Issue Date: September 23, 2013 | Web Date: September 20, 2013
Breaking The Ubiquitin Code
Small proteins called ubiquitins attach individually or in chains to other proteins to mark them for disposal, initiate cell signals, or facilitate protein interactions. Three researchers shared the 2004 Nobel Prize in Chemistry for discovering ubiquitination—the process of protein-ubiquitin conjugation—and its role in protein degradation.
Polyubiquitination, in which ubiquitin chains attach to proteins, is often required for ubiquitin-induced biological activity. But preparing homogeneous polyubiquitinated proteins to study the process systematically has been impossible. Despite decades of effort, polyubiquitination’s biological role at a molecular level largely remains a mystery.
A research team including one of the Nobel Laureates has now synthesized the first di- and tetraubiquitinated proteins in high purity and workable quantities (Proc. Natl. Acad. Sci. USA 2013, DOI: 10.1073/pnas.1315654110). The synthesis has enabled the researchers to evaluate the functional role of polyubiquitination on the protein α-synuclein, which has been linked to Parkinson’s disease.
A nearly endless variety of polyubiquitinated proteins is possible: At least eight types of polyubiquitin chains differing in internal connectivity exist, and they may have different lengths and attach to one of several amino acid residues on the protein. Therefore, a ubiquitination “code”—a system in which specific ubiquitin chains attach to different proteins to produce particular cell signals—may exist. But scientists don’t yet know how to read the code.
Free polyubiquitin chains (unattached to proteins) and monoubiquitinated proteins have been prepared before. Di- and tetraubiquitin chains have also been attached to proteins enzymatically. But the enzymatic process is hard to control and results in heterogeneous mixtures of polyubiquitinated proteins. Pure polyubiquitinated proteins can be isolated from such mixtures, but only in tiny amounts impractical for functional studies.
In the new work, Nobel Laureate Aaron Ciechanover of Technion—Israel Institute of Technology; Hilal A. Lashuel of Swiss Federal Polytechnic Institute of Technology, Lausanne; Ashraf Brik of Israel’s Ben-Gurion University of the Negev; and coworkers used a multistep approach to prepare homogeneous polyubiquitinated α-synucleins. The team used thioester-based linkage reactions to prepare two diubiquitins followed by chemical protein ligation to attach one or both to an α-synuclein modified with a thiolysine. The researchers then desulfurized the thiolysine to form the di- or tetraubiquitinated protein. They used the conjugates to find how ubiquitin chain length affects α-synuclein stability, aggregation, and phosphorylation.
“This is the first time that a target protein has been site-specifically modified with defined ubiquitin chains,” says Eric R. Strieter of the University of Wisconsin, Madison, a specialist in ubiquitination. “It’s a significant accomplishment—something people in the ubiquitin field have been trying to do for a very, very long time.” Nevertheless, the approach is extremely challenging to carry out, Strieter adds. “Additional synthetic advances are still needed in this area, for sure.”
“This is among the largest natural molecules ever synthesized in a lab,” adds protein chemist T. Ashton Cropp of Virginia Commonwealth University. “There is no end to the structure-function experiments that can now be performed to give a better picture of what happens when proteins are modified by polyubiquitins.”
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