The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.

The E3 ligase substrate receptor ankyrin and suppressor of cytokine signaling box protein 9 (ASB9) was shown to bind over 10 different proteins including metabolic enzymes such as creatine kinase, filament proteins such as vimentin, and histones. In previous work, we characterized the ASB9-Cullin 5 E3 ligase (ASB9-CRL5) ubiquitylation of creatine kinase and showed that ubiquitylation required the ring-between-ring ligase, ARIH2. Here, we characterized the ASB9-CRL5 ubiquitylation of histones and show that histones histone 3 (H3) and histone 4 (H4) are polyubiquitylated by the ASB9-CRL5 whereas histones Histone 2A and Histone 2B are much poorer substrates. Many, but not all lysines in the histones are ubiquitylated suggesting some substrate specificity. Binding experiments show that the ligase-histone interaction is highly electrostatic and the neddylated ASB9-CRL5 binds with the highest affinity. Histones in nucleosomes or in complex with the chaperone Asf1, are not ubiquitylated. Only K48 and K63 polyubiquitin chains were observed, suggesting that the ubiquitylation probably drives histone degradation. The presence of ASB9 in specific cell types correlates with situations in which free histones H3 and H4 need to be degraded. In this work, we demonstrate that the ASB9-CRL5 is the ligase that facilitates degradation of histones H3 and H4. In addition, this work represents the first example of Cullin-5 mediated ubiquitylation that does not require a ring-between-ring "helper" ligase.