Isolation and characterization of cold sensitive pex6 mutant of the methylotrophic yeast Hansenula polymorpha

A cold sensitive (cs) suppressor mutant was isolated from the H. polymorpha pex6 strain defective in peroxisome biogenesis. The restored cs pex6 growth on methanol at a permissive temperature was associated with the presence of morphologically normal peroxisomes. The enlarged peroxisomes present at restrictive temperature failed to support methylotrophic growth in the cs pex6 strain. The isolated mutation has no effect on the peroxisome degradation in H. polymorpha.Виділено холодочутливий супресорний мутант зі штаму рех6 Н. polymorpha з пошкодженим біогенезом пероксисом. Відмовлення росту холодочутливого рех6 на метанолі при пермісивній температурі корелювало з наявністю морфологічно нормальних пероксисом. Збільшені у розмірах пероксисоми при рестриктивній температурі не здатні підтримувати метилотрофний ріст холодочутливого штаму рех6. Виділена му­тація не впливає на деградацію пероксисом Н. polymorpha.Выделен холодочувствительный супрессорный мутант на ос­нове штамма рех6 Н. polymorpha с поврежденным биогенезом пероксисом. Восстановление роста холодочувствительного рех6 на метаноле при пермиссивной температуре коррелиро­вало с наличием морфологически нормальных пероксисом. Увеличенные в размерах пероксисомы при рестриктивной темпе­ратуре не способны поддерживать метилотрофный рост холодочувствительного штамма pex6. Выделенная мутация не влияет на деградацию пероксисом Н. polymorpha

Prediction of acute multiple sclerosis relapses by transcription levels of peripheral blood cells

<p>Abstract</p> <p>Background</p> <p>The ability to predict the spatial frequency of relapses in multiple sclerosis (MS) would enable physicians to decide when to intervene more aggressively and to plan clinical trials more accurately.</p> <p>Methods</p> <p>In the current study our objective was to determine if subsets of genes can predict the time to the next acute relapse in patients with MS. Data-mining and predictive modeling tools were utilized to analyze a gene-expression dataset of 94 non-treated patients; 62 patients with definite MS and 32 patients with clinically isolated syndrome (CIS). The dataset included the expression levels of 10,594 genes and annotated sequences corresponding to 22,215 gene-transcripts that appear in the microarray.</p> <p>Results</p> <p>We designed a two stage predictor. The first stage predictor was based on the expression level of 10 genes, and predicted the time to next relapse with a resolution of 500 days (error rate 0.079, p < 0.001). If the predicted relapse was to occur in less than 500 days, a second stage predictor based on an additional different set of 9 genes was used to give a more accurate estimation of the time till the next relapse (in resolution of 50 days). The error rate of the second stage predictor was 2.3 fold lower than the error rate of random predictions (error rate = 0.35, p < 0.001). The predictors were further evaluated and found effective both for untreated MS patients and for MS patients that subsequently received immunomodulatory treatments after the initial testing (the error rate of the first level predictor was < 0.18 with p < 0.001 for all the patient groups).</p> <p>Conclusion</p> <p>We conclude that gene expression analysis is a valuable tool that can be used in clinical practice to predict future MS disease activity. Similar approach can be also useful for dealing with other autoimmune diseases that characterized by relapsing-remitting nature.</p

Selective degradation of peroxisomes in the yeast Hansenula polymorpha requires sterolglucosyltransferase Atg26

UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173) catalyses the biosynthesis of ergosterol glucoside, a minor membrane lipid present in yeasts and plants. The sterol glucosyltransferases from different yeast species, including the methylotrophic yeast Pichia pastoris, display narrow substrate specificity with respect to both activated sugar and glycosyl acceptor. We isolated the mutant of the methanol-utilizing yeast Hansenula polymorpha defective in the ATG26 gene (encoding sterol glucosyltransferase) and analysed its phenotype. Similarly to P. pastoris product of the H. polymorpha ATG26 gene was required for pexophagy, the process of selective autophagic peroxisome degradation in vacuoles. Thus, sterol glucosyltransferases in different species of methylotrophic yeasts have a similar function in pexophagic process probably related to peculiarities of peroxisome morphogenesis and regulation of their homeostasis in these yeasts

Expression of genes of putative hexose transporters in the yeast Hansenula polymorpha are differentially regulated by glucose sensors Hxs1 and Gcr1

