Role of ubiquitin and the HPV E6 oncoprotein in E6AP-mediated ubiquitination.

Deregulation of the ubiquitin ligase E6 associated protein (E6AP) encoded by the UBE3A gene has been associated with three different clinical pictures. Hijacking of E6AP by the E6 oncoprotein of distinct human papillomaviruses (HPV) contributes to the development of cervical cancer, whereas loss of E6AP expression or function is the cause of Angelman syndrome, a neurodevelopmental disorder, and increased expression of E6AP has been involved in autism spectrum disorders. Although these observations indicate that the activity of E6AP has to be tightly controlled, only little is known about how E6AP is regulated at the posttranslational level. Here, we provide evidence that the hydrophobic patch of ubiquitin comprising Leu-8 and Ile-44 is important for E6AP-mediated ubiquitination, whereas it does not affect the catalytic properties of the isolated catalytic HECT domain of E6AP. Furthermore, we show that the HPV E6 oncoprotein rescues the disability of full-length E6AP to use a respective hydrophobic patch mutant of ubiquitin for ubiquitination and that it stimulates E6AP-mediated ubiquitination of Ring1B, a known substrate of E6AP, in vitro and in cells. Based on these data, we propose that E6AP exists in at least two different states, an active and a less active or latent one, and that the activity of E6AP is controlled by noncovalent interactions with ubiquitin and allosteric activators such as the HPV E6 oncoprotein.

Anionic surfactants enhance click reaction-mediated protein conjugation with ubiquitin.

The Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has become increasingly important in the conjugation chemistry of biomolecules. For example, it is an efficient and convenient method to generate defined ubiquitin-protein conjugates. Here, we investigate the effect of surfactants on the efficiency of CuAAC for chemical protein ubiquitylation. We found that anionic surfactants enhance conjugate formation by up to 10-fold resulting in high yields even at low (i.e., micromolar) concentrations of the reactants. Notably, the herein investigated conjugates are functional and thus properly folded.

Selective monitoring of the enzymatic activity of the tumor suppressor Fhit.

Cancer is a leading cause of death worldwide. Functional inactivation of tumor suppressor proteins, mainly by mutations in the corresponding genes, is a key event in cancer development. The fragile histidine triade protein (Fhit) is a tumor suppressor that is frequently affected in different cancer types. Fhit possesses diadenosine triphosphate hydrolase activity, but although reduction of its enzymatic activity appears to be important for exerting its tumor suppressor function, the regulation of Fhit activity is poorly understood. Here, we introduce a novel fluorogenic probe that is suited to selectively analyze the enzymatic activity of Fhit in extracts derived from human cells. This novel method will allow in-depth insight into the mechanisms involved in Fhit regulation in biologically relevant setups and, thus, into its role in the development of cancer.

Dissecting ubiquitin signaling with linkage-defined and protease resistant ubiquitin chains.

Ubiquitylation is a complex posttranslational protein modification and deregulation of this pathway has been associated with different human disorders. Ubiquitylation comes in different flavors: Besides mono-ubiquitylation, ubiquitin chains of various topologies are formed on substrate proteins. The fate of ubiquitylated proteins is determined by the linkage-type of the attached ubiquitin chains, however, the underlying mechanism is poorly characterized. Herein, we describe a new method based on codon expansion and click-chemistry-based polymerization to generate linkage-defined ubiquitin chains that are resistant to ubiquitin-specific proteases and adopt native-like functions. The potential of these artificial chains for analyzing ubiquitin signaling is demonstrated by linkage-specific effects on cell-cycle progression.