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 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.

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.

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.