Expression of fatty-acid-modifying enzymes in the halotolerant black yeast Aureobasidium pullulans (de Bary) G. Arnaud under salt stress.

Multiple tolerance to stressful environmental conditions of the black, yeast-like fungus Aureobasidium pullulans is achieved through different adaptations, among which there is the restructuring of the lipid composition of their membranes. Here, we describe three novel genes encoding fatty-acid-modifying enzymes in A. pullulans, along with the levels of their mRNAs under different salinity conditions. High levels of Delta(9)-desaturase and Delta(12)-desaturase mRNAs were seen at high salinities, which were consistent with an increased desaturation of the fatty acids in the cell membranes. Elevated levels of elongase mRNA were also detected. Surprisingly, increases in the levels of these mRNAs were also seen following hypo-osmotic shock, while hyperosmotic shock had exactly the opposite effect, demonstrating that data that are obtained from up-shift and down-shift salinity studies should be interpreted with caution.

HMG-CoA reductase is regulated by environmental salinity and its activity is essential for halotolerance in halophilic fungi.

The activity and level of HMG-CoA reductase (HMGR) were addressed in halophilic fungi isolated from solar saltpans. Representative fungi belonging to the orders Dothideales, Eurotiales and Wallemiales have a specific pattern of HMGR regulation, which differs from salt-sensitive and moderately salt-tolerant yeasts. In all of the halophilic fungi studied, HMGR amounts and activities were the lowest at optimal growth salinity and increased under hyposaline and hypersaline conditions. This profile paralleled isoprenylation of cellular proteins in H. werneckii. Inhibition of HMGR in vivo by lovastatin impaired the halotolerant character. HMGR may thus serve as an important molecular marker of halotolerance.

Adaptation of extremely halotolerant black yeast Hortaea werneckii to increased osmolarity: a molecular perspective at a glance.

Halophilic adaptations have been studied almost exclusively on prokaryotic microorganisms. Discovery of the black yeast Hortaea werneckii as the dominant fungal species in hypersaline waters enabled the introduction of a new model organism to study the mechanisms of salt tolerance in eukaryotes. Its strategies of cellular osmotic adaptations on the physiological and molecular level revealed novel, intricate mechanisms to combat fluctuating salinity. H. werneckii is an extremely halotolerant eukaryotic microorganism and thus a promising source of transgenes for osmotolerance improvement of industrially important yeasts, as well as in crops.

Nef increases infectivity of HIV via lipid rafts.

Lipid rafts, also known as detergent-resistant membranes (DRM), are microdomains in the plasma membrane enriched in sphingolipids and cholesterol (reviewed in [1, 2]). Human immunodeficiency virus 1 (HIV) buds via lipid rafts [3, 4]. However, the targeting of viral structural components to DRM and its consequences for viral replication are not understood. Moreover, the negative factor Nef from HIV increases viral infectivity (reviewed in [5, 6]). With no apparent differences in structural components and morphology between wild-type and DeltaNef virons, the latter viruses display less efficient reverse transcription in target cells. As Nef is expressed abundantly early in the viral replicative cycle [7], we hypothesized that Nef could affect viral morphogenesis and budding to render viruses more infectious. In this report, we demonstrated first that Nef increases viral budding from lipid rafts. Second, in the presence of Nef, viral envelopes contain more ganglioside (GM1), which is a major component of lipid rafts. This finding correlated directly with the increased infectivity of HIV. Finally, the depletion of exogenous and endogenous cholesterol biochemically and genetically, which disrupted lipid rafts, decreased viral infectivity only in the presence of Nef. Importantly, HIV lacking the nef gene remained unaffected by these manipulations. We conclude that lipids in virions are essential for viral infectivity. Thus, HIV becomes more infectious when it buds from lipid rafts, and Nef plays a major role in this process.

Down-regulation of mammalian 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with highly purified liposomal cholesterol.

Chinese hamster ovary-215 cells (CHO-215) cannot synthesize C27 and C28 sterols because of a defect in the reaction that decarboxylates 4-carboxysterols [Plemenitas, A., Havel, C.M. & Watson, J.A. (1990) J. Biol. Chem. 265, 17012-17017]. Thus, CHO-215 cell growth is dependent on an exogenous metabolically functional source of cholesterol. We used CHO-215 cells to (a) determine whether highly purified (> 99.5%) cholesterol, in egg lecithin liposomes, could down-regulate derepressed 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and if so (b) determine whether the loss in reductase catalytic activity correlated kinetically with the synthesis and accumulation of detectable oxycholesterol derivatives. Liposomal cholesterol (26-39 microM) supported maximum CHO-215 growth and initiated suppression of HMG-CoA reductase activity at concentrations greater than 50 microM. Maximum suppression (50-60%) of reductase activity was achieved with 181.3 microM liposomal cholesterol in 6 h. Also, regulatory concentrations of highly purified liposomal [3H]cholesterol were not converted (biologically or chemically) to detectable levels of oxy[3H]cholesterol derivatives during 3-6 h incubations. Lastly, a broad-spectrum cytochrome P450 inhibitor (miconazole) had no effect on liposomal cholesterol-mediated suppression of HMG-CoA reductase activity. These observations established that (a) highly purified cholesterol, incorporated into egg lecithin liposomes, can signal the down-regulation of derepressed mammalian cell HMG-CoA reductase activity and (b) if oxycholesterol synthesis was required for liposomal cholesterol-mediated down-regulation, the products had to be more potent than 24-, 25-, or 26-/27-hydroxycholesterol.

