Alteration of protein composition in mouse thymocytes by signals through T-cell receptor

To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressin g autoreactive T-cell receptor are deleted in the thymus via an apoptotic p rocess by the signals from the T-cell receptor-CD3 complexes. In order to a nalyze the apoptotic mechansism, we established a cell-free system using th e lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal an tibody (mAb). The soluble cytosolic high molecular mass protein fraction fr om the anti-CD3-treated thymocytes revealed an activity that directly induc es nuclear apoptotic morphological changes and DNA fragmentation. This frag mentation activity was not observed in the fraction from the thymocytes wit hout anti-CD3 treatment. Proteins in both fractions were separated by two-d imensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching o f mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtain ed from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. I n this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T-cell re ceptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyd e 3-phosphate dehydrogenase were found to be decreased by the signals.