Abstract
The cytotoxic Human ribonuclease (hRNase) belongs to RNase A superfamily. It is bactericidal, helminthotoxic, and cytotoxic to some mammalian cells and tissues. In our recent studies, we found that expression of the full length hRNase including its leading sequence in prokaryotic systems caused death of the host cell, but expression of the mature enzyme did not cause cell death. In this study, eGFP fusion proteins containing leading sequence of hRNase3 were generated by inducible Saccharomyces cerevisiae expression system with either CUPI or MET3 promoter. Our results revealed that cells carrying the fusion protein showed normal growth curve under non-inductive condition. Upon induction with Cu2+ or removal of methionine, the fusion protein was successfully expressed in the yeast. However, cells containing the fusion protein revealed a phenomenon of growth retardation. The pulse labeling experiments revealed that the phenomenon of cell growth arrest resulted from defect of protein synthesis. To further investigate the role of the leading sequence of RNase in cell growth arrest in yeast, 2D electrophoresis and mass spectrometry have been performed. Eleven protein spots were identified by MALDI-TOF mass spectrometry. A protein (SSF1) essential for the biogenesis of the large ribosomal subunit was down-regulated upon induction of the fusion eGFP. Hexokinase B increased 5-fold in protein expression level when cells expressed the SPRPPQFTReGFP fusion protein. However, peroxisome biosynthesis protein, PEX6, and a peroxisomal reductase, AHP1, were highly expressed upon induction of the fusion eGFP. The phenomenon implied that the proliferation of peroxisome and a protective mechanism protecting cells from damage may be triggered.