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The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

Shang C, Chen Q, Dell A, Haslam SM, De Vos WH, Van Damme EJ - PLoS ONE (2015)

Bottom Line: In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins.Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula.Our data suggest that one of these pathways involves the induction of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ABSTRACT
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

No MeSH data available.


Related in: MedlinePlus

In cellula protein translation inhibition activity of S. nigra RIPs (A) Merged confocal images of HeLa cells stained with DAPI (blue) and BODIPY (green).Scale bars represent 15 μm. (B) Average numbers of LDs/cell (n>400) (C) Luciferase activity measured in HG1-luc2-IRES-tCD cells incubated with S. nigra RIPs/lectins and controls. The treatment with 1 x PBS was selected as the negative control, and cycloheximide was used as a positive control. The luminescent signal was normalized by the fluorescence signal from the Presto blue assay to correct for variations in cell density. Asterisks denote values significantly different from the cells incubated with control (1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001).
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pone.0132389.g006: In cellula protein translation inhibition activity of S. nigra RIPs (A) Merged confocal images of HeLa cells stained with DAPI (blue) and BODIPY (green).Scale bars represent 15 μm. (B) Average numbers of LDs/cell (n>400) (C) Luciferase activity measured in HG1-luc2-IRES-tCD cells incubated with S. nigra RIPs/lectins and controls. The treatment with 1 x PBS was selected as the negative control, and cycloheximide was used as a positive control. The luminescent signal was normalized by the fluorescence signal from the Presto blue assay to correct for variations in cell density. Asterisks denote values significantly different from the cells incubated with control (1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001).

Mentions: To ascertain protein translation inhibition activity within cells, we used the formation of lipid droplets (LDs) as a proxy [47]. HeLa cells have few small LDs when cultured in normal culture medium [48], but show strong accumulation of LD’s upon treatment with translation inhibitors such as cycloheximide [47]. When treated with 100 nM S. nigra RIPs SNA-I and SNA-V for 22 hours, there were significantly more LDs (Fig 6A and 6B). This was not the case for SNLRP and the non-RIP lectins (SNA-II and SNA-IV), suggesting that the S. nigra RIPs do effectively exert their ribosome inactivating activity inside the cells. Experiments with a bioluminescent reporter cell line (HG1-luc2-IRES-tCD) supported this notion [32] (Fig 6C). SNA-V significantly reduced the luminescent signal compared to the control. The other proteins showed no significant effect on the luminescent signal within the non-toxic range, possibly due to the weak sensitivity of the reporter cell line.


The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

Shang C, Chen Q, Dell A, Haslam SM, De Vos WH, Van Damme EJ - PLoS ONE (2015)

In cellula protein translation inhibition activity of S. nigra RIPs (A) Merged confocal images of HeLa cells stained with DAPI (blue) and BODIPY (green).Scale bars represent 15 μm. (B) Average numbers of LDs/cell (n>400) (C) Luciferase activity measured in HG1-luc2-IRES-tCD cells incubated with S. nigra RIPs/lectins and controls. The treatment with 1 x PBS was selected as the negative control, and cycloheximide was used as a positive control. The luminescent signal was normalized by the fluorescence signal from the Presto blue assay to correct for variations in cell density. Asterisks denote values significantly different from the cells incubated with control (1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493096&req=5

pone.0132389.g006: In cellula protein translation inhibition activity of S. nigra RIPs (A) Merged confocal images of HeLa cells stained with DAPI (blue) and BODIPY (green).Scale bars represent 15 μm. (B) Average numbers of LDs/cell (n>400) (C) Luciferase activity measured in HG1-luc2-IRES-tCD cells incubated with S. nigra RIPs/lectins and controls. The treatment with 1 x PBS was selected as the negative control, and cycloheximide was used as a positive control. The luminescent signal was normalized by the fluorescence signal from the Presto blue assay to correct for variations in cell density. Asterisks denote values significantly different from the cells incubated with control (1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001).
Mentions: To ascertain protein translation inhibition activity within cells, we used the formation of lipid droplets (LDs) as a proxy [47]. HeLa cells have few small LDs when cultured in normal culture medium [48], but show strong accumulation of LD’s upon treatment with translation inhibitors such as cycloheximide [47]. When treated with 100 nM S. nigra RIPs SNA-I and SNA-V for 22 hours, there were significantly more LDs (Fig 6A and 6B). This was not the case for SNLRP and the non-RIP lectins (SNA-II and SNA-IV), suggesting that the S. nigra RIPs do effectively exert their ribosome inactivating activity inside the cells. Experiments with a bioluminescent reporter cell line (HG1-luc2-IRES-tCD) supported this notion [32] (Fig 6C). SNA-V significantly reduced the luminescent signal compared to the control. The other proteins showed no significant effect on the luminescent signal within the non-toxic range, possibly due to the weak sensitivity of the reporter cell line.

Bottom Line: In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins.Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula.Our data suggest that one of these pathways involves the induction of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.

ABSTRACT
Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs) is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry) RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

No MeSH data available.


Related in: MedlinePlus