Substrate Identification of the Adenovirus E3 Ubiquitin Ligase

Abstract

Species C human Adenovirus type 5 (Ad5) E1B-55 kilodalton (kDa) protein is a multifunctional protein that has been implicated in numerous important parts of early viral infections, including repression of interferon-stimulated genes (ISGs), sumoylation of host proteins, export of Late viral mRNA, and ubiquitination of host proteins. Interestingly, E1B-55kDa, along another Early viral transcript (E4orf6), interacts with several human proteins to form a viral E3 ubiquitin ligase. While five targets of this E3 ubiquitin ligase are known (i.e. p53, Bloom Helicase, DNA Ligase IV, Integrin-α3 and Mre11), there are almost certainly more targets to be found. Targets of the viral E3 ubiquitin ligase were isolated by tandem affinity purification of all ubiquitinated proteins in proteasome-inhibited cells. The resulting protein samples were then analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS). Comparison of the resulting interactome of ubiquitinated residues between a wildtype adenovirus and an E1-deleted strain revealed 324 proteins that are likely targets of the viral E3 ubiquitin ligase, which could be confirmed as targets by further analysis. This method demonstrates a rapid and broad method for identifying targets of a ubiquitin ligase, and provides crucial first steps towards identifying the mechanism of action and overall importance of ubiquitination driven by the Adenovirus E3 ubiquitin ligase.