Membrane Sterol Research Articles

Functional characterization of squalene epoxidase genes in the medicinal plant Tripterygium wilfordii

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1–4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1–4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.

Comparative Effects of Cholesterol and β‐Sitosterol on the Liposome Membrane Characteristics

The influence of different phospholipid types (pure phospholipids 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine, POPC, 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine, DPPC, and one commercial phospholipid mixture, Lipoid H100), sterol types (cholesterol vs. β‐sitosterol), and various sterol concentrations (5–50 mol%) on liposomal membrane fluidity, thermotropic properties, liposome size, zeta potential, and lipid oxidation kinetics using fluorescent lipid probe BODIPY 581/591 C11 (4,4‐difluoro‐5‐[4‐phenyl‐1,3‐butadienyl]‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐undecanoic acid) are investigated. DPPC bilayer is more rigid than POPC and phospholipids mixture membranes. Pure DPPC gives the smallest liposomes, while liposomes of Lipoid H100 have the largest diameter. Both sterols reduce membrane fluidity of all liposomes, increase absolute zeta potential, cause significant changes in particle size, and decrease phase transition temperature (Tm) and enthalpy of DPPC. POPC/β‐sitosterol liposomes exhibit the most significant lipid oxidation of the lipophilic probe. Along with beneficial effects of phytosterols on human health, better membrane fluidity, more favorable and stabilizing interactions with phospholipids, smaller vesicle size, and enhanced physical stability in comparison to cholesterol are some of the encouraging results for the use of β‐sitosterol in liposome formulations for potential application in foods, pharmaceutics, and cosmetics.Practical Applications: Adjusting the composition of liposomal membrane (lipid type, sterol type, and concentration) can be used as a tool to control membrane fluidity, permeability, and thermotropic properties, and thus predict release properties, physical, thermal, and oxidative stability. A commercial phospholipid mixture of different natural phospholipids with impurities creates less uniform liposomal membrane that is characterized by higher fluidity in comparison to DPPC. The type of phospholipid has huge influence on MLVs size. β‐sitosterol, which is a phytosterol with beneficial effects on human health can be used as a replacement for cholesterol in liposomal formulations, but with the following in mind: β‐sitosterol reduces fluidity of the phospholipid bilayer to a lesser extent than cholesterol, β‐sitosterol gives smaller MLVs than cholesterol, DPPC/β‐sitosterol SUVs are bigger than 100 nm in diameter (relevant for intravenous administration), MLVs with ≥30 mol% of β‐sitosterol can be considered as physically stable (unlike those with cholesterol), irrespective to the phospholipid type.The influence of different phospholipid types, sterol types, and various sterol concentrations on liposomal membrane fluidity, thermo tropic properties, liposomesize, zeta potential, and lipid oxidation kinetics are investigated.

Overexpression or Deletion of Ergosterol Biosynthesis Genes Alters Doubling Time, Response to Stress Agents, and Drug Susceptibility in Saccharomyces cerevisiae.

ABSTRACTErgosterol (ERG) is a critical sterol in the cell membranes of fungi, and its biosynthesis is tightly regulated by 25 known enzymes along the ERG production pathway. The effects of changes in expression of each ERG biosynthesis enzyme in Saccharomyces cerevisiae were analyzed by the use of gene deletion or plasmid-borne overexpression constructs. The strains overexpressing the ERG pathway genes were examined for changes in doubling time and responses to a variety of stress agents. In addition, ERG gene overexpression strains and ERG gene deletion strains were tested for alterations in antifungal drug susceptibility. The data show that disruptions in ergosterol biosynthesis regulation can affect a diverse set of cellular processes and can cause numerous phenotypic effects. Some of the phenotypes observed include dramatic increases in doubling times, respiratory deficiencies on glycerol media, cell wall insufficiencies on Congo red media, and disrupted ion homeostasis under iron or calcium starvation conditions. Overexpression or deletion of specific enzymes in the ERG pathway causes altered susceptibilities to a variety of classes of antifungal ergosterol inhibitors, including fluconazole, fenpropimorph, lovastatin, nystatin, amphotericin B, and terbinafine. This analysis of the effect of perturbations to the ERG pathway caused by systematic overexpression of each of the ERG pathway genes contributes significantly to the understanding of the ergosterol biosynthetic pathway and its relationship to stress response and basic biological processes. The data indicate that precise regulation of ERG genes is essential for cellular homeostasis and identify several ERG genes that could be exploited in future antifungal development efforts.

Identification and Characterization of Sterol Acyltransferases Responsible for Steryl Ester Biosynthesis in Tomato.

Steryl esters (SEs) serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs) in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid. Until now, only Arabidopsis ASAT and PSAT enzymes (AtASAT1 and AtPSAT1) have been cloned and characterized in plants. Here we report the identification, cloning, and functional characterization of the tomato (Solanum lycopersicum cv. Micro-Tom) orthologs. SlPSAT1 and SlASAT1 were able to restore SE to wild type levels in the Arabidopsis psat1-2 and asat1-1 knock-out mutants, respectively. Expression of SlPSAT1 in the psat1-2 background also prevented the toxicity caused by an external supply of mevalonate and the early senescence phenotype observed in detached leaves of this mutant, whereas expression of SlASAT1 in the asat1-1 mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies using fluorescently tagged SlPSAT1 and SlASAT1 proteins revealed that SlPSAT1 localize in cytoplasmic lipid droplets (LDs) while, in contrast to the endoplasmic reticulum (ER) localization of AtASAT1, SlASAT1 resides in the plasma membrane (PM). The possibility that PM-localized SlASAT1 may act catalytically in trans on their sterol substrates, which are presumably embedded in the ER membrane, is discussed. The widespread expression of SlPSAT1 and SlASAT1 genes in different tomato organs together with their moderate transcriptional response to several stresses suggests a dual role of SlPSAT1 and SlASAT1 in tomato plant and fruit development and the adaptive responses to stress. Overall, this study contributes to enlarge the current knowledge on plant sterol acyltransferases and set the basis for further studies aimed at understanding the role of SE metabolism in tomato plant growth and development.

Cholesterol-Induced Membrane Organization Promotes Influenza Virus Binding

The influenza virus attaches to a cell surface by binding sialic-acid containing receptors with the viral ligand hemagglutinin. Stable viral binding requires more than one viral ligand- host receptor interaction to simultaneously occur. Thus, binding can be modulated by the spatial distribution of target receptors in the host membrane. We show that membrane sterols critically control viral attachment and propose that sterols alter the nanoscale clustering of viral receptors to facilitate binding. Using single virus fluorescence microscopy, we demonstrated that viral binding is dependent on the cholesterol content of the target membrane. Fluorescently labeled viral particles preferentially bound synthetic membranes supplemented with increasing amounts of cholesterol (0-40 mol%). Other sterols exhibited a similar effect on binding, independent of their ability to support liquid-liquid phase separation. To develop a molecular explanation for cholesterol's effect, we ran a series of course grained molecular dynamics simulations of lipid bilayers containing the viral receptor, disialoganglioside GD1a. While simulated GD1a molecules self-associated independent of cholesterol, the dissociation rate between pairs of GD1a molecules was a function of bilayer cholesterol concentration. Additionally, cholesterol increased the order parameter of simulated GD1a lipid tails. We suggest that, by preordering the viral receptor in its monomeric state, cholesterol lowers the entropic penalty of receptor association. This in turn promotes the formation of GD1a multimers and increases the influenza virus binding avidity of the lipid bilayer. Our findings assign a critical role to the host cellular membrane in viral infectivity and reveal sterol-dependent membrane organization not associated with phase separation.

Determining the Target of Membrane Sterols on the Gating of Voltage-Gated Potassium Channels using Voltage-Clamp Fluorometry

Membrane lipids can affect the gating of voltage sensitive ion channels through different non-specific and specific mechanisms. It has been shown that cholesterol and 7-dehydrocholesterol (7DHC) have remarkable effects on the gating of Kv1.3 (shift in voltage-dependency of activation and a slower rate of activation) but it is not clear whether these effects are mediated by the actions through the voltage sensor domain (VSD) or the pore domain (PD). Our aim was to investigate whether the major target of the action of these membrane sterols is the VSD, PD or the coupling between these two domains. To test the specificity of the effect we carried out our measurements on channels with differing structures and gating mechanisms: Kv1.3 and the non-domain-swapped Kv10.1. Current recordings were performed with VCF technique using a Xenopus laevis oocyte expression system. To monitor the movement of the VSDs of the channels the specific cysteine residues on the S3-S4 extracellular linker were labeled with MTS-TAMRA. By electrophysiological measurements we determined typical current parameters of the channels and F-V curves from the fluorescent signal in control and sterol-loaded cells. In the oocyte expression system we were able to successfully reproduce the shift in voltage-dependency of activation and a slower rate of activation in the case of Kv1.3 and as a novel finding we obtained similar results with Kv10.1. We found no voltage shifts in the F-V curves paralleling the G-V shift in either ion channel, only the slopes of these curves were decreased in both cases. These results suggest that cholesterol and 7-DHC exert their effects by acting directly on the PD and/or the coupling mechanism, instead of influencing the VSD. Support: KTIA_NAP_13-2-2015-0009

Impaired sperm function in infertile men relies on the membrane sterol pattern.

Membrane cholesterol removal appears a key step for the gain of fertility potential during sperm maturation. However, the membrane sterol pattern in sperm cells from infertile patients, with impaired sperm parameters, has been poorly investigated. To elucidate a causative link between sperm membrane composition in male fertility, here we have investigated the levels of cholesterol and its oxidized derivatives 7β-hydroxycholesterol and 7-keto-cholesterol in sixteen infertile patients with oligo-asthenozoospermia and 16 normozoospermic (N) fertile subjects. Furthermore, ten of 16N fertile subjects agreed to receive a defined testicular thermal challenge by adhering to a programme of sauna sessions for 1month. Semen samples were obtained from each of the participants, and sperm parameters were assessed according to the World Health Organization criteria. Sperm levels of cholesterol, 7β-hydroxycholesterol and 7-keto-cholesterol were quantified by ultra-pressure liquid chromatography mass spectrometry. The results showed that oligo-asthenozoospermia patients had a huge amount of cholesterol content compared with fertile subjects (12.40±6.05μg/106 cells vs. 0.45±0.28μg/106 cells, p<0.001, N and oligo-asthenozoospermia, respectively). Also, oxidized derivatives were significantly higher in oligo-asthenozoospermia patients (7β-hydroxycholesterol: 1.96±1.03ng/106 cells vs. 0.075±0.05ng/106 cells, p<0.001 and 7-keto-cholesterol: 1.11±0.72ng/106 cells vs. 0.005±0.003ng/106 cells, p<0.001). Moreover, sauna exposure, in parallel with a progressive worsening of sperm motility parameters, was associated with a reversible increase in sperm cholesterol after the third and fourth week of treatment, whilst 7β-hydroxycholesterol and 7-keto-cholesterol levels showed an earlier enhancement starting from the second week. Our data show for the first time in humans a strong difference in the cholesterol and its oxidized derivatives of infertile and fertile subjects. These findings suggest a strict biochemical link relating testis function, sperm membrane status and male fertility potential.

Dark conditions enhance aluminum tolerance in several rice cultivars via multiple modulations of membrane sterols.

Aluminum-sensitive rice (Oryza sativa L.) cultivars showed increased Al tolerance under dark conditions, because less Al accumulated in the root tips (1 cm) under dark than under light conditions. Under dark conditions, the root tip concentration of total sterols, which generally reduce plasma membrane permeabilization, was higher in the most Al-sensitive japonica cultivar, Koshihikari (Ko), than in the most Al-tolerant cultivar, Rikuu-132 (R132), but the phospholipid content did not differ between the two. The Al treatment increased the proportion of stigmasterol (which has no ability to reduce membrane permeabilization) out of total sterols similarly in both cultivars under light conditions, but it decreased more in Ko under dark conditions. The carotenoid content in the root tip of Al-treated Ko was significantly lower under dark than under light conditions, indicating that isopentenyl diphosphate transport from the cytosol to plastids was decreased under dark conditions. HMG2 and HMG3 (encoding the key sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase) transcript levels in the root tips were enhanced under dark conditions. We suggest that the following mechanisms contribute to the increase in Al tolerance under dark conditions: inhibition of stigmasterol formation to retain membrane integrity; greater partitioning of isopentenyl diphosphate for sterol biosynthesis; and enhanced expression of HMGs to increase sterol biosynthesis.

Structural Analogues of Lanosterol from Marine Organisms of the ClassAsteroideaas Potential Inhibitors of Human andCandida albicansLanosterol 14α-demethylases

Lanosterol 14α-demethylases (hemoproteins of the cytochrome P450(51) family) are involved in biosynthesis of different membrane sterols, including animal cholesterol, fungal ergosterol and C24-modified plant and protozoa sterols. In this study we have investigated 10 structural analogs of lanosterol isolated from echinoderms belonging to the class Asteroidea as potential ligands (competitive inhibitors) of human and Candida albicans cytochromes P450(51). The study was performed using the surface plasmon resonance method, spectrophotometric titration and enzyme assay. Among the compounds tested we found several selective ligands for human and Candida albicans cytochromes. Between selective ligands of the human lanosterol 14α-demethylase we found two novel inhibitors of this enzyme: henricioside H1and levisculoside G from Henricia derjugini. With due consideration of obtained data, we conclude that marine organisms from the class Asteroidea can be a valuable source of new lead compounds for creation of selective inhibitors of cytochromes P450(51) family with less side effects due to their selective action on these enzymes in different types of organisms.

CgMED3 Changes Membrane Sterol Composition To Help Candida glabrata Tolerate Low-pH Stress.

Candida glabrata is a promising microorganism for organic acid production. The present study aimed to investigate the role of C. glabrata Mediator complex subunit 3 (CgMed3p) in protecting C. glabrata under low-pH conditions. To this end, genes CgMED3A and CgMED3B were deleted, resulting in the double-deletion Cgmed3ABΔ strain. The final biomass and cell viability levels of Cgmed3ABΔ decreased by 64.5% and 35.8%, respectively, compared to the wild-type strain results at pH 2.0. In addition, lack of CgMed3ABp resulted in selective repression of a subset of genes in the lipid biosynthesis and metabolism pathways. Furthermore, C18:1, lanosterol, zymosterol, fecosterol, and ergosterol were 13.2%, 80.4%, 40.4%, 78.1%, and 70.4% less abundant, respectively, in the Cgmed3ABΔ strain. In contrast, the concentration of squalene increased by about 44.6-fold. As a result, membrane integrity, rigidity, and H+-ATPase activity in the Cgmed3ABΔ strain were reduced by 62.7%, 13.0%, and 50.3%, respectively. In contrast, overexpression of CgMED3AB increased the levels of C18:0, C18:1, and ergosterol by 113.2%, 5.9%, and 26.4%, respectively. Moreover, compared to the wild-type results, dry cell weight and pyruvate production increased, irrespective of pH buffering. These results suggest that CgMED3AB regulates membrane composition, which in turn enables cells to tolerate low-pH stress. We propose that regulation of CgMed3ABp may provide a novel strategy for enhancing low-pH tolerance and increasing organic acid production by C. glabrataIMPORTANCE The objective of this study was to investigate the role of Candida glabrata Mediator complex subunit 3 (CgMed3ABp) and its regulation of gene expression at low pH in C. glabrata We found that CgMed3ABp was critical for cellular survival and pyruvate production during low-pH stress. Measures of the levels of plasma membrane fatty acids and sterol composition indicated that CgMed3ABp could play an important role in regulating homeostasis in C. glabrata We propose that controlling membrane lipid composition may enhance the robustness of C. glabrata for the production of organic acids.

Abstract 4500: Inhibition of PERK by small molecule inhibitors enhances the response to ionizing radiation in vitro and animal tumor models

Abstract Endoplasmic reticulum (ER) is a well organized membranous network, responsible for synthesis and folding of secretory and membrane proteins, lipid and sterol biosynthesis and intracellular calcium storage. Perturbations in ER lumen caused by depletion of Ca2+ levels, hypoxia, nutrient deprivation, affect the ER homeostasis, a condition known as an ER stress. In order to cope with this stress, cells have developed an orchestrated biochemical response, termed Unfolded Protein Response (UPR). Hypoxia, an ER stress inducer, is an important and unique characteristic of the solid tumor microenvironment, promotes resistance to radiotherapy and contributes to poor patient prognosis. Cancer cells overcome these stress conditions through activation the PERK-eIF2α arm of the UPR, leading to inhibition of global protein synthesis and cell survival. We hypothesize that the use of small molecule PERK inhibitors will decrease the survival of hypoxic cells and eventually sensitize them to irradiation. In this study we used highly radioresistant melanoma and head and neck squamous cell carcinoma cell lines B16F10-ova and SQ20B, under nomroxia and hypoxia, respectively. We used two specific PERK inhibitors (PERKi) and tested for their efficacy both in vitro and in vivo. Induction of ER stress in both cell lines, either by treatment with the pharmacologic agents thapsigargin (TG) and tunicamycin (TN), or by exposure to hypoxic conditions (0.2%), was evaluated by activation of PERK and its downstream targets. Pre-treatment of stressed B16F10-ova and SQ20B cells for 2h with PERKi (1μM), caused complete inhibition of PERK-eIF2a-ATF4-CHOP arm of the UPR. Moreover, both cell lines pre-treated with PERKi presented a significant decrease in survival fraction under ER-stress induced by TG (0.5μM) as well as under hypoxia (0.2%) along with increasing doses of irradiation (IR). For the in vivo experiments, nude and C57BL/6 mice were treated with either 100mg/kg of PERKi by oral gavage, twice a day for 3weeks, or IR or a combination of PERKi and IR. Mice treated with PERKi showed a small but not significant delay of tumor growth in both backgrounds of mice. However, combined treatment with IR (12 and 15Gy) caused a significant reduction of tumor volume as well as a delayed tumor growth which was significantly more pronounced in the immunocompetent mice. Data from flow cytometry analysis of one of the the PERKi+IR treated tumors from C57BL/6 mice showed that there is a significant infiltration of CD3+ cells as well as significant higher number of CD3+CD8+ cells compared to the untreated mice or mice treated with either PERKi or IR alone. Collectively, our results show that PERKi effectively reduces the survival of hypoxic B16F10-ova and SQ20B cells and sensitize them to IR in vitro and in tumors. Further experiments will clarify whether PERKi have a dual role on reducing the hypoxic fraction and direct recruitment of the cytotoxic T-cells. Citation Format: Ioannis Verginadis, Joel Encarnación, Souvik Dey, Constantinos Koumenis. Inhibition of PERK by small molecule inhibitors enhances the response to ionizing radiation in vitro and animal tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4500. doi:10.1158/1538-7445.AM2017-4500

Trans-Cinnamic and Chlorogenic Acids Affect the Secondary Metabolic Profiles and Ergosterol Biosynthesis by Fusarium culmorum and F. graminearum Sensu Stricto.

Plant-derived compounds limiting mycotoxin contamination are currently of major interest in food and feed production. However, their potential application requires an evaluation of their effects on fungal secondary metabolism and membrane effects. In this study, different strains of Fusarium culmorum and F. graminearum sensu stricto were exposed to trans-cinnamic and chlorogenic acids on solid YES media. Fusaria produced phenolic acids, whose accumulation was lowered by exogenous phenolic compounds. In addition, fungi reduced exogenous phenolic acids, leading either to their conversion or degradation. trans-Cinnamic acid was converted to caffeic and ferulic acids, while chlorogenic acid was degraded to caffeic acid. The latter underwent further degradation to protocatechuic acid. Fungal-derived trans-cinnamic acid, as the first intermediate of the shikimate pathway, increased after chlorogenic acid treatment, presumably due to the further inhibition of the conversion of trans-cinnamic acid. Exogenous trans-cinnamic and chlorogenic acid displayed the inhibition of mycotoxin production by Fusaria, which appeared to be largely dependent on the phenolic compound and its concentration and the assayed strain. Exogenous phenolic acids showed different effects on ergosterol biosynthesis by fungi. It was found that the production of this membrane sterol was stimulated by trans-cinnamic acid, while chlorogenic acid negatively impacted ergosterol biosynthesis, suggesting that phenolic acids with stronger antifungal activities may upregulate ergosterol biosynthesis by Fusaria. This paper reports on the production of phenolic acids by Fusaria for the first time.

Heme deficiency sensitizes yeast cells to oxidative stress induced by hydroxyurea

Hydroxyurea (HU) has a long history of clinical and scientific use as an antiviral, antibacterial, and antitumor agent. It inhibits ribonucleotide reductase and reversibly arrests cells in S phase. However, high concentrations or prolonged treatment with low doses of HU can cause cell lethality. Although the cytotoxicity of HU may significantly contribute to its therapeutic effects, the underlying mechanisms remain poorly understood. We have previously shown that HU can induce cytokinesis arrest in the erg11-1 mutant of fission yeast, which has a partial defect in the biosynthesis of fungal membrane sterol ergosterol. Here, we report the identification of a new mutant in heme biosynthesis, hem13-1, that is hypersensitive to HU. We found that the HU hypersensitivity of the hem13-1 mutant is caused by oxidative stress and not by replication stress or a defect in cellular response to replication stress. The mutation is hypomorphic and causes heme deficiency, which likely sensitizes the cells to the HU-induced oxidative stress. Because the heme biosynthesis pathway is highly conserved in eukaryotes, this finding, as we show in our separate report, may help to expand the therapeutic spectrum of HU to additional pathological conditions.

Differential profiles of membrane proteins, fatty acids, and sterols associated with genetic variations in heat tolerance for a perennial grass species, hard fescue (Festuca Trachyphylla)

Cellular membrane composition controls membrane stability, affecting plant tolerance to abiotic stress. The objectives of this study were to examine differential changes in membrane constituents (sterol, fatty acid and membrane protein) in response to heat stress for a cool-season perennial grass species, hard fescue (Festuca trachyphylla) and identify major membrane constituents associated with the genetic variations in heat tolerance for two varieties contrasting in heat tolerance. Plants of heat-tolerant ‘Reliant IV’ and heat-sensitive ‘Predator’ were exposed to heat stress at 38/33°C (day/night) in growth chambers. Under heat stress, ‘Reliant IV’ showed better turf quality, greater membrane stability and higher photochemical efficiency compared to ‘Predator’. Leaf membranes of ‘Reliant IV’ showed greater increase of ethyl sterols (fucosterol, stigmasterol, sitosterol and avenasterol), and two fatty acids [oleic acid (18:1) and linoleic acid (18:2)] under heat stress compared to those for ‘Predator’. Several membrane proteins were less down-regulated or more up-regulated by heat stress in ‘Reliant IV’ than in ‘Predator’, including down-regulated proteins involved in electron transport of photosynthesis (ATP synthase subunits and cytochrome b6-f complex iron-sulfur subunit), signaling (lectin-domain containing receptor kinase A4.2), and protein modification (S-acyltransferase), and up-regulated proteins involved in carboxylation in photosynthesis (Rubisco activase beta form) and stress defense (disease resistance protein 1). These differentially accumulated sterols, fatty acids, and proteins in cellular membranes could contribute to the differential level of heat tolerance between the two varieties of hard fescues. These compounds could be used as biomarkers to select for heat-tolerant germplasm in hard fescues.

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication.IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication.

Expression of C-5 sterol desaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance.

Bioethanol is an environment friendly and renewable source of energy produced by the fermentation of agricultural raw material by a variety of microorganisms including yeast. Obtaining yeast strains that are tolerant to stresses like high levels of ethanol and high temperature is highly desirable as it reduces cost and increases yield during bioethanol production. Here, we report that heterologous expression of C-5 Sterol desaturase (FvC5SD)—an ergosterol biosynthesis enzyme from an edible mushroom Flammulina velutipes in fission yeast, not only imparts increased thermotolerance but also tolerance towards high ethanol concentration and low pH. This tolerance could be attributed to an increase of ≈1.5 fold in the level of ergosterol and oleic acid (C-18 unsaturated fatty acid) as analysed by gas chromatography- mass spectrometry. FvC5SD is a membrane localized iron binding enzyme that introduces double bond at C-5 position into the Δ7-sterol substrates to yield Δ5, 7- sterols as products. In F. velutipes, FvC5SD transcript was observed to be upregulated by ≈5 fold under low pH condition and by ≈ 9 folds and ≈5 fold at 40°C and 4°C respectively when compared to normal growth temperature of 23°C. Besides, susceptibility to cell wall inhibiting drugs like Congo red and Calcoflour white was also found to increase in FvC5SD expressing S. pombe strain. Alteration in membrane sterol and fatty acid composition could also lead to increase in susceptibility to cell wall inhibiting drugs. Thus, this study has immense industrial application and can be employed to ensure competitiveness of fermentation process.

Yeasts from Scarlet ibises (Eudocimus ruber): A focus on monitoring the antifungal susceptibility of Candida famata and closely related species.

This study aimed to identify yeasts from the gastrointestinal tract of scarlet ibises (Eudocimus ruber) and from plant material collected from the environment where they live. Then, the isolates phenotypically identified as Candida famata were submitted to molecular identification of their closely related species and evaluated for their antifungal susceptibility and possible resistance mechanisms to antifungal drugs. Cloacal swabs from 20 scarlet ibises kept in captivity at Mangal das Garças Park (Brazil), pooled stool samples (n = 20) and samples of trunks and hollow of trees (n = 20) obtained from their enclosures were collected. The samples were seeded on Sabouraud agar supplemented with chloramphenicol. The 48 recovered isolates were phenotypically identified as 15 Candida famata, 13 Candida catenulata, 2 Candida intermedia, 1 Candida lusitaniae, 2 Candida guilliermondii, 1 Candida kefyr, 1 Candida amapae, 1 Candida krusei, 8 Trichosporon spp., and 4 Rhodotorula spp. The C. famata isolates were further identified as 3 C. famata, 8 Debaryomyces nepalensis, and 4 C. palmioleophila. All C. famata and C. palmioleophila were susceptible to caspofungin and itraconazole, while one D. nepalensis was resistant to fluconazole and voriconazole. This same isolate and another D. nepalensis had lower amphotericin B susceptibility. The azole resistant strain had an increased efflux of rhodamine 6G and an alteration in the membrane sterol content, demonstrating multifactorial resistance mechanism. Finally, this research shows that scarlet ibises and their environment harbor C. famata and closely related species, including antifungal resistant isolates, emphasizing the need of monitoring the antifungal susceptibility of these yeast species.

Lipid Phase Heterogeneity and Size Dependence in Quarternary Lipid Bilayer System: A Coarse-Grained Molecular Dynamics Study

The spatial organization of phospholipids and sterols in cellular membranes is believed to play an important role in membrane function and cell activities. Both recent experimental and simulation studies have suggested various possible heterogeneous spatial distributions of lipids and cholesterol, including the formation of nanoscale lipid domains, in multi-component bilayer systems. In this study, we use coarse-grained Molecular Dynamics simulations to study the phase behavior of quarternary lipid mixtures as a function of membrane composition. We focus on quantifying the phase composition of heterogeneous membranes using model facilitated tatistical approaches, as well as identifying the nature of lipid heterogeneity by measuring signatures in observables such as partial density correlation functions, as well as local lipid density distributions and their dependence on system size.

The molecular mechanism of Nystatin action is dependent on the membrane biophysical properties and lipid composition.

Nystatin (Nys) is a pore forming broad-spectrum and efficient antifungal drug with significant toxicity in mammalian organisms. In order to develop a non-toxic and more effective Nys formulation, its molecular mechanism of action at the cell membrane needs to be better understood. It is widely accepted that Nys activity and toxicity depend on the presence and type of membrane sterols. Taking advantage of multiple biophysical methodologies, we now show that the formation and stabilization of Nys aqueous pores, which are associated with Nys cytotoxicity, occur in the absence of membrane sterols. Our results suggest that the Nys mechanism of action is driven by the presence of highly ordered membrane domains capable of stabilizing the Nys oligomers. Moreover, Nys pore formation is accompanied by strong Nys-induced membrane reorganization that depends on membrane lipid composition and seems to underlie the Nys cytotoxic effect. Accordingly, in membranes enriched in a gel-phase forming phospholipid, Nys incorporates within the phospholipid-enriched gel domains, where it forms pores able to expand the gel domains. In contrast, in membranes enriched in gel domain forming sphingolipids, Nys-induced pore formation occurs through the destabilization of the gel phase. These results show that the Nys mechanism of action is complex and not only dependent on membrane sterols, and provide further insight into the molecular details governing Nys activity and toxicity.

Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes.

The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1’s acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1’s membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1’s ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.