Predominant Identification of RNA-binding Proteins in Fas-induced Apoptosis by Proteome Analysis

Bernd Thiede,Christiane Dimmler,Frank Siejak,Thomas Rudel

doi:10.1074/jbc.m101062200

Abstract

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.

Highlights

Apoptosis, a genetically determined form of cellular suicide, is an essential and complex process involved in the development and maintenance of cell homeostasis in multicellular organisms
In order to identify proteins that are modified during apoptosis, we used the twodimensional gel electrophoresis data base as a reference to investigate the proteome of Jurkat T cells that had been induced to undergo apoptosis. 37 apoptosis-modified spots of 21 different proteins were identified
2000 spots were resolved and detected by silver staining. 10 twodimensional gel electrophoresis gels of apoptotic cells were compared with 10 two-dimensional gel electrophoresis gels of control cells (Fig. 2). 24 spots were detected in patterns of apoptotic Jurkat T cells that were not present in patterns on nonapoptotic cells. 21 additional spots were observed in the pattern of control cells

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The Jurkat T cell line E6 was maintained in RPMI tissue culture medium (Life Technologies, Inc.) supplemented with 10%. The cells were homogenized and centrifuged at 3500 units/min for 1 min at 4 °C (Rotor SS-34; Sorvall RC5B, Hanau, Germany). The interphase that contained the mitochondria was collected, suspended in 4 volumes of MSM buffer, and centrifuged again at 15,500 units/min for 10 min at 4 °C (Rotor SS-34; Sorvall RC5B). The pellet with the nucleus was suspended in 5 ml of phosphatebuffered saline and centrifuged for 2 min at 3500 units/min at 4 °C (Rotor SS-34; Sorvall RC5B). The pellet was suspended in NB buffer (10 mM Hepes, pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, and 1 mM pefabloc) (1 ml/108 cells) and incubated for 1 h on ice, subsequently homogenized, and applied to 10 ml of 30% sucrose in NB buffer. Data Base Searching—The proteins were identified by using the peptide mass fingerprinting analysis software MS-Fit (available on the World Wide Web).

RESULTS

RNP 2 RNP

DISCUSSION