SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

How to find information on national food and nutrient consumption surveys across Europe: systematic literature review and questionnaires to selected country experts are both good strategies.

The present research was conducted within the framework of the EURopean micronutrient RECommendations Aligned project. In order to identify the best practice in assessing nutrient intakes, a search strategy for collecting data from national food consumption surveys/studies in Europe was developed. Systematic literature searches were carried out on twenty-eight European and the four European Free Trade Association countries. A questionnaire was also sent to two to five experts in each country. Systematic reviews using PubMed yielded 12 703 abstracts that were reduced to 200 studies using inclusion and exclusion criteria. Similarly, a search of ministry web sites yielded 3033 hits, and subsequently reduced to nine surveys. Belgium, France, Germany, Ireland, Sweden, Spain and the United Kingdom were the countries with most data and Slovenia and Liechtenstein were those with the least. Seventy-eight expert questionnaires were obtained from all countries except for Liechtenstein, Luxembourg and Slovakia. Detailed results and references are given. A systematic search and questionnaires are equally good at identifying national surveys across countries. Literature searching provides globally accessible and objective information albeit limited, whereas the questionnaire provides information that, depending upon responders, can be more complete. A combination of both strategies is recommended.

How does the quality of surveys for nutrient intake adequacy assessment compare across Europe? A scoring system to rate the quality of data in such surveys.

Research was conducted within the EURopean micronutrient RECommendations Aligned (EURRECA) Network of Excellence, to find the best practice in assessing nutrient intakes. Objectives include: to search for and use data on individual nutrient intake adequacy (NIA) assessment collected in twenty-eight European countries and the four European Free Trade Association countries; to design and test innovative tools for data quality analysis. The information was obtained using the method described by Blanquer et al. in the present issue. The best-practice criteria were devised to select the most appropriate survey in each country. Then a survey quality scoring system was developed in consultation with experts and tested on these surveys. Weights were allocated according to a variable priority order agreed by consultation. The thirty-two countries yielded twenty-four national surveys (eight countries excluded). Data collection techniques: eleven countries/surveys used personal interviews only; six used combinations of techniques. Dietary assessment methods: two used repeated 24 h recalls only; eleven used combinations. NIA assessment methods: two used probabilistic approach and sd/Z-scores only; eleven used comparison with estimated average requirements/RDA only. Countries were ranked according to the survey quality scoring, but careful interpretation is needed because of incomplete data from some surveys; bearing this in mind, the information quality is high in 37.5 % countries, medium in 50.0 % and low in 12.5 %. Although there is room for improvement and caution should be taken when drawing conclusions and recommendations from these results, the lessons learned and tools developed at this first attempt form the basis for future work within the EURRECA framework for aligning European micronutrient recommendations.