Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with 75 Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10 ؊8 to 10 ؊6 M selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10 ؊6 M selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.
Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty. We now report the shifty tRNA(Phe) contains 1-methylguanosine (m(1)G) in place of Y and that the m(1)G form from rabbit reticulocytes stimulates frameshifting more efficiently than its m(1)G-containing counterpart from mouse neuroblastoma cells. The latter tRNA contains unmodified C and G nucleosides at positions 32 and 34, respectively, while the former tRNA contains the analogous 2'-O-methylated nucleosides at these positions. The data suggest that not only does the loss of a highly modified base from the 3' side of the anticodon render tRNA(Phe) shifty, but the modification status of the entire anticodon loop contributes to the degree of shiftiness. Possible biological consequences of these findings are discussed.
Effects of human selenoprotein SelK on the adhesion and migration ability of human gastric cancer BGC-823 cells using Matrigel adhesion and transwell migration assays, respectively, were investigated in this study. The Matrigel adhesion ability of BGC-823 cells that overexpressed SelK declined extremely significantly (p < 0.01) compared with that of the cells not expressing the protein. The migration ability of BGC-823 cells that overexpressed SelK also declined extremely significantly (p < 0.01). On the other hand, the Matrigel adhesion ability and migration ability of the cells that overexpressed C-terminally truncated SelK did not decline significantly. The Matrigel adhesion ability and migration ability of human embryonic kidney HEK-293 cells that overexpressed SelK did not show significant change (p > 0.05) with the cells that overexpressed the C-terminally truncated protein. In addition to the effect on Matrigel adhesion and migration, the overexpression of SelK also caused a loss in cell viability (as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) colorimetric assay) and induced apoptosis as shown by confocal microscopy and flow cytometry. The cytosolic free Ca2+ level of these cells was significantly increased as detected by flow cytometry. But the overexpression of SelK in HEK-293 cells caused neither significant loss in cell viability nor apoptosis induction. Only the elevation of cytosolic free Ca2+ level in these cells was significant. Taken together, the results suggest that the overexpression of SelK can inhibit human cancer cell Matrigel adhesion and migration and cause both the loss in cell viability and induction of apoptosis. The release of intracellular Ca2+ from the endoplasmic reticulum might be a mechanism whereby the protein exerted its impact. Furthermore, only the full-length protein, but not C-terminally truncated form, was capable of producing such impact. The embryonic cells were not influenced by the elevation of free Ca2+ level in cytosol, probably due to their much greater tolerance to the variation.
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