Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen

Aquaporins and aquaglyceroporins mediate the transport of water and solutes across biological membranes. Saccharomyces cerevisiae Fps1 is an aquaglyceroporin that mediates controlled glycerol export during osmoregulation. The transport function of Fps1 is rapidly regulated by osmotic changes in an apparently unique way and distinct regions within the long N- and C-terminal extensions are needed for this regulation. In order to learn more about the mechanisms that control Fps1 we have set up a genetic screen for hyperactive Fps1 and isolated mutations in 14 distinct residues, all facing the inside of the cell. Five of the residues lie within the previously characterized N-terminal regulatory domain and two mutations are located within the approach to the first transmembrane domain. Three mutations cause truncation of the C-terminus, confirming previous studies on the importance of this region for channel control. Furthermore, the novel mutations identify two conserved residues in the channel-forming B-loop as critical for channel control. Structural modelling-based rationalization of the observed mutations supports the notion that the N-terminal regulatory domain and the B-loop could interact in channel control. Our findings provide a framework for further genetic and structural analysis to better understand the mechanism that controls Fps1 function by osmotic changes.

Synthesis of sulforaphane and inhibition of cytochrome P450 enzymes as a basis for antigenotoxicity

Many dietary factors have been associated with a decreased risk of developing cancer. One potential mechanism by which these factors, chemopreventors, protect against cancer may be via alteration of carcinogen metabolism. The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulinylbutane) (CH3-S0-(CH2)4-NCS) has been isolated as a potential inducer of phase II detoxification enzymes and also protects rodents against 9,10-dimethyl-1,2-benz[aJanthracene-induced mammary tumours. The ability of sulforaphane to also modulate phase I activation enzymes (cytochrome P450) (CYP450) was studied here. Sulforaphane was synthesised with an overall yield of 15%, essentially via 1-methylsulfinylphthalimidobutane, which was oxidised to the sulfoxide moiety. Deprotective removal of phthalimide yielded the amine, which was converted into sulforaphane by reaction with N,N'-thionocarbonyldiimidazole. Purity (95 %) was checked by 1H-NMR,13C-NMR and infrared and mass spectrometry.Sulforaphane was a competitive inhibitor of CYP2E1 in acetone-induced Sprague-Dawley rat microsomes (Ki 37.9 ± 4.5μM), as measured by the p-nitrophenol hydroxylase assay. Ethoxyresorufin deethylase activity (EROD), a measurement of CYP1A activity, was also inhibited by sulforaphane (100μM) but was not competitive, and a preincubation time-dependence was observed. In view of these results, the capacity of sulforaphane to inhibit N-nitrosodimethylamine (NDMA)-induced genotoxicity (CYP2E1-mediated) was studied using mouse liver activation systems. Sulforaphane (>0.8μM) inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with acetone-induced liver 9000 g supernatants from Balb/c mice. Unscheduled DNA synthesis induced by NDMA (33μ5 M) in mouse hepatocytes was also reduced by sulforaphane in a concentration-dependent manner (0.064-20μM). Sulforaphane was not genotoxic itself in any of these systems and cytotoxic only at high concentrations (>0.5 mM and > 40μM respectively). The ability of sulforaphane to modulate the orthologous human enzymes was studied using a human epithelial liver cell line (THLE) expressing individual human CYP450 isoenzymes. Using the Comet assay (a measurement of DNA strand breakage under alkaline conditions), NDMA (0.01-1μg/ml) and IQ (0.1-10μg/ml) were used to produce strand breaks in T5-2E1 cells (expressing human CYP2E1) and T5-1A2 cells (expressing human CYP1A2) respectively, however no response was observed in T5-neo cells (without CYP450 cDNA transfection). Sulforaphane inhibited both NDMA and IQ-induced DNA strand breakage in a concentration-dependent manner (0.1-10μM).The inhibition of metabolic activation as a basis for the antigenotoxic action of sulforaphane in these systems (bacteria, rodent hepatocytes and human cells) is further supported by the lack of this chemopreventor to influence NaN3 mutagenicity in S. typhimurium and H202-induced DNA strand breakage in T5-neo cells. These findings suggest that inhibition of CYP2E1 and CYP1A by sulforaphane may contribute to its chemoprotective potential.

Production of membrane proteins in yeast

Background Yeast is an important and versatile organism for studying membrane proteins. It is easy to cultivate and can perform higher eukaryote-like post-translational modifications. S. cerevisiae has a fully-sequenced genome and there are several collections of deletion strains available, whilst P. pastoris can produce very high cell densities (230 g/l). Results We have used both S. cerevisiae and P. pastoris to over-produce the following His6 and His10 carboxyl terminal fused membrane proteins. CD81 – 26 kDa tetraspanin protein (TAPA-1) that may play an important role in the regulation of lymphoma cell growth and may also act as the viral receptor for Hepatitis C-Virus. CD82 – 30 kDa tetraspanin protein that associates with CD4 or CD8 cells and delivers co-stimulatory signals for the TCR/CD3 pathway. MC4R – 37 kDa seven transmembrane G-protein coupled receptor, present on neurons in the hypothalamus region of the brain and predicted to have a role in the feast or fast signalling pathway. Adt2p – 34 kDa six transmembrane protein that catalyses the exchange of ADP and ATP across the yeast mitochondrial inner membrane. Conclusion We show that yeasts are flexible production organisms for a range of different membrane proteins. The yields are such that future structure-activity relationship studies can be initiated via reconstitution, crystallization for X-ray diffraction or NMR experiments.

The effect of antifoams upon recombinant protein production in yeast

Foaming during fermentation reduces the efficiency of the process leading to increased costs and reduced productivity. Foaming can be overcome by the use of chemical antifoaming agents, however their influence upon the growth of organisms and protein yield is poorly understood. The objective of this work was to evaluate the effects of different antifoams on recombinant protein production. Antifoam A, Antifoam C, J673A, P2000 and SB2121 were tested at different concentrations for their effect on the growth characteristics of Pichia pastoris producing GFP, EPO and A2aR and the yield of protein in shake flasks over 48 h. All antifoams tested increased the total GFP in the shake flasks compared to controls, at higher concentrations than would normally be used for defoaming purposes. The highest yield was achieved by adding 1 % P2000 which nearly doubled the total yield followed by 1 % SB2121, 1 % J673A, 0.6 % Antifoam A and lastly 0.8 % Antifoam C. The antifoams had a detrimental effect upon the production of EPO and A2aR in shake flasks, suggesting that their effects may be protein specific. The mechanisms of action of the antifoams was investigated and suggested that although the volumetric mass oxygen transfer coefficient (kLa) was influenced by the agents, their effect upon the concentration of dissolved oxygen did not contribute to the changes in growth or recombinant protein yield. Findings in small scale also suggested that antifoams of different compositions such as silicone polymers and alcoxylated fatty acid esters may influence growth characteristics of host organisms and the ability of the cells to secrete recombinant protein, indirectly affecting the protein yield. Upon scale-up, the concentration effects of the antifoams upon GFP yield in bioreactors was reversed, with lower concentrations producing a higher yield. These data suggest that antifoam can affect cells in a multifactorial manner and highlights the importance of screening for optimum antifoam types and concentrations for each bioprocesses.

Novel characteristics of the biological properties of the yeast Saccharomyces cerevisiae eukaryotic initiation factor 2A

Eukaryotic initiation factor 2A (eIF2A) has been shown to direct binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-dependent manner, in contrast to eIF2, which requires GTP but not the AUG codon to bind initiator tRNA to 40 S subunits. We show here that yeast eIF2A genetically interacts with initiation factor eIF4E, suggesting that both proteins function in the same pathway. The double eIF2A/eIF4E-ts mutant strain displays a severe slow growth phenotype, which correlated with the accumulation of 85% of the double mutant cells arrested at the G(2)/M border. These cells also exhibited a disorganized actin cytoskeleton and elevated actin levels, suggesting that eIF2A might be involved in controlling the expression of genes involved in morphogenic processes. Further insights into eIF2A function were gained from the studies of eIF2A distribution in ribosomal fractions obtained from either an eIF5BDelta (fun12Delta) strain or a eIF3b-ts (prt1-1) strain. It was found that the binding of eIF2A to 40 and 80 S ribosomes was not impaired in either strain. We also found that eIF2A functions as a suppressor of Ure2p internal ribosome entry site-mediated translation in yeast cells. The regulation of expression from the URE2 internal ribosome entry site appears to be through the levels of eIF2A protein, which has been found to be inherently unstable with a half-life of approximately 17 min. It was hypothesized that this instability allows for translational control through the level of eIF2A protein in yeast cells.

Cell surface analysis of the basidiomycete yeast cryptococcus neoformans

Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry. Confocal imaging of surface exposed carbohydrate and protein revealed common localised areas of vivid fluorescence associated with buds, bud scars and nascent daughter cells. Carbohydrate and protein fluorescence often varied between strains, culture age and capsule status of cells examined. Finally, extension of protein tagging techniques resulted in the isolation and extraction of two biotinylated proteins from the yeast cell wall surface of an acapsulate strain of C.neoformans.

Influence of iron deprivation and sub-inhibitory concentrations of antifungal antibiotics on surface antigens of candida albicans yeast cells

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

Conditional osmotic stress in yeast:a system to study transport through aquaglyceroporins and osmostress signaling

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Δ gpd2Δ mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Δ gpd2Δ mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-AΔ of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Δ1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Δ1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Δ gpd2Δ mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Δ gpd2Δ mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Investigating G protein signalling bias at the glucagon-like peptide-1 receptor in yeast

Background and Purpose The glucagon-like peptide 1 (GLP-1) receptor performs an important role in glycaemic control, stimulating the release of insulin. It is an attractive target for treating type 2 diabetes. Recently, several reports of adverse side effects following prolonged use of GLP-1 receptor therapies have emerged: most likely due to an incomplete understanding of signalling complexities. Experimental Approach We describe the expression of the GLP-1 receptor in a panel of modified yeast strains that couple receptor activation to cell growth via single Gα/yeast chimeras. This assay enables the study of individual ligand-receptor G protein coupling preferences and the quantification of the effect of GLP-1 receptor ligands on G protein selectivity. Key Results The GLP-1 receptor functionally coupled to the chimeras representing the human Gαs, Gαi and Gαq subunits. Calculation of the dissociation constant for a receptor antagonist, exendin-3 revealed no significant difference between the two systems. We obtained previously unobserved differences in G protein signalling bias for clinically relevant therapeutic agents, liraglutide and exenatide; the latter displaying significant bias for the Gαi pathway. We extended the use of the system to investigate small-molecule allosteric compounds and the closely related glucagon receptor. Conclusions and Implications These results provide a better understanding of the molecular events involved in GLP-1 receptor pleiotropic signalling and establish the yeast platform as a robust tool to screen for more selective, efficacious compounds acting at this important class of receptors in the future. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast

Copyright © 2015 John Wiley & Sons, Ltd. Funded by College of Life Science and Medicine, University of Aberdeen, UK This work was funded by a start-up grant from the College of Life Science and Medicine, University of Aberdeen, UK. I am grateful to J. Bähler, E. Hartsuiker, F. Klein, J. Kohli, K. Nasmyth, M. C. Whitby, the Leibniz Institute – German Collection of Microorganisms and Cell Cultures (DMSZ) and the National BioResource Project Japan (NBRP) for providing materials used in this study. I thank Alistair J. P. Brown and Takashi Kubota for critically reading this manuscript.

New cassettes for single-step drug-resistance and prototrophic marker switching in fission yeast

Copyright © 2015 John Wiley & Sons, Ltd. Funded by College of Life Science and Medicine, University of Aberdeen, UK This work was funded by a start-up grant from the College of Life Science and Medicine, University of Aberdeen, UK. I am grateful to J. Bähler, E. Hartsuiker, F. Klein, J. Kohli, K. Nasmyth, M. C. Whitby, the Leibniz Institute – German Collection of Microorganisms and Cell Cultures (DMSZ) and the National BioResource Project Japan (NBRP) for providing materials used in this study. I thank Alistair J. P. Brown and Takashi Kubota for critically reading this manuscript.

Predicting Transcript Production Rates in Yeast With Sparse Linear Models

To provide biological insights into transcriptional regulation, a couple of groups have recently presented models relating the promoter DNA-bound transcription factors (TFs) to downstream gene’s mean transcript level or transcript production rates over time. However, transcript production is dynamic in response to changes of TF concentrations over time. Also, TFs are not the only factors binding to promoters; other DNA binding factors (DBFs) bind as well, especially nucleosomes, resulting in competition between DBFs for binding at same genomic location. Additionally, not only TFs, but also some other elements regulate transcription. Within core promoter, various regulatory elements influence RNAPII recruitment, PIC formation, RNAPII searching for TSS, and RNAPII initiating transcription. Moreover, it is proposed that downstream from TSS, nucleosomes resist RNAPII elongation.

Here, we provide a machine learning framework to predict transcript production rates from DNA sequences. We applied this framework in the S. cerevisiae yeast for two scenarios: a) to predict the dynamic transcript production rate during the cell cycle for native promoters; b) to predict the mean transcript production rate over time for synthetic promoters. As far as we know, our framework is the first successful attempt to have a model that can predict dynamic transcript production rates from DNA sequences only: with cell cycle data set, we got Pearson correlation coefficient Cp = 0.751 and coefficient of determination r2 = 0.564 on test set for predicting dynamic transcript production rate over time. Also, for DREAM6 Gene Promoter Expression Prediction challenge, our fitted model outperformed all participant teams, best of all teams, and a model combining best team’s k-mer based sequence features and another paper’s biologically mechanistic features, in terms of all scoring metrics.

