Biochemical analysis of the E6AP ubiquitin ligase and its role in Angelman syndrome

E6AP was identified as the cellular factor which cooperates with the human papillomavirus E6 oncoprotein to stimulate the ubiquitin-mediated degradation of the tumor suppressor p53. In this process, the E6/E6AP complex comprises the E3 ubiquitin ligase activity in the ubiquitin thiolester cascade. E6AP was actually the first known member of a family of ubiquitin ligases called hect proteins, all of which contain a 350--400 amino acid domain h&barbelow;omologous to the E&barbelow;6AP c&barbelow;arboxy t&barbelow;erminus. These are modular proteins in which the conserved hect domains catalyze ubiquitin transfer, whereas other, divergent domains confer substrate specificity.; Recently, UBE3A, the gene encoding E6AP, was identified as the gene affected in a rare neurological disorder called Angelman syndrome. Affected individuals have symptoms including severe mental retardation, absent speech, ataxia, seizures, and hyperactivity. Most patients have chromosomal deletions that remove the entire maternal allele of UBE3A. However, a small subset of patients have E6AP point mutations that result in single amino acid changes or short, in-frame deletions that still allow translation of a full-length protein. By studying these E6AP point mutations, we have found a strong correlation between Angelman-associated mutations and a loss of E3 ubiquitin ligase activity. Interestingly, the point mutations affect E6AP activity in different ways. Some mutant proteins cannot form thiolester intermediates with ubiquitin, others retain the thiolester formation activity but cannot efficiently transfer ubiquitin to a substrate, and still others are unstable in cells. Our results suggest that the loss of E6AP catalytic activity and, likely, the improper regulation of E6AP substrate(s), are important in the development of Angelman syndrome.; Furthermore, our studies suggest that E6AP may undergo a novel biochemical step before accomplishing substrate ubiquitylation and is subject to an, as yet, unidentified modification. We discuss hypotheses to explain these two novel findings and their implications for affecting E6AP activity.