Salt stress affects sterol biosynthesis in the halophilic black yeast Hortaea werneckii.

Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on sterol biosynthesis in eukaryotes.

Activation of Ste20 by Nef from human immunodeficiency virus induces cytoskeletal rearrangements and downstream effector functions in Saccharomyces cerevisiae.

The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome. Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase. In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively. These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections. These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade.

CDC42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1.

BACKGROUND: The negative factor (Nef) of human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) is required for high levels of viremia and progression to AIDS. Additionally, Nef leads to cellular activation, increased viral infectivity and decreased expression of CD4 on the cell surface. Previously, we and others demonstrated that Nef associates with a cellular serine kinase (NAK) activity. Recently, it was demonstrated that NAK bears structural and functional similarity to p21-activated kinases (PAKs). RESULTS: In this study, we demonstrate that Nef not only binds to but also activates NAK via the small GTPases CDC42 and Rac1. First, the dominant-negative PAK (PAKR), via its GTPase-binding domain, and dominant-negative GTPases (CDC42Hs-N17 and Rac1-N17) block the ability of Nef to associate with and activate NAK. Second, constitutively active small GTPases (CDC42Hs-V12 and Rac1-V12) potentiate the effects of Nef. Third, interactions between Nef and NAK result in several cellular effector functions, such as activation of the serum-response pathway. And finally, PAKR, CDC42Hs-N17 and Rac1-N17 decrease levels of HIV-1 production to those of virus from which the nef gene is deleted. CONCLUSIONS: By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.

Purification and characterization of 17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) from the filamentous fungus Cochliobolus lunatus was purified in three steps, yielding a protein of an apparent molecular mass of 28 kDa. According to the obtained experimental data, the native form of the enzyme could be a dimer (60 kDa) and/or a tetramer (120 kDa). The enzyme was found to catalyse preferentially the reduction of steroid substrates using NADPH as an electron donor. Both androgens and estrogens are substrates for 17beta-HSD. Kinetic studies revealed the equilibrium ordered kinetic mechanism with NADPH as the first ligand to be bound to the enzyme followed by the addition of the substrate androstenedione. The purification and characterization of 17beta-HSD from Cochliobolus lunatus represents a step towards the elucidation of the role of this enzyme in fungal metabolism.

Steroid hormone signalling system and fungi.

Three components of the steroid hormone signalling system, 17 beta-hydroxysteroid dehydrogenase, androgen binding proteins and steroid hormone signalling molecule testosterone were determined in the filamentous fungus Cochliobolus lunatus for the first time in a fungus. Their possible role in C. lunatus is discussed in comparison with their role in mammalian steroid hormone signalling system. The results are in accordance with the hypothesis, that the elements of primordial signal transduction system should exist in present day eukaryotic microorganisms.

A hydroxymethylglutaryl-CoA reductase inhibitor synthesized by yeasts.

Screening of different yeast species showed that they are able to synthesize hydroxymethylglutaryl-CoA (HMGCoA) reductase inhibitors. Crude methanol extracts and the purified inhibitors from Pichia labacensis and Candida cariosilignicola were tested for their biological activity on the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells. Identification of the inhibitors was studied by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.

Isolation and identification of testosterone and androstenedione in the fungus Cochliobolus lunatus.

The cytosol of the filamentous fungus Cochliobolus lunatus was found to contain binding proteins for testosterone and androst-4-ene-3,17-dione. Both of these steroids were also found to be good exogenous substrates for the constitutive 17 beta-hydroxysteroid dehydrogenase, also found in this fungus. We were looking for the possible endogenous substrate. The procedures for isolation and identification of the endogenous steroid molecules in the fungus C. lunatus are described in this paper. The lipids were extracted from the cells and from the growth medium and purified by column and thin-layer chromatography. Analysis of the steroids, isolated from the cells of C. lunatus by gas chromatography and combination of gas chromatography-mass spectrometry, revealed the presence of testosterone and androst-4-ene-3,17-dione. Their structures were confirmed by comparison with the standards on the basis of chromatographic behavior and mass spectra. No such structures were found in the growth medium. Endogenous synthesis of androgens in this fungus was independently confirmed by the detection of radioactively labeled testosterone when the growing cells of C. lunatus were labeled with radioactive precursor molecule [5-3H] mevalonate.

Pleurotus fungi produce mevinolin, an inhibitor of HMG CoA reductase.

Fungi of the genus Pleurotus were shown to produce the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor mevinolin. Crude methanol extracts and the purified inhibitor from three different species, P. sapidus, P. saca and P. ostreatus, were tested using the solubilized microsomal HMGCoA reductase from Chinese hamster ovary cells. The identity of the inhibitor was also confirmed by thin layer chromatography, high pressure liquid chromatography and mass spectroscopy.

Identification of progesterone binding sites in the plasma membrane of the filamentous fungus Cochliobolus lunatus.

Plasma membrane associated binding sites for progesterone have been identified in the filamentous fungus Cochliobolus lunatus (C. lunatus). The Kd for progesterone determined by Scatchard analysis was 13.9 +/- 5.7 nM and the Bmax was 250-360 fmol/mg protein. A broad ligand specificity of these binding sites is suggested by the observation that all tested steroids, regardless of their capability to act as inducers of the 11 beta-steroid hydroxylase, competed at 250-fold excess with [3H]progesterone binding. A biological role of these plasma membrane associated steroid binding sites is nevertheless suggested since in protoplasts which were devoid of them, 11 beta-steroid hydroxylase could not be induced. Progesterone binding sites were present in the plasma membrane as well as in the cytosol and were detected in this fraction, in contrast to the plasma membrane fraction, only under special experimental conditions in respect to redox state. Kd and Bmax of cytosol binding sites were of the same order of magnitude compared to the plasma membrane progesterone binding sites. Ethisterone and 4-cholesten-3-one which cannot induce 11 beta-hydroxylase competed efficiently for plasma membrane binding sites; ethisterone, however also competed for cytosol binding sites and acted, in contrast with 4-cholesten-3-one, as antagonist in the induction of 11 beta-steroid hydroxylase in C. lunatus. On the basis of presented evidence we concluded that C. lunatus contains binding sites for steroids in the plasma membrane and in the cytosol and that both types of binding site are involved in the process of induction of enzymes which transform steroids in this fungus.

Sterol-mediated regulation of mevalonic acid synthesis. Accumulation of 4-carboxysterols as the predominant sterols synthesized in a Chinese hamster ovary cell cholesterol auxotroph (mutant 215).

Chinese hamster ovary-215 (CHO-215) mutant cells are auxotrophic for cholesterol. Berry and Chang (Berry, D. J., and Chang, T. Y. (1982) Biochemistry 21, 573-580) suggested that the metabolic lesion was at the level of 4-methyl sterol oxidation. However, the observed cellular accumulation of lanosterol was not consistent with a defect at this metabolic site. With the use of a novel Silica Sep Pak sterol separation procedure, we demonstrated that 60-80% of the acetonesoluble lipid radioactivity in [5-3H]mevalonate-labeled CHO-215 cells was incorporated into acidic sterols. 7(8),Cholesten-4 beta-methyl,4 alpha-carboxy,3 beta-ol was the dominant end product. In addition to this acidic sterol, 7(8),24-cholestadien,4 beta-methyl,4 alpha-carboxy,3 beta-ol and 7(8),24-cholestadien,4 alpha-carboxy,3 beta-ol were also isolated. Incubation of cell-free extracts with [3H]7(8)-cholesten-4 beta-methyl, 4 alpha-carboxy,3 beta-ol and pyridine nucleotides confirmed that CHO-215 4-carboxysterol decarboxylase activity was less than 1% of that for wild type cells. Thus, a correspondence between decreased 4-carboxysterol decarboxylase activity and the spectrum of accumulated sterol products by intact CHO-215 cells was demonstrated. No detectable cholesterol was synthesized by CHO-215 cells. 3H-Product accumulation studies demonstrated that 7(8),24-cholestadien, 4 beta-methyl,4 alpha-carboxy,3 beta-ol increased prior to its subsequent saturation at the delta 24 carbon. Furthermore, the steady state ratio for delta 24-saturated acidic sterols/unsaturated acidic sterols was dependent on media cholesterol source and amount. Finally, the accumulated acidic sterol(s) were not regulatory signal molecules for the modulation of 3-hydroxy-3-methyl-glutaryl coenzyme. A reductase activity in response to cholesterol availability.

11 beta-hydroxylation of steroids by Cochliobolus lunatus.

The substrate specificity of 11 beta-hydroxylase of Cochliobolus lunatus was studied and a close parallelism to the results obtained with 11 alpha-hydroxylase of Rhizopus nigricans was observed. It was found that the cell wall does not differentiate between the steroid substrates used and the absence of the cell wall increases the bioconversion.

Hydroxysteroid dehydrogenase of Cochliobolus lunatus.

Cochliobolus lunatus is known to be able to hydroxylate steroids at position 11 beta. Besides this inducible enzyme, we found a constitutive hydroxysteroid dehydrogenase activity which is strongly regioselective with the highest activity at position 17, and the best substrate was found to be androstenedione. Using different substrates, no such activities were observed at positions 3 or 11. The enzyme is membrane bound and NADH or NADPH dependent. The protoplasts of Cochliobolus lunatus show the same activity as intact cells, which means that the cell wall does not influence the reaction.