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A High Affinity hRpn2-Derived Peptide That Displaces Human Rpn13 from Proteasome in 293T Cells
Xiuxiu Lu1, Fen Liu1, Sarah E Durham2
1Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America.
Abstract:
Rpn13 is a proteasome ubiquitin receptor that has emerged as a therapeutic target for human cancers. Its ubiquitin-binding activity is confined to an N-terminal Pru (pleckstrin-like receptor for ubiquitin) domain that also docks it into the proteasome, while its C-terminal DEUBAD (DEUBiquitinase ADaptor) domain recruits deubiquitinating enzyme Uch37 to the proteasome. Bis-benzylidine piperidone derivatives that were found to bind covalently to Rpn13 C88 caused the accumulation of polyubiquitinated proteins as well as ER stress-related apoptosis in various cancer cell lines, including bortezomib-resistant multiple myeloma lines. We find that a 38-amino acid peptide derived from the C-terminus of proteasome PC repeat protein hRpn2/PSMD1 binds to hRpn13 Pru domain with 12 nM affinity. By using NMR, we identify the hRpn13-interacting amino acids in this hRpn2 fragment, some of which are conserved among eukaryotes. Importantly, we find the hRpn2-derived peptide to immunoprecipitate endogenous Rpn13 from 293T cells, and to displace it from the proteasome. These findings indicate that this region of hRpn2 is the primary binding site for hRpn13 in the proteasome. Moreover, the hRpn2-derived peptide was no longer able to interact with endogenous hRpn13 when a strictly conserved phenylalanine (F948 in humans) was replaced with arginine or a stop codon, or when Y950 and I951 were substituted with aspartic acid. Finally, over-expression of the hRpn2-derived peptide leads to an increased presence of ubiquitinated proteins in 293T cells. We propose that this hRpn2-derived peptide could be used to develop peptide-based strategies that specifically target hRpn13 function in the proteasome.
Insights
A peptide from hRpn2 binds Rpn13, a proteasome receptor targeted in cancer. This peptide disrupts Rpn13’s proteasome interaction, offering a novel strategy for cancer therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Rpn13 is a proteasome ubiquitin receptor and a therapeutic target in human cancers.
- Rpn13 has an N-terminal Pru domain for ubiquitin binding and proteasome docking, and a C-terminal DEUBAD domain for recruiting Uch37.
Purpose of the Study:
- To identify the binding site of Rpn13 within the proteasome.
- To investigate the potential of targeting Rpn13-proteasome interactions for therapeutic strategies.
Main Methods:
- Nuclear Magnetic Resonance (NMR) spectroscopy to identify interacting amino acids.
- Peptide binding assays to determine affinity (12 nM).
- Immunoprecipitation to assess Rpn13 displacement from the proteasome.
Main Results:
- A 38-amino acid peptide from hRpn2 binds the Rpn13 Pru domain with high affinity.
- NMR identified key interacting residues, including conserved ones.
- The hRpn2 peptide immunoprecipitates endogenous Rpn13 and displaces it from the proteasome.
- Mutations in conserved residues (F948, Y950, I951) abolish peptide interaction.
- Overexpression of the peptide increases ubiquitinated proteins.
Conclusions:
- The C-terminal region of hRpn2 is the primary binding site for Rpn13 in the proteasome.
- This hRpn2-derived peptide can disrupt Rpn13 function within the proteasome.
- The peptide holds potential for developing novel, peptide-based cancer therapeutics targeting Rpn13.
