein profiling (ABPP) system and apply it to uncover a new class of E3. We show that MYCBP2 is an E3 ligase with a novel mechanism of action that ubiquitylates threonine residues. MYCBP2 contains a RING domain, that recruits the ubiquitin-loaded E2, and a novel Zn-binding fold that contains two catalytic cysteine residues which relay the Ub to substrate via two thioester intermediates (RING-Cys-Relay, RCR). This discovery demonstrates the power and potential of our E3 activity based protein profiling (ABPP) system.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2018

Author dc:creator

• Pao, Kuan-Chuan

Advisor dc:contributor.advisor

• Virdee, Satpal

• E3
• Activity-based probe
• MYCBP2
• Ring-Cys-Relay

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/6239e172-60b3-47c3-81e1-f4b0a577f1a4

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/6239e172-60b3-47c3-81e1-f4b0a577f1a4