title: Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications.
abstract: Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States.
authors.forename: Kelly
authors.initials: K
authors.lastname: Kyro
authors.forename: Surya P
authors.initials: SP
authors.lastname: Manandhar
authors.forename: Daniel
authors.initials: D
authors.lastname: Mullen
authors.forename: Walter K
authors.initials: WK
authors.lastname: Schmidt
authors.forename: Mark D
authors.initials: MD
authors.lastname: Distefano
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D010782:Photolysis; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000302:isolation & purification; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D010455:Peptides; D010649:Phenylalanine; C053764:benzoylphenylalanine; D010450:Endopeptidases
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