Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

A ubiquitin variant-based affinity approach selectively identifies substrates of the ubiquitin ligase E6AP in complex with HPV-11 E6 or HPV-16 E6.

The E6 protein of both mucosal high-risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low-risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6-associated protein (E6AP). This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, whereas several substrate proteins of the high-risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low-risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole-cell extracts and high-resolution MS. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination but not by E6AP alone. By this approach, we identified ∼190 potential substrate proteins for low-risk HPV-11 E6 and high-risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP.