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.
Vande Walle
Lieselotte
L
Department of Molecular Biomedical Research, VIB, 9052, Ghent, Belgium.
Van Damme
Petra
P
Lamkanfi
Mohamed
M
Saelens
Xavier
X
Vandekerckhove
Joël
J
Gevaert
Kris
K
Vandenabeele
Peter
P
D017209:Apoptosis; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010446:Peptide Fragments; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics*
D003598:Cytoskeletal Proteins; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D020543:Proteome; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2