The recently cloned small RWD-domain containing protein RSUME was shown to increase protein levels of hypoxia-inducible factor-1a (HIF-1a). The latter is the oxygen-regulated subunit of HIF-1, the most important transcription factor of the cellular adaptive processes to hypoxic conditions. It is also a major regulator of vascular endothelial growth factor-A (VEGF-A), which is critically involved in the complex process of tumour neovascularisation. In this study, the expression and role of RSUME in pituitary tumours was studied. We found that RSUME mRNA was up-regulated in pituitary adenomas and significantly correlated with HIF-1a mRNA levels. Hypoxia (1% O 2 ) or treatment with hypoxia-mimicking CoCl 2 enhanced RSUME and HIF-1a expression, induced translocation of HIF-1a to the nuclei and stimulated VEGF-A production both in pituitary tumour cell lines and primary human pituitary adenoma cell cultures. When RSUME expression was specifically down-regulated by siRNA, the CoCl 2 -induced increase VEGF-A secretion was strongly reduced which was shown to be a consequence of the RSUME knockdown-associated reduction of HIF-1a synthesis. Thus, RSUME plays an important role in initiating pituitary tumour neovascularisation through regulating HIF-1a levels and subsequent VEGF-A production and may therefore be critically involved in pituitary adenoma progression.

Research performed on the pituitary has proven that cytokines play an important role in maintaining pituitary physiology, affecting not only cell proliferation but also hormone secretion. The effects of cytokines can be autocrine or paracrine. This review gives an overview on the effects of the most studied cytokines in the pituitary. Special interest is focused on interleukin-6 (IL-6) because it has the distinctive characteristic of stimulating pituitary tumor cell growth, but has the opposite effect on normal pituitary cells. On the other hand, IL-6 is a cytokine of interest in the pituitary because recent work has shown that it promotes and maintains senescence in certain types of tumors. Given that the majority of pituitary adenomas are microadenomas and the fact that clinically inapparent pituitary tumors are quite common, senescence, perhaps mediated by IL-6, is an attractive mechanism for explaining the benign nature of pituitary tumors.

Alontaga et al. (1) analyzed the structure of RSUME/RWDD3 and confirmed our findings (2, 3) that RWD is the only well structured domain in RSUME and participates in its binding to Ubc9 and SUMOylation action. Based on experimental data, we proposed that RSUME interacts with Ubc9 prior to the Ubc9-SUMO complex formation (2). The absence of NMR chemical shifts on Ubc9 induced by RWD after the Ubc9-SUMO complex is already formed and the higher K d of RSUME for Ubc9 than SUMO are to be expected and are fully consistent with our model. The authors obtained crystals of a loose heterotrimeric complex bearing two Ubc9 molecules and one RWD domain. The NMR results and analysis of the Ubc9-RWD interaction based on the HSQC (hetero-nuclear single quantum coherence) data do not exclude other possible Ubc9-RWD complexes that are also compatible with all NMR data. Moreover, the NMR shift histogram proves that the N-terminal helix of RSUME interacts with Ubc9, as we predicted (2). An evolutionarily conserved interaction for Ubc9-RSUME similar to established E2-UEV structures (4), as we previously proposed, remains likely. The authors confirmed that RSUME stimulates the formation of the Ubc9-SUMO thioester conjugate and added the stimulation of the SAE2-SUMO thioester and SAE2-SUMO isopeptide. The inference from the global SUMOylation assay performed in cells different from those reported, without Ubc9 co-transfection (optimal condition) and without confirmation of the SUMO identity of the bands, is inconclusive. In vitro, RSUME showed no effect on the SUMOylation of sp100, supporting the notion that RSUME acts on several, but specific, targets as HIF-1, IB, GR, and pVHL (2, 3, 5). (2015) RWD Domain as an E2 (Ubc9)-interaction module. In silico structural and functional characterization of the RSUME splice variants. PloS ONE 8, e57795 4. Eddins, M. J., Carlile, C. M., Gomez, K. M., Pickart, C. M., and Wolberger, C. (2006) Mms2-Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation. Nat. Struct. Mol. Biol. 13, 915-920 5

Renal cell carcinoma (RCC) is the major cause of death among patients with von Hippel-Lindau (VHL) disease. Resistance to therapies targeting tumor angiogenesis opens the question about the underlying mechanisms. Previously we have described that RWDD3 or RSUME (RWD domain-containing protein SUMO Enhancer) sumoylates and binds VHL protein and negatively regulates HIF degradation, leading to xenograft RCC tumor growth in mice. In this study, we performed a bioinformatics analysis in a ccRCC dataset showing an association of RSUME levels with VHL mutations and tumor progression, and we demonstrate the molecular mechanism by which RSUME regulates the pathologic angiogenic phenotype of VHL missense mutations. We report that VHL mutants fail to downregulate RSUME protein levels accounting for the increased RSUME expression found in RCC tumors. Furthermore, we prove that targeting RSUME in RCC cell line clones carrying missense VHL mutants results in decreased early tumor angiogenesis. The mechanism we describe is that RSUME sumoylates VHL mutants and beyond its sumoylation capacity, interacts with Type 2 VHL mutants, reduces HIF-2α-VHL mutants binding, and negatively regulates the assembly of the Type 2 VHL, Elongins and Cullins (ECV) complex. Altogether these results show RSUME involvement in VHL mutants deregulation that leads to the angiogenic phenotype of RCC tumors.

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.