The first and limiting step of metabolism of glucose, the most widespread carbon and energy source for majority of cells, is the transport of this sugar across cytoplasmic membrane. In the eukaryotic cells glucose controls and ensures its own effective metabolism, acting as extracellular effector, regulating on transcriptional and translational levels amount, type and activity of corresponding hexose transporters. In this study we investigated regulation of expression of genes encoding hexose transporter homologues – НрHxt1, НрHxt2, НрHxt3 and putative fructose transporter – НрFrt1 by two glucose sensors НрGcr1 and НрHxs1 in methylotrophic yeast Hansenula polymorpha. We have demonstrated that putative glucose sensor НрGcr1 is involved in repression of the gene of functional low affinity hexose transporter НрHxt1 under glucose deficient conditions and participates in repression of genes of putative high affinity glucose transporters НрHxt2 and НрHxt3 under excess of this carbon source. It was shown that glucose sensor НрHxs1 is involved in induction of НрHxt1 expression. As a result of deletion of HpGCR1, but not НрHxS1, the gene of putative high affinity fructose transporter НрFRT1 is constitutively expressed independently of glucose pre­sence and its concentration in growth medium. Therefore, regulation of hexose (glucose and fructose) transport in methylotrophic yeast H. polymorpha at the level of gene expression is a complicated system of interac­ting regulatory and transporting elements where glucose sensors НрGcr1 and НрHxs1 act as the key factors

Prooxidant-antioxidant balance in the methylotrophic yeast Ogataea polymorpha exoposed to spermidine

Background. Many molecular compounds used by eukaryotic cells as antioxidant agents can directly interact with reactive oxygen/nitrogen species. Among them are polyamines – natural substances with pronounced antioxidant properties. The main polyamines are putrescine, spermidine, spermine. Spermidine is a positively charged polyamine that is synthesized from putrescine and serves a precursor of spermine. In particular, spermidine is distinguished by its anti-aging properties. When administered to several model organisms, it prolonged their lifespan and provided resistance to stress. Spermidine also limits overproduction of reactive oxigene species and reduces age-related oxidative protein damage, which accounts for its antioxidant activity. The aim of our study was to investigate the effects of exogenous spermidine in different concentrations on the prooxidant-antioxidant balance in the methylotrophic yeast Ogataea polymorpha naturally adapted to stressful conditions such as growth on toxic methanol-containing medium and cultivation at high temperatures (42–45 °C). Materials and Methods. In this work, O. polymorpha wild-type strain was used. Yeast cells were cultivated in the mineral medium with 0.1 % glucose for creating stressful conditions. Spermidine was added in 1 and 2 mM concentrations immediately before the start of yeast cultivation. Reactive oxygen species, the level of oxidative modifications of proteins and lipids, the concentration of reduced gluthatione, superoxide dismutase and catalase activities were assayed to examine the prooxidant-antioxidant state of the yeast cells. Results. In this study, we analyzed the effects of exogenous spermidine in different concentrations on the enzymatic (superoxide dismutase and catalase activity) and non-enzymatic (reduced glutathione) antioxidant defense systems and markers of oxidative injury (products of proteins and lipids oxidation) in the methylotrophic yeast O. polymorpha starved for glucose. It was revealed that 1 mM spermidine had a protective effect on O. polymorpha cells and decreased the content of products of the oxidative modification of proteins. At the same time, the superoxide dismutase and catalase activities and content of the reduced glutathione remained almost unchanged in the cells cultivated in the glucose-depleted medium with 1 mM spermidine compared to the medium without spermidine. Adverse effects of 2 mM spermidine (increased levels of carbonyl groups of proteins, lipid peroxidation products, disregulated superoxide dismutase and catalase activities, reduced glutathione levels, growth inhibition and cells vacuolization) were observed in the exponential growth phase of the yeast culture. During a long-term cultivation, these effects diminished, and the corresponding values approximated those of the cells grown in the control medium with the low concentration of glucose without spermidine. Conclusions. The data suggest a concentration-dependent effect of spermidine on the physiology of O. polymorpha that can be used in further studies on compounds able to mitigate negative effects of the oxidative stress in this yeast and other model organisms. At 1 mM concentration, spermidine had an apparent protective effect, whereas at the elevated 2 mM concentration this polyamine exacerbated stress load in this yeast

Modeling of molecular processes underlying Parkinson’s disease in cells of methylotrophic yeast Hansenula polymorpha

Abnormal oligomerisation and aggregation of the protein called alpha-synuclein (α-syn) are the key events in the pathogenesis of Parkinson’s disease (PD). Recent discoveries revealed cellular pathways that potentially relate neurodegenerative disease (ND) to abnormal functioning of mitochondria or anomalous glucose metabolism. In this study we describe for the first time strains of the thermotolerant methylotrophic yeast Hansenula polymorpha that produce human GFP-tagged α-syn as a new model of molecular processes leading to PD. We observed that NCYC495-SNCA wild-type strain did not form visible α-syn amyloid-like aggregates but exhibited plasma membrane perforations and cytoplasm leakage. gcr1-2-SNCA mutant strain deficient in catabolite repression and glucose transport exhibited enhanced aggregation of fluorescently tagged α-syn. However, the observed differences did not result from the impaired glucose metabolism as were observed in both α-syn-producing strains grown on glycerol. Production of α-syn was detrimental for both strains and decreased their growth rate on alternative carbon sources. Our data suggests that H. polymorpha may serve as an informative new yeast model for deciphering molecular mechanisms of PD that regulate amyloid formation and degradation under the influence of various extra- and intracellular factors

Peculiarities of hexose transport and catabolite repression regulation by hexose sensors HpGcr1 and HpHxs1 in the yeast Hansenula polymorpha

In the yeasts, as in the majority of microorganisms, glucose is the main source of carbon and energy, as well as a key effector molecule involved in transcriptional regulation. Many genes are repressed in the presence of glucose, others are instead induced by glucose. In the mutants of bakers’ yeast Sacharomyces cerevisiae impaired in hexo­se transport, the proficiency of transport determins the strength of the repression signal. In the mutants of methylotrophic yeast Hansenula polymorpha with deleted glucose sensors НрGcr1 and НрHxs1, efficiency of glucose and fructose uptake and the effect of hexose transport on catabolite repression of peroxisomal alcohol oxidase was investigated. Contrary to S. cerevisiae, in the mutants ∆gcr1 and ∆hxs1 impairment glucose and fructose transport did not always corelated with the rate of catabolite repression of metabolic genes of alternative carbon sources. For instance, in the recessive gcr1-2 mutant with one amino acid substitution S85F glucose transport was less impaired relative to corresponding deletion mutant, whereas defect of glucose repression was more pronounced. Also, in the mutants ∆hxs1 and ∆gcr1, the defect of fructose transport was similar, when fructose repression defect was stronger in ∆hxs1. Therefore, specific impairment of hexose transport upon deletion of alternative hexose sensors in H. polymorpha is not an only cause that determins profiociency of hexose catabolite repression

Influence of arginine metabolites on human tumor cell viability upon arginine deprivation in vitro

In human organism arginine is necessary not only for protein synthesis but it also serves as a precursor for a number of important biologically active substances that influence cell growth and viability. These substances include such arginine metabolites as polyamines, nitric oxide, agmatine, ornithine and urea. It is known that in vitro tumor cells substantially differ in the level of sensitivity to arginine deprivation, which is reflected in the dynamics of apoptotic manifestations under these conditions. Identification of the molecular reasons of this phenomenon is important for optimization of the regimes of arginine deprivation-based anticancer enzymotherapy. One of possible reasons of the various level of sensitivity of tumor cells to arginine deprivation may reside in differences in their susceptibility to arginine catabolites withdrawal. Therefore, we aimed to investigate the effect of exogenous arginine metabolites (polyamines putrescine and spermine, as well as agmatine, nitric oxide, ornithine and urea) under arginine deprived conditions on viability and proliferative potential of two human cancer cell lines (A549 lung carcinoma and HepG2 hepatocarcinoma) that differ in the level of sensitivity to arginine depletion. It was revealed that none of the studied arginine catabolite affected cancer cell viability and proliferative potential in arginine-free medium, independently of the level of their sensitivity to arginine starvation. Thus, despite the complexity and versatility of arginine metabolic networks, the depletion of metabolites of this amino acid is not a key reason that determines differences in cell response to arginine deprivation. Therefore, identification of signaling mechanisms that underlie apoptosis induction in cancer cells upon arginine starvation needs to be further elucidated

Expression of recombinant human argininosuccinate synthetase in Escherichia coli

Escherichia coli strain BL21(DE3)/pET42a/ASS – an efficient producer of recombinant human argininosuccinate synthetase (rhASS) – was constructed, and preparations of purified rhASS were obtained using His-tag affinity chromatography. The effect of specific inhibitor, α-methyl-DL-aspartate, and nitric oxide donor, sodium nitroprusside, on the ASS specific activity was evaluated with purified rhASS protein and in mouse liver lysates. The developed expression platform is a useful tool in search for new ASS inhibitors efficient under in vitro and in vivo conditions