Proteomic analysis of ubiquitin-proteasome effects:

The global effect of ubiquitinproteasome (UP) inhibitors on leukemic cell proteome was analysed. A total of 39 protein spots, affected by UP inhibitors, were identi.ed, including 11 new apoptosis-associated proteins. They are involved in different cellular functions and four were associated with caspase-3 activation. Eukaryotic initiation factor (eIF-) was identi.ed in two spots; however, the peptide mass-.ngerprinting for the accumulated one included a peptide with lysine50, indicating that hypusine formation was suppressed during UP inhibitor-induced apoptosis. Hypusine modi.cation ensues immediately following translation of eIF- precursor, unless cells are treated with the modi.cation inhibitors diaminoheptane. However, UP inhibitors induced a much stronger accumulation of unmodi.ed eIF- compared to the effect of diaminoheptane. We further showed the unmodi.ed eIF- was regulated in a proteasome-dependent manner. Inhibition of hypusine formation by diaminoheptane triggered apoptosis, but of particular interest is the finding that eIF- expression inhibition by antisense oligodeoxynucleotides signi.cantly enhanced the stimulating effect of GM-CSF on cell growth. Therefore, the eIF- accumulation played important roles in the apoptosis induced by UP inhibitors. Moreover, hypusine inhibition in apoptosis was further revealed to be associated with the subcellular localization of eIF-. Our data pave the way to a better understanding of the mechanisms by which UP system has been linked to apoptosis.