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 tw o-dimensional gels, comparison of control versus apoptotic cells revealed t hat 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 loc alization by metabolic labeling and analysis of subcellular protein fractio ns in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that ag. hsp27, hsp70B, calmodulin, a nd H-ras synthesis was induced upon Fas signaling. 34 proteins were affecte d by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-lar 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 wer e compared with those of other cell death inducers, indicating specific phy siological characteristics of different cell death mechanisms, consequent t o as well as independent of caspase activation. Characteristic proteome alt erations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.