Proteome-wide Identification of HtrA2/Omi Substrates
- 1 February 2007
- journal article
- Published by American Chemical Society (ACS) in Journal of Proteome Research
- Vol. 6 (3), 1006-1015
- https://doi.org/10.1021/pr060510d
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. Keywords: COFRADIC • HtrA2 • Omi • apoptosis • substrateKeywords
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