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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 HIF-1 signaling pathway.A. COS-7 cells were co-transfected with 500 ng of HRE-LUC report vector, 300 ng of Gaussia report vector and different concentrations of RSUME195 or 267 (100 or 300 ng) to evaluate their effect in HIF-1 transcriptional activity. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then 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) (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, under hypoxia, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. B. COS-7 cells were co-transfected with 300 ng of Flag-HIF-1alpha and/or 500 ng of V5-RSUME195 or V5-RSUME267, or the corresponding empty vector (were both are absent). Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Cell extracts were subjected to western blot. NMX, normoxia; HPX, hypoxia; Vect, cells transfected with the corresponding empty vectors. C. COS-7 cells were co-transfected with RSUME and HIF-1alpha. Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. RIPA cell extracts were inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. D. COS-7 cells were co-transfected with 500 ng of VEGF-LUC report vector, 300 ng of CMV-β Gal report vector and RSUME195 or 267. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to β-galactosidase 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) under hypoxia (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267, under hypoxia (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. E. Semi-quantitative RT-PCR of endogenous VEGF and β-actin mRNA in HepG2 cells transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. F. VEGF mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HeLa cell stimulated with hypoxia for 16 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). G. HepG2 cells were transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. VEGF protein levels were analysed by western blot.
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pone-0057795-g006: Effect of RSUME195 and RSUME267 over HIF-1 signaling pathway.A. COS-7 cells were co-transfected with 500 ng of HRE-LUC report vector, 300 ng of Gaussia report vector and different concentrations of RSUME195 or 267 (100 or 300 ng) to evaluate their effect in HIF-1 transcriptional activity. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then 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) (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, under hypoxia, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. B. COS-7 cells were co-transfected with 300 ng of Flag-HIF-1alpha and/or 500 ng of V5-RSUME195 or V5-RSUME267, or the corresponding empty vector (were both are absent). Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Cell extracts were subjected to western blot. NMX, normoxia; HPX, hypoxia; Vect, cells transfected with the corresponding empty vectors. C. COS-7 cells were co-transfected with RSUME and HIF-1alpha. Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. RIPA cell extracts were inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. D. COS-7 cells were co-transfected with 500 ng of VEGF-LUC report vector, 300 ng of CMV-β Gal report vector and RSUME195 or 267. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to β-galactosidase 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) under hypoxia (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267, under hypoxia (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. E. Semi-quantitative RT-PCR of endogenous VEGF and β-actin mRNA in HepG2 cells transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. F. VEGF mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HeLa cell stimulated with hypoxia for 16 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). G. HepG2 cells were transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. VEGF protein levels were analysed by western blot.

Mentions: Since we have shown that RSUME195 modulates HIF-1 transcriptional activity, we tested if RSUME195 and 267 differ in their stimulatory ability on this signaling pathway. We observed the same result that on the NF-κB pathway (Fig. 6A). RSUME195 and 267 increase HIF-1 transcriptional activity, but lower doses of RSUME267 are necessary to produce the highest effect of the RSUME195 (100 ng vs. 300 ng). To confirm this result, we tested the ability of RSUME267 to increase HIF-1alpha expression. We found that RSUME267 increases HIF-1alpha protein at greater levels than RSUME195 (Fig. 6B). Also, we demonstrate by inmunoprecipitation that RSUME267 has a higher interaction with HIF-1alpha than RSUME195 (Fig. 6C). Analyzing the expression of the HIF-1 target VEGF by a reporter plasmid VEGF-LUC assay, semi-quantitative RT-PCR, quantitative real time RT-PCR and western blot, we conclude that the longest RSUME isoform is more effective in increasing VEGF expression levels (Fig. 6D, 6E, 6F and 6G).


