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

Abstract

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.

CPProtectides: Rapid uptake of well‐folded β‐hairpin peptides with enhanced resistance to intracellular degradation

“…It was hypothesized that the number of positively charged residues on the peptides identified by Cline and co‐workers and Houston and co‐workers could facilitate their behavior as potential CPPs. A library of β‐hairpin peptides was synthesized using the sequences previously published by Houston and co‐workers with different amino acid substitutions between ornithine and arginine at positions 1, 4, and 11 (Figure A; Supporting Information Figure S1).…”

“…Moreover, RWRWR exhibits a maximum near 225 nm that arises from exciton coupling of the indole rings of the cross‐strand Trp residues, further supporting the correct‐fold . These spectra are consistent with the CD spectrum of related peptides WKWK, WRWR, OWRWR, OWOWO, and S‐OWOWO that have been previously characterized by both NMR and CD and shown to adopt a well‐folded β‐hairpin.…”

“…A library of β‐hairpin peptides was synthesized using the sequences previously published by Houston and co‐workers with different amino acid substitutions between ornithine and arginine at positions 1, 4, and 11 (Figure A; Supporting Information Figure S1). Ornithine was selected because it is similar in structure to lysine, yet was demonstrated to exhibit greater stability . Five iterations of the β‐hairpin sequences (RWRWR, RWOWR, OWRWR, OWOWO, and WKWK) were found to enter intact HeLa cells (Figure B).…”

“…Five iterations of the β‐hairpin sequences (RWRWR, RWOWR, OWRWR, OWOWO, and WKWK) were found to enter intact HeLa cells (Figure B). Uptake of the β‐hairpin peptides was compared to an unstructured, negative control peptide (III‐67B) that was previously demonstrated to be rapidly degraded under cytosolic conditions and was initially found here to be impermeable in intact cells and thus not a CPP. Statistical t ‐tests were performed against this peptide to illustrate the significance of intracellular fluorescent signals for the β‐hairpin peptides (Figure B).…”

“…Coupling arginine‐ and proline‐rich sequences for the generation of structured CPPs with enhanced intracellular stability has also been explained by Daniels and Schepartz . Recent work by Houston and co‐workers found that replacing the lysine residues with ornithine or arginine further increased intracellular stability and also converted the β‐hairpin sequence into a primary degron capable of intracellular ubiquitination . The goal of this work is to determine if these β‐hairpin protectides, which are rich in positively charged residues, could also function as CPPs.…”