Abstract
The ubiquitin–proteasome system is an essential cellular process that plays a fundamental role in the regulation of protein stability. This pathway is tightly controlled by a sequential cascade of enzymatic steps that culminates in the formation of a poly-ubiquitin chain onto the substrate protein targeted for 26S proteasome degradation. Through a process of co-evolution viruses have evolved mechanisms to utilize or suppress this pathway in order to enhance their replication and spread. One of the first proteins to be expressed during herpes simplex virus 1 (HSV-1) infection is ICP0, a viral RING-finger E3 ubiquitin ligase that targets a variety of cellular proteins for ubiquitination and proteasome-dependent degradation. This activity is required in order for ICP0 to efficiently stimulate the onset of HSV-1 lytic infection and viral reactivation from latency. While it is clear that the RING-finger domain of ICP0 plays an important role in the biology of HSV-1, methods for accurately quantifying its biochemical activity are currently lacking. Here we describe a protocol that enables the quantitative measurement of the ubiquitin ligase activity of ICP0 using near-infrared (IR) western blot imaging. The use of such imaging technology provides an accurate means to examine the biochemical and kinetic parameters of RING-finger ubiquitin ligases in solution, and may provide significant application for inhibitor studies.
Highlights
The ubiquitin pathway is an essential cellular process in all eukaryotes
In order to examine the application of such imaging technology to study the biochemical properties of ICP0 in solution, we performed a series of in vitro assays monitoring the ability of ICP0 to catalyze the formation of unanchored poly-ubiquitin chains and monoubiquitinated products in the presence of recombinant human UBA1 (E1) and UBE2D1 (E2) enzymes under established assay conditions [9]
Using standard chemiluminescence methods we have previously shown that ethylenediaminetetraacetic acid (EDTA) is able to competitively inhibit the RING-finger ubiquitin ligase activity of ICP0 in a dose-dependent manner in vitro [43]
Summary
The ubiquitin pathway is an essential cellular process in all eukaryotes. The attachment of ubiquitin typically occurs through the formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and the amino side chain of a solvent exposed lysine residue within the substrate protein. This initial monoubiquitination event can be subsequently extended through the sequential addition of ubiquitin molecules onto lysine residues
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