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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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RSUME267 and RSUME195 similar actions on the SUMO pathway.A. 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from E.coli transformed with the empty pQE30 vector. B. 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. C. Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.
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pone-0057795-g004: RSUME267 and RSUME195 similar actions on the SUMO pathway.A. 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from E.coli transformed with the empty pQE30 vector. B. 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. C. Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.

Mentions: In order to study the functional contribution of the RSUME267 C-terminal domain to the SUMO enhancer properties of this protein, we tested and compared the ability of RSUME195 and 267 isoforms for Ubc9 binding and SUMO-attachment stimulation. As shown in Figure 4A, by in vitro pull-down assays, RSUME267 and the shorter isoform bind GST-Ubc9 to similar extents. As an internal control, no unspecific binding was detected when GST alone was used to pull-down any endogenously present RSUME isoforms. We tested the RSUME267 ability to enhance overall protein SUMOylation in cells, and found that this variant increases SUMO conjugation similarly to RSUME195 (Fig. 4B). We also tested the SUMO enhancer properties of this variant on a specific substrate of SUMOylation, and found that RSUME195 and RSUME267 increase SUMO conjugation to Topoisomerase I (TOPOI) to similar extents, as shown in an in vitro SUMOylation assay (Fig. 4C). Taken together, these results indicate that these RSUME isoforms do not show differences when evaluating functionally the SUMO pathway.


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)

RSUME267 and RSUME195 similar actions on the SUMO pathway.A. 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from E.coli transformed with the empty pQE30 vector. B. 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. C. Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585135&req=5

pone-0057795-g004: RSUME267 and RSUME195 similar actions on the SUMO pathway.A. 1 µg of recombinant RSUME195 or RSUME267 was co-precipitated by GST-Ubc9 in vitro. Pull down experiments were performed and the samples were subjected to Western blot. To compare, the ratio between input and pull-down intensities measured by Image-J Software was calculated. As internal control, to detect unspecific binding, GST alone was used to pull-down any RSUME isoform. Ø, control elution extract purified from E.coli transformed with the empty pQE30 vector. B. 300 ng HA-SUMO-1, expression vector was co-transfected with 500 ng of V5-RSUME195 or V5-RSUME267 and V5-Ubc9 expression vectors. 48 h post-transfection, cell extracts were subjected to western blot with anti-HA to detect sumoylated proteins. RSUME expression was confirmed with anti-RSUME antibodies. C. Sumoylation assay of Topoisomerase I (TOPOI) to test enhancer properties of RSUME isoforms. Experiments were performed in triplicates.
Mentions: In order to study the functional contribution of the RSUME267 C-terminal domain to the SUMO enhancer properties of this protein, we tested and compared the ability of RSUME195 and 267 isoforms for Ubc9 binding and SUMO-attachment stimulation. As shown in Figure 4A, by in vitro pull-down assays, RSUME267 and the shorter isoform bind GST-Ubc9 to similar extents. As an internal control, no unspecific binding was detected when GST alone was used to pull-down any endogenously present RSUME isoforms. We tested the RSUME267 ability to enhance overall protein SUMOylation in cells, and found that this variant increases SUMO conjugation similarly to RSUME195 (Fig. 4B). We also tested the SUMO enhancer properties of this variant on a specific substrate of SUMOylation, and found that RSUME195 and RSUME267 increase SUMO conjugation to Topoisomerase I (TOPOI) to similar extents, as shown in an in vitro SUMOylation assay (Fig. 4C). Taken together, these results indicate that these RSUME isoforms do not show differences when evaluating functionally the SUMO pathway.

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