Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

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 essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples

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

In recent years the ubiquitin proteasome system (UPS) has garnered increasing interest as a target for chemotherapeutics. Due to the success of the proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma, several new compounds have been developed to target E3 ubiquitin ligases and the proteasome in numerous human cancers. This has increased the need for new analytical methods to precisely measure intracellular enzyme activity in cells. A key component of a desired analytical method is a substrate that is capable of rapid intracellular ubiquitination yet easily incorporated into the next generation of more sophisticated UPS reporters. Portable degradation sequences, or degrons, have the ability to bind to E3 ligases and promote substrate ubiquitination when the sequence is presented in isolation or appended to other entities such as fluorescent peptide-based reporters. Previous work identified an E3 ligase (MDM2)-binding element at p53 amino acids 92-112, which was later demonstrated to be rapidly ubiquitinated in cytosolic lysates effectively functioning as a transportable degron. In this work, a shortened p53 sequence within amino acids 92-112 that displayed rapid ubiquitination kinetics was identified. A nine-member peptide library was synthesized using sequence elements of various sizes and lengths, all based on the initial 22 amino acid long sequence, containing a single ubiquitination site lysine. The ubiquitination kinetics were determined using a combination of gel electrophoresis and analytical high performance liquid chromatography (HPLC) to rank the members of the library and identify the optimal ubiquitination sequence. This analysis identified the five amino acid sequence, KGSYG, corresponding to residues 105-108 with an added N-terminal lysine, as a portable degron since this sequence demonstrated the most rapid ubiquitination kinetics

Quantification of substrate ubiquitination.

<p>Eight members of the substrate library were subjected to a ubiquitin pull down assay to confirm substrate ubiquitination. Ubiquitin-conjugated substrates were purified using ubiquitin-binding beads and then visualized using the fluorescein tag. Three substrates are depicted: Bonger-based (A), p53-based (B), and iNOS-based (C) substrates. Relative protein sizes are compared to values obtained from a fluorescent protein marker (numerical text on the left of A–C). Suspected mono-, di-, tri-,and tetra-ubiquitin conjugated substrates are labeled accordingly. The fluorescence intensity of the bands was measured for Bonger (D), p53 (E), and iNOS (F) -based substrates. The data points represent the average (n = 3) and the error bars the standard deviation of the data points.</p

Kinetic analysis of substrate ubiquitination implicates multi-monoubiquitination of degron-based substrates.

<p>(A) Schematic representing proposed model for multi-monoubiquitination of the degron-based substrates. Rate constants correspond to Equations S1–S6 representing substrate ubiquitination and peptidase-dependent degradation. Model output (lines) for the three degron-based substrates: Bonger (B), p53 (C), and iNOS (D) demonstrates a good fit between model prediction and observed data. Model fit for mono-, di-, tri-, and tetra-ubiquitinated substrates are labeled accordingly. Model output is depicted as relative ubiquitination (C_i/C_o) to permit comparison across substrates (Eq. S7). Multiple ubiquitins conjugated to a substrate are denoted by according to the legend in the upper left corner.</p

First order rate constants determined by kinetic analysis of degron-based substrates.

<p>Rate constants for ubiquitination (k₁–k₄) and peptidase-dependent degradation (k₅–k₉) determined by Markov Chain Monte Carlo algorithm solving the series of kinetic differential equations modeling substrate ubiquitination (Equations S1–S6). Sum of squared difference calculated for each substrate according to Equation S8–S9. Values contained in table are multiplied by 10³.</p

Preliminary validation of degron-based substrate ubiquitination.

<p>(A) Schematic of the degron-based substrate consisting of four essential components: the E3-ligase recognition site (degron), a proximal lysine to be ubiquitinated, a short spacing region so that the degron-bound E3 ligase can access the lysine, and a fluorescein tag for visualization. Three substrates, Bonger-based (B), p53-based (C), and IFNAR1-based (D) were incubated with either ubiquitin (Ub) or methylated ubiquitin (MeUb) in an <i>in vitro</i> ubiquitination reaction mixture for varying times. Unmodified substrate was loaded into the first lane of each gel to compare unreacted vs. degraded substrate. Arrow indicates unmodified substrate.</p

Development of β‑Hairpin Peptides for the Measurement of SCF-Family E3 Ligase Activity in Vitro via Ornithine Ubiquitination

Regulation of the ubiquitin–proteasome system (UPS) to treat select types of cancer has become a popular area of drug discovery research. The FDA approval of proteasome inhibitors Bortezomib and Carfilzomib in the treatment of multiple myeloma has led to an increased need for chemical reporters capable of detecting and quantifying protein ubiquitination and the activity of members of the UPS including E3 ubiquitin ligases and the proteasome in the tumor cells of the patients. One limitation of peptide-based reporters is their rapid degradation in the cellular environment by cytosolic peptidases. Conversely, β-hairpin “protectides” exhibit a pronounced secondary structure that significantly increases their lifetime under cellular conditions. The goal of this work was to develop a family of novel, ornithine-rich protectides that could act as primary degrons serving as substrates for in vitro ubiquitination. The fluorescent peptide-based reporters were demonstrated to be highly resistant to degradation in multiple myeloma cell lysates. The most stable β-hairpin primary degron, containing a single ornithine residue at the N-terminus, OWRWR [Ac-OWVRVpGO­(FAM)­WIRQ-NH₂], demonstrated rapid ubiquitination kinetics and a 20-fold increase in stability when compared with an unstructured primary degron. A screen of E1 and E3 enzyme inhibitors in cell lysates showed that ubiquitination of OWRWR was significantly impaired by inhibitors of the SCF family of E3 ligases. Furthermore, this is the first report demonstrating the use of an ornithine residue on a primary degron as a ubiquitination site. This study serves as a strong foundation for the development of stable, fluorescent, peptide-based reporters capable of quantifying protein ubiquitination and the enzymatic activity of members of the UPS

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

<div><p>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.</p></div

Variations in E3 ligase activity result in fluxuations in degron-based substrate ubiquitination.

<p>(A) To evaluate the impact of different levels of E3 ligase activity, the four top performing degron-based substrates (β-Catenin, Bonger, iNOS, and p53) were incubated in lysates from four different cell lines (HeLa, U937, THP-1, and HL60) in the ubiquitin pull down assay. Further, the assay was supplemented with wild type ubiquitin to examine polyubiquitin chain formation. All reaction mixtures were incubated for 2 hours at 37°C prior to continuation with the pull down assay. (B) The effects of a NEDD8 inhibitor (MLN-4924) and an E1 inhibitor (PYR-41) were examined on the TAZ-based substrate (a target of the SCF^βTrCP E3 ligase). The ubiquitin pull down was performed as previously described with the reactions incubated for 2 hours with the listed inhibitor concentrations at 37°C prior to continuation with the pull down assay. (C) A cell-free ubiquitination assay was performed on the p53-based substrate using an enzymatic cascade culminating with 1 µM Hdm2 and supplemented with wild type ubiquitin. Lane 1 contains all assay components, lane 2–4 were control experiments removing a member of the E1–E3 enzymatic cascade. The assay was then supplemented with two commerical Mdm2 inhibitors: HLI-373 (1,10 µM, lane 5–6 respectively) and NSC-66811 (1,10 µM, lane 7–8 respectively). All reactions were incubated for 2 hours at 30°C prior to halting the reaction with sample buffer. Arrow indicates unmodified substrate.</p