Proteomic Profiling of Potential E6AP Substrates via Ubiquitin-based Photo-Crosslinking Assisted Affinity Enrichment.

The ubiquitin (Ub) ligase E6AP, encoded by the UBE3A gene, has been causally associated with human diseases including cervical cancer and Angelman syndrome, a neurodevelopmental disorder. Yet, our knowledge about disease-relevant substrates of E6AP is still limited, presumably because at least some of these interactions are rather transient, a phenomenon observed for many enzyme-substrate interactions. Here, we introduce a novel approach to trap such potential transient interactions by combining a stable E6AP-Ub conjugate mimicking the active state of this enzyme with photo-crosslinking (PCL) followed by affinity enrichment coupled to mass spectrometry (AE-MS).

Biochemical and Structural Consequences of NEDD8 Acetylation.

Similar to ubiquitin, the ubiquitin-like protein NEDD8 is not only conjugated to other proteins but is itself subject to posttranslational modifications including lysine acetylation. Yet, compared to ubiquitin, only little is known about the biochemical and structural consequences of site-specific NEDD8 acetylation. Here, we generated site-specifically mono-acetylated NEDD8 variants for each known acetylation site by genetic code expansion.

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.

Interactome of intact chromatosome variants with site-specifically ubiquitylated and acetylated linker histone H1.2.

Post-translational modifications (PTMs) of histones have fundamental effects on chromatin structure and function. While the impact of PTMs on the function of core histones are increasingly well understood, this is much less the case for modifications of linker histone H1, which is at least in part due to a lack of proper tools. In this work, we establish the assembly of intact chromatosomes containing site-specifically ubiquitylated and acetylated linker histone H1.