Proteome-wide Identification of HtrA2/Omi Substrates

J Proteome Res. 2007 Mar;6(3):1006-15. doi: 10.1021/pr060510d. Epub 2007 Feb 1.

Abstract

To identify apoptotic targets of HtrA2/Omi, we purified recombinant HtrA2/Omi and its catalytically inactive S306A mutant. Lysates of human Jurkat T lymphocytes incubated with either wild-type recombinant HtrA2/Omi or the S306A mutant were screened using the gel-free COFRADIC approach that isolates peptides covering the N-terminal parts of proteins. Analysis of the 1162 proteins identified by mass spectrometry yielded 15 HtrA2/Omi substrates of potential physiological relevance together holding a total of 50 cleavage sites. Several processing events were validated by incubating purified recombinant HtrA2/Omi with in vitro translated substrates or with Jurkat cell lysates. In addition, the generated set of cleavage sites was used to assess the protein substrate specificity of HtrA2/Omi. Our results suggest that HtrA2/Omi has a rather narrow cleavage site preference and that cytoskeletal proteins are prime targets of this protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism*
  • High-Temperature Requirement A Serine Peptidase 2
  • Humans
  • Jurkat Cells
  • Mass Spectrometry
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Mutation, Missense
  • Peptide Fragments
  • Proteome
  • Proteomics / methods*
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism*
  • Substrate Specificity / genetics*

Substances

  • Cytoskeletal Proteins
  • Mitochondrial Proteins
  • Peptide Fragments
  • Proteome
  • Serine Endopeptidases
  • HTRA2 protein, human
  • High-Temperature Requirement A Serine Peptidase 2