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In silico structural and functional characterization of the RSUME splice variants.

Gerez J, Fuertes M, Tedesco L, Silberstein S, Sevlever G, Paez-Pereda M, Holsboer F, Turjanski AG, Arzt E - PLoS ONE (2013)

Bottom Line: Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain.We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME.The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET, Buenos Aires, Argentina.

ABSTRACT
RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.

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Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.A. COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. *, band corresponding to an IgG used in the inmunoprecipitation. B. and C. COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (B), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (C), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). D. IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).
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pone-0057795-g005: Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.A. COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. *, band corresponding to an IgG used in the inmunoprecipitation. B. and C. COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (B), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (C), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). D. IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).

Mentions: In order to study the functional contribution of the C-terminal domain of RSUME267 to NF-κB signaling pathways, we first compared the interaction of the RSUME isoforms with IκBα, a subunit of the NF-κB inhibitor, by inmunoprecipitation experiments. Previously, we have demonstrated a direct interaction of RSUME195 with IκBα. Now, we probe that RSUME267 shows a greater interaction with IκBα than RSUME195 (Fig. 5A). Based on this result, we expect that RSUME267 has a greater impact on the inhibition of the NF-κB transcriptional activity. COS-7 cells were co-transfected with NF-κB-LUC reporter plasmid and different concentrations of RSUME195 or RSUME267 expression vectors. As shown in Figure 5B, although both RSUME isoforms inhibit NF-κB transcriptional activity, low doses of RSUME267 are sufficient for a strong inhibition (65%); 5-folds higher amounts of RSUME195 expression vectors are needed to reach a similar effect (100 ng of RSUME267 vs. 500 ng of RSUME195). Additionally, RSUME267 also has a stronger inhibitory effect on IL-8 promoter activity (Fig. 5C, upper panel). This action of RSUME is absent when the NF-κB binding site of the promoter of IL-8 was mutated (Fig. 5C, lower panel). In addition, we show by quantitative real time RT-PCR that RSUME195 decreases the endogenous mRNA expression of IL-8, and this effect is significantly higher for the RSUME267 isoform, although this effect is very small (Fig. 5D).


In silico structural and functional characterization of the RSUME splice variants.

Gerez J, Fuertes M, Tedesco L, Silberstein S, Sevlever G, Paez-Pereda M, Holsboer F, Turjanski AG, Arzt E - PLoS ONE (2013)

Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.A. COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. *, band corresponding to an IgG used in the inmunoprecipitation. B. and C. COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (B), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (C), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). D. IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).
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getmorefigures.php?uid=PMC3585135&req=5

pone-0057795-g005: Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.A. COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. *, band corresponding to an IgG used in the inmunoprecipitation. B. and C. COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (B), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (C), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). D. IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).
Mentions: In order to study the functional contribution of the C-terminal domain of RSUME267 to NF-κB signaling pathways, we first compared the interaction of the RSUME isoforms with IκBα, a subunit of the NF-κB inhibitor, by inmunoprecipitation experiments. Previously, we have demonstrated a direct interaction of RSUME195 with IκBα. Now, we probe that RSUME267 shows a greater interaction with IκBα than RSUME195 (Fig. 5A). Based on this result, we expect that RSUME267 has a greater impact on the inhibition of the NF-κB transcriptional activity. COS-7 cells were co-transfected with NF-κB-LUC reporter plasmid and different concentrations of RSUME195 or RSUME267 expression vectors. As shown in Figure 5B, although both RSUME isoforms inhibit NF-κB transcriptional activity, low doses of RSUME267 are sufficient for a strong inhibition (65%); 5-folds higher amounts of RSUME195 expression vectors are needed to reach a similar effect (100 ng of RSUME267 vs. 500 ng of RSUME195). Additionally, RSUME267 also has a stronger inhibitory effect on IL-8 promoter activity (Fig. 5C, upper panel). This action of RSUME is absent when the NF-κB binding site of the promoter of IL-8 was mutated (Fig. 5C, lower panel). In addition, we show by quantitative real time RT-PCR that RSUME195 decreases the endogenous mRNA expression of IL-8, and this effect is significantly higher for the RSUME267 isoform, although this effect is very small (Fig. 5D).

Bottom Line: Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain.We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME.The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET, Buenos Aires, Argentina.

ABSTRACT
RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.

Show MeSH
Related in: MedlinePlus