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.

Three different methods of collecting preputial material for bacteriological examination were compared using 3 bulls infected with Campylobacter fetus subsp. fetus. The first method utilised a specially designed instrument to scrape the preputial and penile mucosa, int he second method plastic pipettes were used to aspirate material and the third method involved washing the preputial cavity with sterile peptone water. Bacteriological examination of the samples showed conclusively that scraping was the method of choice because more C. fetus positive samples were identified and there was less interference from contaminating organisms.

The factors triggering pancreatic neuroendocrine tumor (PanNET) progression are largely unknown. Here we investigated the role and mechanisms of the sumoylation enhancing protein RSUME in PanNET tumorigenesis. Immunohistochemical studies showed that RSUME is strongly expressed in normal human pancreas, in particular in β-cells. RSUME expression is reduced in insulinomas and is nearly absent in other types of PanNETs suggesting a role in PanNET tumorigenesis. In human pancreatic neuroendocrine BON1 cells, RSUME stimulates hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A), which are key components of tumor neovascularisation. In contrast, RSUME suppresses nuclear factor-κB (NF-κB) and its target interleukin-8 (IL-8). Correspondingly, PanNET cells with RSUME knockdown showed decreased HIF-1α activity and increased NF-κB and IL-8 production leading to a moderate reduction of VEGF-A release as reduced HIF-1α/VEGF-A production is partly compensated by NF-κB/IL-8-induced VEGF-A. Notably, RSUME stabilizes the tumor suppressor PTEN, which is frequently lost in PanNETs and whose absence is associated with metastasis formation. In vivo orthotopic transplantation of PanNET cells with or without RSUME expression into nude mice showed that PanNETs without RSUME have reduced PTEN expression, grow faster and form multiple liver metastases. In sum, RSUME differentially regulates key components of PanNET formation suggesting that the observed loss of RSUME in advanced PanNETs is critically involved in PanNET tumorigenesis, particularly in metastasis formation.