Date Published: October 29, 2013
Publisher: Public Library of Science
Author(s): Adam T. Melvin, Gregery S. Woss, Jessica H. Park, Lukas D. Dumberger, Marcey L. Waters, Nancy L. Allbritton, James P. Brody.
The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.
The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing upstream roles in the signaling pathways governing DNA repair, cell cycle regulation, cell migration, and the immune response . Posttranslational protein modification by ubiquitin requires a cascade of three increasingly diverse enzymes: an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Protein ubiquitination starts with an E1 enzyme forming a high energy thioester bond with free ubiquitin, which is recognized and transferred to an E2 enzyme. Next, an E3 ubiquitin ligase forms a complex with the E2 enzyme to mediate the transfer of ubiquitin to the target protein. The E3 recognizes its target protein by binding to a specific amino acid degradation sequence, or degron. These degrons, normally in close proximity to a ubiquitin-accepting lysine residue, impart specificity to protein degradation since each E3 binds to a subset of degrons. The large number of E3 ligases (>600 in humans) permits recognition of a wide variety of degrons including phospho-degrons, oxygen dependent degrons, and N-terminal degrons . The manner in which ubiquitin is transferred to a protein can occur either directly from E2 to the target protein, as is the case with RING family (Really Interesting New Gene) E3 ligases, or through an E3 ligase-bound intermediate, as is the case with HECT family (Homologous to the E6AP Carboxyl Terminus) E3 ligases . Following initial ubiquitin-protein conjugation, additional ubiquitin subunits are added via one of seven different lysine residues found on ubiquitin (e.g. K48, K63, or K11) to form a polyubiquitin chain or through the N-terminal methionine residue to form a linear ubiquitin chain . The residue to which the polyubiquitin chain is linked determines the fate of the conjugated protein where K48-linked chains are targeted for proteasomal degradation and K63-linked chains play a role in regulating cell signaling and DNA damage repair . A polyubiquitinated protein targeted for degradation is recognized by the 19S cap of the 26S proteasome, where the target protein is deubiquitinated, unfolded, and degraded by the 20 s core particle . Further, a single ubiquitin can be conjugated to the target protein, termed mono-ubiquitination, or multiple individual ubiquitins can be conjugated to multiple lysine residues near the degron, termed multi mono-ubiquitination. These differences in the number and location of protein ubiquitination significantly impact the fate of the target protein. Control of polyubiquitin chain formation is further mediated by another class of protein, deubiquitinating enzymes (DUBs), which are capable of cleaving the isopeptide bond between ubiquitin and the target protein.
Novel substrates capable of intracellular ubiquitination are a highly successful class of reporters capable of measuring cellular proteasome activity. With the growing trend of therapeutics targeted against the proteasome, it is imperative to develop the next generation of proteasome reporters that are both rapidly targeted to the proteasome and compatible with single cells. While there exist a few successful proteasome reporters, many of these established methods are not compatible with single cells and require large populations of cells and complex genetic manipulation . In recent years it has become apparent that analysis of single cells, especially those obtained directly from a patient, provide a great deal more information than analysis of bulk cell lysates due to the heterogeneous nature of tumor biopsies . To address the need for single cell-compatible proteasome reporters, this study focused on the characterization and analysis of multiple degrons to identify those with rapid ubiquitination kinetics for the eventual incorporation into next generation proteasome reporters. Ultimately, three candidates were selected that show a preference for ubiquitination over peptidase-dependent degradation and further study identified the preferred location of the ubiquitination site lysine.