Alteration of protein composition in mouse thymocytes by signals through T-cell receptor.
To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressing autoreactive T-cell receptor are deleted in the thymus via an apoptotic process by the signals from the T-cell receptor-CD3 complexes. In order to analyze the apoptotic mechanism, we established a cell-free system using the lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti-CD3-treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti-CD3 treatment. Proteins in both fractions were separated by two-dimensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. In 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 receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3-phosphate dehydrogenase were found to be decreased by the signals.
Kawakami
T
T
Core Technology Department, Glaxo Wellcome K.K. Tsukuba Research Laboratories, Ibaraki, Japan.
Nagata
T
T
Muraguchi
A
A
Nishimura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism*; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology
D011506:Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell