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Your search keyword '"Oreb, Mislav"' showing total 193 results
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193 results on '"Oreb, Mislav"'

21. Functional expression of the human glucose transporters GLUT2 and GLUT3 in yeast offers novel screening systems for GLUT-targeting drugs

Author

Schmidl, Sina, Tamayo Rojas, Sebastian A., Iancu, Cristina V., Choe, Jun-Yong, and Oreb, Mislav

Subjects
endocrine system, ddc:570, hxt0 yeast strain, drug screening system, Molecular Biosciences, ddc:610, GLUT3, Original Research, GLUT2, Glucose transport inhibitor
Abstract

Human GLUT2 and GLUT3, members of the GLUT / SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.

Published
2021

35. A label-free real-time method for measuring glucose uptake kinetics in yeast.

Author

Schmidl, Sina, Iancu, Cristina V, Reifenrath, Mara, Choe, Jun-yong, and Oreb, Mislav

Subjects
GREEN fluorescent protein, GLUCOSE, RADIOACTIVE wastes, YEAST, SACCHAROMYCES cerevisiae
Abstract

Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system. [ABSTRACT FROM AUTHOR]

Published
2021
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45. A comparative analysis of NADPH supply strategies in Saccharomyces cerevisiae:production of d-xylitol from d-xylose as a case study

Author

Regmi, Priti, Knesebeck, Melanie, Boles, Eckhard, Weuster-Botz, Dirk, and Oreb, Mislav

Abstract

Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In S. cerevisiae, several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L-1xylitol from 20 g L-1xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase ALD6gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from Kluyveromyces lactisacted synergistically with ZWF1overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies – ZWF1overexpression and deletion of ALD6- also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.

Published
2024
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49. Nucleotides and Substrates Trigger the Dynamics of the Toc34 GTPase Homodimer Involved in Chloroplast Preprotein Translocation

Author

Lumme, Christina, primary, Altan-Martin, Hasret, additional, Dastvan, Reza, additional, Sommer, Maik S., additional, Oreb, Mislav, additional, Schuetz, Denise, additional, Hellenkamp, Björn, additional, Mirus, Oliver, additional, Kretschmer, Jens, additional, Lyubenova, Sevdalina, additional, Kügel, Wolfgang, additional, Medelnik, Jan P., additional, Dehmer, Manuela, additional, Michaelis, Jens, additional, Prisner, Thomas F., additional, Hugel, Thorsten, additional, and Schleiff, Enrico, additional

Published
2014
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