Cullin-5 RING Ligases ubiquitylate histones.

Abstract

The ubiquitin proteasome system is the primary means by which proteins are degraded. In this system, ubiquitin is activated by thioester linkage to an E1, transferred to an E2, and finally transferred to a substrate protein by E3. Consecutive rounds of ubiquitin transfer leads to formation of a polyubiquitin chain sufficient for proteasomal recognition and subsequent substrate degradation. We have been studying the Cullin-5 RING Ligases (CRLs), one of the largest groups of E3 ligases. These ligases are composed of several proteins; Cullin-5 scaffold protein, an interchangeable substrate receptor protein, two adaptor proteins, and a ring box protein that recruits an E2. We recently showed that the Cullin-5, when neddylated at K724, can recruit an additional E3 ligase called ARIH2 that with its E2 ubiquitylates substrates bound to the substrate receptor protein, ASB9. Herein, we have experimentally verified a new Cullin-5 substrate, histones, which were previously identified as putative substrates of ASB9-EloB/C-Cullin5-Rbx2 (AECR) through proteomics experiments. I have demonstrated that AECR polyubiquitylates histones and I have confirmed the preferred oligomeric state of the histones through in vitro ubiquitinassays. I have also measured the binding affinity of the histones to various components of the AECR. Finally, I have identified histone binding sites on AECR using hydrogen-deuterium exchange mass spectrometry.