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 HIF-1 signaling pathway.A. COS-7 cells were co-transfected with 500 ng of HRE-LUC report vector, 300 ng of Gaussia report vector and different concentrations of RSUME195 or 267 (100 or 300 ng) to evaluate their effect in HIF-1 transcriptional activity. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then 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) (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, under hypoxia, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. B. COS-7 cells were co-transfected with 300 ng of Flag-HIF-1alpha and/or 500 ng of V5-RSUME195 or V5-RSUME267, or the corresponding empty vector (were both are absent). Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Cell extracts were subjected to western blot. NMX, normoxia; HPX, hypoxia; Vect, cells transfected with the corresponding empty vectors. C. COS-7 cells were co-transfected with RSUME and HIF-1alpha. Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. RIPA cell extracts were inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. D. COS-7 cells were co-transfected with 500 ng of VEGF-LUC report vector, 300 ng of CMV-β Gal report vector and RSUME195 or 267. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to β-galactosidase 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) under hypoxia (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267, under hypoxia (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. E. Semi-quantitative RT-PCR of endogenous VEGF and β-actin mRNA in HepG2 cells transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. F. VEGF mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HeLa cell stimulated with hypoxia for 16 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). G. HepG2 cells were transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. VEGF protein levels were analysed by western blot.
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pone-0057795-g006: Effect of RSUME195 and RSUME267 over HIF-1 signaling pathway.A. COS-7 cells were co-transfected with 500 ng of HRE-LUC report vector, 300 ng of Gaussia report vector and different concentrations of RSUME195 or 267 (100 or 300 ng) to evaluate their effect in HIF-1 transcriptional activity. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then 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) (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, under hypoxia, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. B. COS-7 cells were co-transfected with 300 ng of Flag-HIF-1alpha and/or 500 ng of V5-RSUME195 or V5-RSUME267, or the corresponding empty vector (were both are absent). Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Cell extracts were subjected to western blot. NMX, normoxia; HPX, hypoxia; Vect, cells transfected with the corresponding empty vectors. C. COS-7 cells were co-transfected with RSUME and HIF-1alpha. Twenty-four hours post-transfection, cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. RIPA cell extracts were inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. D. COS-7 cells were co-transfected with 500 ng of VEGF-LUC report vector, 300 ng of CMV-β Gal report vector and RSUME195 or 267. Twenty-four hours after transfection cells were subjected to hypoxic conditions (1% O2, 5% CO2 and 94% N2) for 16 h. Then LUC activity was measured in the cell extracts. Each value was normalized to β-galactosidase 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) under hypoxia (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267, under hypoxia (ANOVA with Scheffè’s test). NMX, normoxia; HPX, hypoxia. E. Semi-quantitative RT-PCR of endogenous VEGF and β-actin mRNA in HepG2 cells transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. F. VEGF mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HeLa cell stimulated with hypoxia for 16 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). G. HepG2 cells were transfected with empty vector (Vect), RSUME195 or RSUME267, and subjected to hypoxia (1% O2, 5% CO2 and 94% N2) for 16 h, twenty-four hours after transfection. VEGF protein levels were analysed by western blot.
Mentions: Since we have shown that RSUME195 modulates HIF-1 transcriptional activity, we tested if RSUME195 and 267 differ in their stimulatory ability on this signaling pathway. We observed the same result that on the NF-κB pathway (Fig. 6A). RSUME195 and 267 increase HIF-1 transcriptional activity, but lower doses of RSUME267 are necessary to produce the highest effect of the RSUME195 (100 ng vs. 300 ng). To confirm this result, we tested the ability of RSUME267 to increase HIF-1alpha expression. We found that RSUME267 increases HIF-1alpha protein at greater levels than RSUME195 (Fig. 6B). Also, we demonstrate by inmunoprecipitation that RSUME267 has a higher interaction with HIF-1alpha than RSUME195 (Fig. 6C). Analyzing the expression of the HIF-1 target VEGF by a reporter plasmid VEGF-LUC assay, semi-quantitative RT-PCR, quantitative real time RT-PCR and western blot, we conclude that the longest RSUME isoform is more effective in increasing VEGF expression levels (Fig. 6D, 6E, 6F and 6G).

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