A Comparative Analysis of the Ubiquitination Kinetics of Multiple Degrons to Identify an Ideal Targeting Sequence for a Proteasome Reporter

Abstract

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.

TAZ Protein Accumulation Is Negatively Regulated by YAP Abundance in Mammalian Cells

“…2B). These results indicate that upon YAP induction, TAZ is proteasomally degraded within the cytoplasm, which is consistent with the established mechanism of TAZ degradation (32,33,42).…”

Identification of a p53-based portable degron based on the MDM2-p53 binding region

“…Moreover, the fact that peptides 1 and 2 exhibited decreased kinetics even with the GSYG binding sequence would suggest that substrate length and the location of the ubiquitination site lysine both play a role in substrate ubiquitination. The fact that this sequence element was found in the peptide substrate previously characterized by Melvin et al 13 highlights the importance of this specific sequence in the recognition by the ubiquitination machinery. Taken together, the kinetic analysis performed here demonstrates that peptide 7 behaves as a shortened portable degron that is ubiquitinated in an MDM2-dependent manner.…”

“…13 The peptide library presented in this study was based on the sequence but with the three C-terminal lysines replaced with arginines to prevent ubiquitination on these residues and to eliminate multi-monoubiquitination of the peptides. 13 The side chain of the lysine at the −4 position (from the C-terminus) of each peptide was used as a site for fluorescein conjugation leaving the N-terminal lysine as the only possible ubiquitination site. The N-terminal lysine was not a part of the sequence element identified by Gu et al , but was incorporated by Melvin et al and identified to be the preferential site of ubiquitination.…”

“…The N-terminal lysine was not a part of the sequence element identified by Gu et al , but was incorporated by Melvin et al and identified to be the preferential site of ubiquitination. 13 Nine peptides were designed based on these key sequence elements (Figure 1A). The shortened sequences were designed around the internal lysine residue (+11 in peptide 1 ) as this was the only lysine residue in the p53 sequence element.…”

“…11, 12 While several degrons have been identified by researchers, the ability of each sequence to serve as a portable degron varies greatly depending on the ubiquitination kinetics, the number of amino acids (e.g., size and length), and need for further post-translational modification (e.g., phosphorylation). 13 A broader suite of portable degrons would significantly enhance efforts to generate sensors for both the ligases and proteasome.…”