Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: Improved labeling and structural implications

Kelly Kyro, Surya P. Manandhar, Daniel Mullen, Walter K. Schmidt, Mark D. Distefano

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)7559-7569
Number of pages11
JournalBioorganic and Medicinal Chemistry
Volume19
Issue number24
DOIs
StatePublished - Dec 15 2011

Keywords

  • Benzophenone
  • Bpa
  • CAAX processing
  • Farnesyl
  • Photoaffinity labeling
  • Prenyl protease
  • Protein prenylation
  • Rce1

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