title: A High Affinity hRpn2-Derived Peptide That Displaces Human Rpn13 from Proteasome in 293T Cells.
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.
authors.affiliation: Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Xiuxiu
authors.initials: X
authors.lastname: Lu
authors.affiliation: Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Fen
authors.initials: F
authors.lastname: Liu
authors.affiliation: Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America; Middletown High School, Middletown, Maryland, United States of America.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Durham
authors.affiliation: Biophysics Resource, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Sergey G
authors.initials: SG
authors.lastname: Tarasov
authors.affiliation: Protein Processing Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, United States of America.
authors.forename: Kylie J
authors.initials: KJ
authors.lastname: Walters
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D016415:Sequence Alignment
chemical_list: C500781:ADRM1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C484162:PSMD1 protein, human; D010446:Peptide Fragments; D046988:Proteasome Endopeptidase Complex