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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

S. nigra proteins induce autophagy.(A) Confocal images of HeLa cells treated with S. nigra proteins, immuno-stained for p62 (red) and counterstained with DAPI (blue). (B) Quantification of p62 puncta numbers/cell (n>600 cells). Asterisks denote values significantly different from the cells incubated in the control (medium containing 1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001). (C) representative images of HeLa cells transfected with a tandem fluorescent mRFP-GFP-LC3 treated with 1x PBS (control) or SNA-I, (D) Quantification of yellow (autophagosomes) and red (autophagolysosomes) puncta reveals increased autophagy (total number of spots) as well as autophagic flux (red spots) in SNA-I treated cells. The asterisk indicates a significant difference compared to the control with P-value < 0.05. Scale bars in panels A and C represent 10 μm and 15 μm, respectively.
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pone.0132389.g005: S. nigra proteins induce autophagy.(A) Confocal images of HeLa cells treated with S. nigra proteins, immuno-stained for p62 (red) and counterstained with DAPI (blue). (B) Quantification of p62 puncta numbers/cell (n>600 cells). Asterisks denote values significantly different from the cells incubated in the control (medium containing 1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001). (C) representative images of HeLa cells transfected with a tandem fluorescent mRFP-GFP-LC3 treated with 1x PBS (control) or SNA-I, (D) Quantification of yellow (autophagosomes) and red (autophagolysosomes) puncta reveals increased autophagy (total number of spots) as well as autophagic flux (red spots) in SNA-I treated cells. The asterisk indicates a significant difference compared to the control with P-value < 0.05. Scale bars in panels A and C represent 10 μm and 15 μm, respectively.

Mentions: Given the fairly high load of proteins in the lysosomes, we reasoned that cellular uptake of some S. nigra proteins may trigger alternative degradation pathways such as autophagy. p62 is a direct substrate for autophagy that becomes included in autophagosomes, which is why we used it to monitor formation of autophagosomes [45]. A quantitative analysis of the number of p62 puncta per cell revealed that the autophagosome formation increased significantly after administration of any of the different elderberry proteins (Fig 5A and 5B). To determine whether the autophagic flux was altered, a tandem mRFP-GFP-tagged LC3 construct [34,46] was used. This fusion construct relies on the properties of mRFP to withstand the acidic environment of the lysosomes and maintain its fluorescence, whereas GFP does not. A strong increase in red over yellow (green+red) foci suggested enrichment of LC3 positive autophagolysosomes and thus an increase in autophagic flux in HeLa cells incubated with SNA-I (Fig 5C and 5D).


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)

S. nigra proteins induce autophagy.(A) Confocal images of HeLa cells treated with S. nigra proteins, immuno-stained for p62 (red) and counterstained with DAPI (blue). (B) Quantification of p62 puncta numbers/cell (n>600 cells). Asterisks denote values significantly different from the cells incubated in the control (medium containing 1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001). (C) representative images of HeLa cells transfected with a tandem fluorescent mRFP-GFP-LC3 treated with 1x PBS (control) or SNA-I, (D) Quantification of yellow (autophagosomes) and red (autophagolysosomes) puncta reveals increased autophagy (total number of spots) as well as autophagic flux (red spots) in SNA-I treated cells. The asterisk indicates a significant difference compared to the control with P-value < 0.05. Scale bars in panels A and C represent 10 μm and 15 μm, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493096&req=5

pone.0132389.g005: S. nigra proteins induce autophagy.(A) Confocal images of HeLa cells treated with S. nigra proteins, immuno-stained for p62 (red) and counterstained with DAPI (blue). (B) Quantification of p62 puncta numbers/cell (n>600 cells). Asterisks denote values significantly different from the cells incubated in the control (medium containing 1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001). (C) representative images of HeLa cells transfected with a tandem fluorescent mRFP-GFP-LC3 treated with 1x PBS (control) or SNA-I, (D) Quantification of yellow (autophagosomes) and red (autophagolysosomes) puncta reveals increased autophagy (total number of spots) as well as autophagic flux (red spots) in SNA-I treated cells. The asterisk indicates a significant difference compared to the control with P-value < 0.05. Scale bars in panels A and C represent 10 μm and 15 μm, respectively.
Mentions: Given the fairly high load of proteins in the lysosomes, we reasoned that cellular uptake of some S. nigra proteins may trigger alternative degradation pathways such as autophagy. p62 is a direct substrate for autophagy that becomes included in autophagosomes, which is why we used it to monitor formation of autophagosomes [45]. A quantitative analysis of the number of p62 puncta per cell revealed that the autophagosome formation increased significantly after administration of any of the different elderberry proteins (Fig 5A and 5B). To determine whether the autophagic flux was altered, a tandem mRFP-GFP-tagged LC3 construct [34,46] was used. This fusion construct relies on the properties of mRFP to withstand the acidic environment of the lysosomes and maintain its fluorescence, whereas GFP does not. A strong increase in red over yellow (green+red) foci suggested enrichment of LC3 positive autophagolysosomes and thus an increase in autophagic flux in HeLa cells incubated with SNA-I (Fig 5C and 5D).

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