Proteome-wide Identification of HtrA2/Omi Substrates.
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.
Mesh Terms:
Apoptosis, Cytoskeletal Proteins, Humans, Jurkat Cells, Mass Spectrometry, Mitochondrial Proteins, Mutation, Missense, Peptide Fragments, Proteome, Proteomics, Serine Endopeptidases, Substrate Specificity
Apoptosis, Cytoskeletal Proteins, Humans, Jurkat Cells, Mass Spectrometry, Mitochondrial Proteins, Mutation, Missense, Peptide Fragments, Proteome, Proteomics, Serine Endopeptidases, Substrate Specificity
J. Proteome Res.
Date: Mar. 01, 2007
PubMed ID: 17266347
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