Moreover, our framework shows its capability of identifying generalizable fea- tures by interpreting the highly predictive models, and thereby provide support for associated hypothesized mechanisms about transcriptional regulation. With the learned sparse linear models, we got results supporting the following biological insights: a) TFs govern the probability of RNAPII recruitment and initiation possibly through interactions with PIC components and transcription cofactors; b) the core promoter amplifies the transcript production probably by influencing PIC formation, RNAPII recruitment, DNA melting, RNAPII searching for and selecting TSS, releasing RNAPII from general transcription factors, and thereby initiation; c) there is strong transcriptional synergy between TFs and core promoter elements; d) the regulatory elements within core promoter region are more than TATA box and nucleosome free region, suggesting the existence of still unidentified TAF-dependent and cofactor-dependent core promoter elements in yeast S. cerevisiae; e) nucleosome occupancy is helpful for representing +1 and -1 nucleosomes’ regulatory roles on transcription.

Biofuels and high value added products from the yeast Rhodosporidium toruloides NCYC 921: Strategies towards a true cost-effective and environmentally sustainable integrated multiproduct driven biorefinery [Poster]

Single-cell oils (SCO) have been considered a promising source of 3rd generation biofuels mainly in the final form of biodiesel. However, its high production costs have been a barrier towards the commercialization of this commodity. The fast growing yeast Rhodosporidium toruloides NCYC 921 has been widely reported as a potential SCO producing yeast. In addition to its well-known high lipid content (that can be converted into biodiesel), is rich in high value added products such as carotenoids with commercial interest. The process design and integration may contribute to reduce the overall cost of biofuels and carotenoid production and is a mandatory step towards their commercialization. The present work addresses the biomass disruption, extraction, fractionation and recovery of products with special emphasis on high added valued carotenoids (beta-carotene, torulene, torularhodin) and fatty acids directed to biodiesel. The chemical structure of torularhodin with a terminal carboxylic group imposes an additional extra challenge in what concern its separation from fatty acids. The proposed feedstock is fresh biomass pellet obtained directly by centrifugation from a 5L fed-batch fermentation culture broth. The use of a wet instead of lyophilised biomass feedstock is a way to decrease processing energy costs and reduce downstream processing time. These results will contribute for a detailed process design. Gathered data will be of crucial importance for a further study on Life-Cycle Assessment (LCA).

Improvement of the yeast slide culture technique

Este trabalho tem como objetivo melhorar a técnica de cultura em lâmina para ser usada na avaliação da viabilidade de leveduras sob diferentes condições fisiológicas. Inicialmente, foram otimizadas as condições ideais para o cultivo em lâmina de uma estirpe laboratorial (BY4741) e de uma estirpe industrial (NCYC 1214) da levedura Saccharomyces cerevisiae. O melhor protocolo foi obtido utilizando: YEPD agar com uma espessura de cerca de 2 mm; 20 μL de uma suspensão de 1 x 105 células/mL para a estirpe BY4741 ou de 5 x 104 células/mL para a estirpe NCYC 1214; uma câmara de humedecimento com 100 μL de água desionizada e um tempo de incubação de 24 h, a 25 ° C. Com o objetivo de facilitar a contagem das microcolónias, foi adicionado um corante (calcofluor white, CFW) ao meio YEPD agar. Ensaios preliminares, em YEPD líquido, contendo diferentes concentrações de CFW, permitiram verificar que o corante, até 5,0 μg/L, não inibe o crescimento da levedura. Uma concentração de 2,5 μg/L de CFW permitiu a coloração da parede das leveduras, não se observando células com morfologia alterada, sendo esta a concentração de CFW selecionado nos estudos subsequentes. A técnica de cultura em lâmina, com ou sem CFW, foi aplicada para avaliar a viabilidade de células saudáveis (células em fase exponencial de crescimento), células submetidas a stress de etanol [células expostas a 20% (v/v) de etanol, a 25 ºC, durante 2 h] e células envelhecidas (células incubadas em água, a 25 ° C, durante 48 h), da estirpe laboratorial. A percentagem de células viáveis não foi significativamente diferente entre as duas técnicas (com ou sem CFW), após uma incubação de 24 horas. Finalmente, a técnica de cultura de lâmina, contendo CFW, foi comparada com duas técnicas habitualmente usadas na indústria cervejeira: fermentação de curta duração e determinação da percentagem de células gemuladas. Os resultados obtidos através da técnica de cultura de lâmina, desenvolvida, seguem um padrão similar aos obtidos nos ensaios de fermentação de curta duração e aos da determinação da percentagem de células gemuladas. Os resultados obtidos sugerem que a técnica de cultura em lâmina, combinada com CFW, parece ser uma alternativa, fácil, rápida (em 24 h) e reprodutível, relativamente ao método convencional (técnica de plaqueamento), para a avaliação da viabilidade de células de levedura. Deverá ser realizado trabalho adicional a fim de validar o método com estirpes industriais.