Jenny Veide Vilg

Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen‐activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo‐osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr‐197 and this phosphorylation requires LmjMPK2 activity. Lys‐42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild‐type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild‐type cells. This is the first report where a parasite aquaglyceroporin activity is post‐translationally modulated by a mitogen‐activated protein kinase.

Design, Synthesis, and Characterization of a Highly Effective Hog1 Inhibitor: A Powerful Tool for Analyzing MAP Kinase Signaling in Yeast

The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G1 checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

Seasonal and spatial variation in biochemical composition of Saccharina latissima during a potential harvesting season for Western Sweden

Abstract This study monitored the biomass composition of Saccharina latissima during a potential harvesting season on the West coast of Sweden, in order to find suitable harvest times for biorefinery purposes. Specimens of S. latissima were sampled at three locations in June, August and October and the biomass was analysed for its macromolecular composition, as well as for the content of several specific compounds, e.g. sugars and fatty acids. PERMANOVA analyses showed that there was a significant difference in the biomass composition among time points. The total carbohydrate concentration was lowest in June and peaked at 360 mg g-1 dry weight in August, while the mannitol content was highest, 90 mg g-1, in June and decreased throughout the sampling period. Total protein and fatty acid concentrations were found to be approximately 80 and 3 mg g-1, respectively, with relatively little variation over time. Overall, there was little spatial variation in the macromolecular composition, although the concentration of some specific monosaccharides and fatty acids, as well as the total phenolic content, differed among localities. We discuss the implications of the observed variation in biomass composition of S. latissima for future biorefinery purposes.

Application of a peptide-based assay to characterize inhibitors targeting protein kinases from yeast

Abstract Chemical molecules that inhibit protein kinase activity are important tools to assess the functions of protein kinases in living cells. To develop, test and characterize novel inhibitors, a convenient and reproducible kinase assay is of importance. Here, we applied a biotinylated peptide-based method to assess adenosine triphosphate-competitive inhibitors that target the yeast kinases Hog1, Elm1 and Elm1-as. The peptide substrates contained 13 amino acids, encompassing the consensus sequence surrounding the phosphorylation site. To test whether the lack of distal sites affects inhibitor efficacy, we compared the peptide-based assay with an assay using full-length protein as substrate. Similar inhibitor efficiencies were obtained irrespective of whether peptide or full-length protein was used as kinase substrates. Thus, we demonstrate that the peptide substrates used previously (Dinér et al. in PLoS One 6(5):e20012, 2011) give accurate results compared with protein substrates. We also show that the peptide-based method is suitable for selectivity assays and for inhibitor screening. The use of biotinylated peptide substrates provides a simple and reliable assay for protein kinase inhibitor characterization. The utility of this approach is discussed.

Elucidating the response of Kluyveromyces lactis to arsenite and peroxide stress and the role of the transcription factor KlYap8

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8Δ cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8Δ mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function.

Strain improvement of Pichia kudriavzevii TY13 for raised phytase production and reduced phosphate repression.

In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate‐degrading capacity. The mutant strain TY1322 shows a biomass‐specific phytate degradation of 1.26 mmol g−1 h−1 after 8 h of cultivation in a high‐phosphate medium, which is about 8 times higher compared with the wild‐type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46°C) and in the presence of ox bile (2% w/v), indicating this strains ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55°C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non‐genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification.