Ubiquitin proteomics
Proteomics

Over the last decade, ubiquitin has emerged to rival phosphorylation as one of the cell's most broadly utilized protein modifications. Different from phosphorylation, however, is the inherent protein nature of ubiquitin that allows it to be used in an array of different configurations, which appear to be used in distinct ways to regulate a myriad of activities. And though the full extent of ubiquitin's action in the cell is unknown, it's easy to imagine that every cellular process is controlled in some way by ubiquitination. This is underscored by the large number of genes that encode for known or predicted ubiquitination-associated proteins in eukaryotic genomes - more than 900 in humans alone. It's true that functions for a considerable number of these ubiquitination-associated proteins have been identified in a variety of organisms. But we've only scratched the surface as most remain functionally uncharacterized. In fact, even for those with known functions, only a handful of substrates have been identified for each even though it's likely that they target many more substrates.

Of the proteins that function in the ubiquitination enzymatic hierarchy, the ubiquitin-protein ligases impart the substrate specificity either by directly binding the substrate or by recruiting ancillary factors that bind substrates. But we know very little about which substrates are targeted by which ubiquitin-protein ligases. The holy grail of protein ubiquitination is to identify which proteins in the cell are substrates for a particular ubiquitin-protein ligase, thus characterizing the total regulatory action for an individual ligase. This is especially important when considering drug therapies that would target specific ubiquitin-protein ligases; it is essential to understand what other proteins and pathways would be impacted by such targeted manipulation. To this point, identifying target substrates for a particular ubiquitin-protein ligase has been done on an individual basis. That is, a particular protein under study found to be ubiquitinated is often employed in a screen or selection to discover the ubiquitin-protein ligase responsible. Unfortunately, the case by case method is often laborious and fails to give insight into the breadth of action for a particular ubiquitin-protein ligase.

To that end, we've devised a method to identify the total pool of ubiquitinated proteins in yeast. Now that we have a catalogue of substrates, we're eliminating particular ubiquitin-protein ligases to assess which proteins disappear from the ubiquitination profile. This should allow us to identify the particular ligase's set of substrates. In addition, the set of ubiquitinated proteins likely changes depending upon the stage of the cell cycle, or the environmental conditions of growth, so we're also using this method to define how the cell modulates ubiquitination of all the different proteins under various different growth and stress conditions. Of course, the combinations and variables are endless and, once established, the technique will have great employ for many different fields of research.

The basic technique is simple. To ubiquitin, we've attached a molecular hook that will bind to a bait held stationary on a matrix. Using a yeast strain obtained from Dan Finley that has all of the genomic ubiquitin genes deleted, we've replaced the plasmid-borne copy of ubiquitin with the tagged version. The strain now expressing the ubiquitin attached to the molecular hook is grown under whatever conditions are of interest, the cells are lysed, and the lysate is applied to the affinity column. In this way, ubiquitinated proteins are separated and purified from non-ubiquitinated proteins. The subsequent purified pool of ubiquitinated proteins is digested with trypsin and subject to mass spectroscopy to identify the proteins that comprise this pool. By coupling this method with various quantitation methods, we will be able to determine the substrates for a particular ubiquitin-protein ligase or ubiquitin hydrolase through comparison of the ubiquitinated protein profile from a wild-type strain with a strain deleted for a particular ubiquitin-protein ligase or ubiquitin hydrolase.

The other projects
Intro text NPQC text Chromatin text
Introduction NPQC Chromatin
There are a variety of projects going on in the lab from devising global ubiquitin proteomic strategies that will allow us to elucidate the functions of ubiquitin-protein ligases and ubiquitin proteases, to understanding the roles ubiquitination and deubiquitination play in gene transcription and silencing, to uncovering the ways in which the cell destroys aberrant proteins in the nucleus for the purposes of protein quality control. Move mouse over pictures to learn more...