The Fas-induced apoptosis analyzed by high throughput proteome analysis.
The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.
Gerner
C
C
Institute of Cancer Research, University of Vienna, A-1090 Vienna, Austria. Christopher.Gerner@univie.ac.at
Frohwein
U
U
Gotzmann
J
J
Bayer
E
E
Gelbmann
D
D
Bursch
W
W
Schulte-Hermann
R
R
D000595:Amino Acid Sequence; D017209:Apoptosis; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D016923:Cell Death; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000378:metabolism; D010766:Phosphorylation; D021381:Protein Transport; D020543:Proteome / Q000378:metabolism*; D016622:Silver Staining; D019014:fas Receptor / Q000378:metabolism*
D020543:Proteome; D019014:fas Receptor; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins; D018995:Myosin Heavy Chains