Multipotent Adult Germline Stem Cells and Embryonic Stem Cells Functional Proteomics Revealed an Important Role of Eukaryotic Initiation Factor 5A (Eif5a) in Stem Cell Differentiation

Abstract

Multipotent adult germline stem cells (maGSCs) are pluripotent cells that can be differentiated into somatic cells of the three primary germ layers. To highlight the protein profile changes associated with stem cell differentiation, retinoic acid (RA) treated mouse stem cells (maGSCs and ESCs) were compared to nontreated stem cells. 2-DE and DIGE reference maps were created, and differentially expressed proteins were further processed for identification. In both stem cell types, the RA induced differentiation resulted in an alteration of 36 proteins of which 18 were down-regulated and might be potential pluripotency associated proteins, whereas the other 18 proteins were up-regulated. These might be correlated to stem cell differentiation. Surprisingly, eukaryotic initiation factor 5A (Eif5a), a protein which is essential for cell proliferation and differentiation, was significantly down-regulated under RA treatment. A time-dependent investigation of Eif5a showed that the RA treatment of stem cells resulted in a significant up-regulation of the Eif5a in the first 48 h followed by a progressive down-regulation thereafter. This effect could be blocked by the hypusination inhibitor ciclopirox olamine (CPX). The alteration of Eif5a hypusination, as confirmed by mass spectrometry, exerts an antiproliferative effect on ESCs and maGSCs in vitro, but does not affect the cell pluripotency. Our data highlights the important role of Eif5a and its hypusination for stem cell differentiation and proliferation.

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“…The protein extraction was performed as described previously (Dihazi et al, 2011). To reduce the salt contamination and to enrich the proteins, chloroform-methanol precipitation was performed according to Wessel and Flügge (Wessel and Flügge, 1984).…”

“…Large scale 2-DE and gel staining were carried out as described previously (Dihazi et al, 2011). For protein identification, 2-DE gels were additionally stained with collodial Coomassie blue, Roti-Blue (Roth, Karlsruhe, Germany) overnight.…”

“…The resulting pellet was solubilized in labelling buffer (30 mM Tris/HCl pH 8.5, 9.5 M urea, 2% CHAPS, 10 mM PMSF), centrifuged (5 min, 13,000 g), and the protein concentration of the supernatant was determined as described above. The protein labelling with CyDyes and the 2-DE were performed as described previously (Dihazi et al, 2011). Fluorescent images were captured in 16-bit TIFF files format.…”

“…In-gel digestion and peptide extraction were carried out as described previously (Dihazi et al, 2011). Subsequently the extracted peptides were subjected to peptide sequence analysis.…”

“…Subsequently the extracted peptides were subjected to peptide sequence analysis. The samples were dissolved in 0.1% formic acid and processed as described previously (Dihazi et al, 2011). Processed data were searched against MSDB and Swissprot databases through the Mascot search engine using a peptide mass tolerance of 50 ppm (parts per million) and fragment tolerance of 100 mmu (millimass unit).…”