| The ubiquitin-proteasome pathway (UP) is responsible for the specific degration of abnormal, short-lived, or regulatory proteins in the cytoplasm or nucleus of the eukaryotic cells. The pathway is implicated in many different cellular processes such as cell progression, signal transduction, protein quality control and enzymatic regulation. Recently, a plethora of cell proteins implicated in the regulation of basic cellular processes, such as proliferation, differentiation, cell cycling, and apoptosis have been discovered to undergo processing and functional limitation by entering the UP pathway with the final destination to be proteolytically degraded by the 26S proteasome. Because both negative and positive regulators of proliferation and apoptosis undergo proteasomal degradation in a tightly regulated and temporally controlled fashion, the 26S proteasome can play opposite roles in the regulation of proliferation and apoptosis. Inhibition of the pathway strongly induced apoptosis in some types of cells, however, it had less or no effects or even protected cells from apoptosis in others. Little is known hitherto about the mechanisms by which components of the UP pathway have been linked to apoptosis, and it is currently unknown how various types of cells showed so great different response to the same inhibition of the pathway. Further, the mechanisms of eIF-5A in the regulation of cell proliferation and apoptosis are elusive.In this paper, treatment with the proteasome inhibitors, MG-132 or Lactacystin, resulted in concentration- and time-dependent apoptosis and activation of caspase-3 in MoVe cells. Using two-dimensional electrophoresis gels (2-DE) followed by identifications with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and electrospray ionization MS/MS (ESI-MS/MS), we analyzed the proteomic response of Mo7e cells to inhibition of ubiquitin-proteasome pathway. Thirty-nine protein spots, affected by UP inhibitors, were identified and clusteredaccording to their functions. Using a caspase-3-specific inhibitor acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO), we showed the up-regulation of profilin 1 and Rho GDI proteins were associated with the activation of caspase-3, which was activated by the pathway inhibitors. We demonstrated that eIF-5A without hypusine modification was accumulated as an early response to UP inhibitor stimulation. It is reported that unmodified eIF-5A could rarely be detected as hypusine modification ensues immediately following translation of eIF-5A precursor unless cells are treated with the modification inhibitor diaminoheptane(DAH). Treatment of Mo7e cells with DAH resulted the inhibition of cell proliferation in a concentration-dependent manner, while apoptosis was also induced at the same time. Upon treatment of the cell lines with DAH, cell growth was inhibited. Cell cycle analysis showed that DAH induced cell growth arrest at the Gl/S boundary of the cell cycle. In synchronized MCF7 cells, the expression level of eIF-5A peaked at Gl phase but very low at S and G2/M phases. However, we found UP inhibitors induced unmodified eIF-5A accumulation even far more strongly than DAH. Of particular interest is the finding that eIF-5A expression inhibition by antisense oligodeoxynucleotides significantly stimulated Mo7e cell growth. Further, eIF-5A overexpression through transfection in COS? cells markedly promoted UP inhibitor-induced apoptosis. We also found that eIF-5A could homodimerize and associate with ubiquitin, but the mechanism remains to be investigated. Our data pave the way to a better understanding of the mechanisms by which UP system and eIF-5A are linked to cell cycle regulation and apoptosis.
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