具細胞毒性的人類核醣核酸水解酶屬於核醣核酸水解酵素A族之成員，它具有抗細菌和抗寄生蟲等特性，此外亦對部分哺乳類細胞及組織造成細胞毒性。在近期研究中我們發現在大腸桿菌中大量表現全長含前導序列（leading sequence）的此核醣核酸水解酶時會造成寄主細胞的死亡，而此現象在表現不含前導序列之成熟型的酵素時不會發生。
本次研究係利用可調節的CUPI和MET3啟動子，分別建立麵包酵母菌之誘導表現系統，以分泌表達含前導序列的綠色螢光蛋白（LS-eGFP）。本研究發現，在未誘導的條件下，含有含前導序列的綠色螢光蛋白麵包酵母菌的生長正常，無遲緩的現象；然而，在誘導的條件下，表達含前導序列的綠色螢光蛋白分別在兩組誘導表現系統皆顯示了生長遲緩的現象。另外，我們證實了在誘導出重組蛋白後，新生蛋白合成的能力受損，導致生長遲緩的現象。利用二維電泳及質譜的分析方法，我們鑑定出11個蛋白的表現量在反應中產生變化。其中有一牽涉核酸醣小體生合成之蛋白（SSF1）與表現受到抑制，而六碳醣激酶（HXKB）的蛋白表現量提升。另外烷基過氧化物還原酶（AHP）及過氧化體生合成蛋白（PEX6）的蛋白表現量提升，意味著過氧化體的增生以及酵母菌菌體內的一種保護機制已被啟動，以致酵母菌僅生長遲緩而不走向死亡。

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.

1. Rosenberg, H.F., Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. J Biol Chem, 1995. 270(14): p. 7876-81.
2. Rosenberg, H.F., S.J. Ackerman, and D.G. Tenen, Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J Exp Med, 1989. 170(1): p. 163-76.
3. C. M. Wu, et al., Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase. BBRC accepted at 2004. 7. 22
4. Parsons AB, Geyer R, Hughes TR, Boone C, Yeast genomics and proteomics in drug discovery and target validation. Prog Cell Cycle Res., 2003. 5: p. 159-66.
5. Grant SG, Blackstock WP, Proteomics in neuroscience: from protein to network. J Neurosci., 2001. 21(21): p. 8315-8.
6. Martoglio, B., R. Graf, and B. Dobberstein, Signal peptide fragments of preprolactin and HIV-1 p-gp160 interact with calmodulin. Embo J, 1997. 16(22): p. 6636-45.
7. Wicher, K.B., et al., Deletion of a cytotoxic, N-terminal putatitive signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus. Appl Microbiol Biotechnol, 2001. 55(5): p. 578-84.
8. Belin, D., et al., Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors. J Mol Biol, 2004. 335(2): p. 437-53.
9. Burgers, P.M., Overexpression of multisubunit replication factors in yeast. Methods, 1999. 18(3): p. 349-55.
10. Hottiger, T., et al., 2-micron vectors containing the Saccharomyces cerevisiae metallothionein gene as a selectable marker: excellent stability in complex media, and high-level expression of a recombinant protein from a CUP1-promoter-controlled expression cassette in cis. Yeast, 1995. 11(1): p. 1-14.
11. Mao, X., et al., MET3 promoter: a tightly regulated promoter and its application in construction of conditional lethal strain. Curr Microbiol, 2002. 45(1): p. 37-40.
12. Breter, H.J., M.T. Knoop, and H. Kirchen, The mapping of chromosomes in Saccharomyces cerevisiae. I. A cosmid vector designed to establish, by cloning into cdc-mutants, numerous start loci for chromosome walking in the yeast genome. Gene, 1987. 53(2-3): p. 181-90.
13. Shevchenko, A., et al., Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem, 1996. 68(5): p. 850-8.
14. Jungblut, P.R., et al., Comparative proteome analysis of Helicobacter pylori. Mol Microbiol, 2000. 36(3): p. 710-25.
15. Bogengruber, E., et al., eds. Functional analysis in yeast of the Brix protein superfamily involved in the biogenesis of ribosomes. FEMS Yeast Res. Vol. 3. 2003. 35-43.
16. Veenhuis, M. and W. Harder, Microbodies in yeasts: structure, function and biogenesis. Microbiol Sci, 1988. 5(11): p. 347-51.
17. Daniel, T., et al., Expression and regulation of the AMP-activated protein kinase-SNF1 (sucrose non-fermenting 1) kinase complexes in yeast and mammalian cells: studies using chimaeric catalytic subunits. Biochem J, 2002. 365 (3): p. 629-38.
18. Sanz, P., et al., Snf1 protein kinase: a key player in the response to cellular stress in yeast. Biochem Soc Trans, 2003. 31 (1): p. 178-81.
19. Ahuatzi, D., et al., The glucose-regulated nuclear localization of hexokinase 2 in Saccharomyces cerevisiae is Mig1-dependent. J Biol Chem, 2004. 279 (14): p. 14440-6.
20. Lee, J., et al., A new antioxidant with alkyl hydroperoxide defense properties in yeast. J Biol Chem, 1999. 274(8): p. 4537-44.
21. Erdmann, R., et al., PAS1, a yeast gene required for peroxisome biogenesis, encodes a member of a novel family of putative ATPases. Cell, 1991. 64(3): p. 499-510.
22. Stroobants, A.K., et al., Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ire1p. FEBS Lett, 1999. 453(1-2): p. 210-4.
23. Hegde, R.S., Targeting and beyond: new roles for old signal sequences. Mol Cell, 2002. 10(4): p. 697-8.
24. Weihofen, A., et al., Identification of signal peptide peptidase, a presenilin-type aspartic protease. Science, 2002. 296(5576): p. 2215-8.
25. Willis, K.A., et al., The global transcriptional activator of Saccharomyces cerevisiae, Gcr1p, mediates the response to glucose by stimulating protein synthesis and CLN-dependent cell cycle progression. Genetics, 2003. 165(3): p. 1017-29.
26. Kim, J. and J.P. Hirsch, A nucleolar protein that affects mating efficiency in Saccharomyces cerevisiae by altering the morphological response to pheromone. Genetics, 1998. 149(2): p. 795-805.
27. Middleton JL, Marth EH, Fennema O, Dichlorofluoromethane inactivates Saccharomyces cerevisiae. Appl Microbiol., 1975. 29(2): p. 195-200.
.