In this study we analyzed how exogenous glucose levels affect enzymatic and non-enzymatic antioxi... more In this study we analyzed how exogenous glucose levels affect enzymatic and non-enzymatic antioxidant defense systems and markers of oxidative stress in cells of the methylotrophic yeast Ogataea polymorpha producing recombinant human α-synuclein, implicated in pathogenesis of neurodegenerative Parkinson's disease (PD). We found that glucose depletion up-induced activity of antioxidant enzymes superoxide dismutase, and catalase, and increased content of reduced and oxidized glutathione in the cells cultivated in the medium with 0,1% glucose, as compared to physiological growth condition (1% glucose-containing medium). In addition, low glucose concentration in the medium upregulated content of proteins carbonyl groups and of products of lipid peroxidation. Notably, the shift in the equilibrium toward pro-oxidant changes was similar for recombinant α-synuclein producer and parental wild-type strain. Thus, glucose limitation leads to the overproduction of reactive oxygen species in the methylotrophic yeast cells independently of the recombinant human α-synuclein production.
Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic po... more Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-ad...
Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of P... more Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of Parkinson’s disease. The most important factor influencing the process of α-synuclein aggregation is the level of this protein in neurons which depends on the balance between its synthesis, degradation and secretion. Under certain conditions, when α-synuclein is synthesized at a high level, monomers of this protein can aggregate on the lipid membrane, which leads to the formation of amyloids, fibrils and protofibrils unable to perform their physiological functions. Since it is virtually impossible to study the properties of α-synuclein in vivo, researchers are actively using model biological systems (single-celled microorganisms, human cell lines, animal models etc.). The aim of this study was to construct a recombinant strain of Saccharomyces cerevisiae with controlled expression of human α-synuclein to study the regulation and properties of this protein and for screening for new low mol...
Results. In this study, we analyzed the effects of exogenous spermidine in different concentratio... more Results. In this study, we analyzed the effects of exogenous spermidine in different concentrations on the enzymatic (superoxide dismutase and catalase activity) and nonenzymatic (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.
Abnormal oligomerisation and aggregation of the protein called alpha-synuclein (α-syn) are the ke... more 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.
It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arg... more It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities. k e y w o r d s: arginase, canavanine, murine leukemia, animal model.
A cold sensitive (cs) suppressor mutant was isolated from the H. polymorpha рехб strain defective... more A cold sensitive (cs) suppressor mutant was isolated from the H. polymorpha рехб strain defective in peroxisome biogenesis. The restored cs рехб 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 рехб strain. The isolated mutation has no effect on the peroxisome degradation in H. polymorpha.
Для генно-інженерного конструювання надпродуцента глутатіонзалежної формальдегіддегідро генази (Ф... more Для генно-інженерного конструювання надпродуцента глутатіонзалежної формальдегіддегідро генази (ФдДГ) обрано термотолерантні метилотрофні дріжджі Н. polymorpha NCYC 495 (leul-l). Ген FLD1 з власним промотором введено у плазміду інтегративного типу pYTl, яка містила ген LEU2 Saccharomyces cerevisiae, з подальшим включенням цього гена у геном штаму-реципіента leul-J. Здійснено селекцію інтегративних трансформантів за ознаками прототрофності по лейцину та резистентності до підвищених концентрацій формальдегіду (до 15 мМ) у ростовому середовищі. Визначено оптимальні умови культивування штамів для досягнення максимального рівня синтезу ФдДГ. Відібрано кращий трансформант як перспективний продуцент ФдДГ з активністю до 4 мкмольхв' 1 мг' 1 білка у безклітинному екстракті.
Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane... more Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAs), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. however, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35Δ mutant was not affected in macropexophagy, cytoplasm-tovacuole targeting or general autophagy. however, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MiPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral eR to the nuclear envelope was required to organize a peri-nuclear structure (PNs), the site of Atg35 colocalization during micropexophagy. in summary, Atg35 is a new Atg protein that relocates to the PNs and specifically regulates MiPA formation during micropexophagy.
Arginine deprivation has been recently suggested as a therapeutic approach against difficult to c... more Arginine deprivation has been recently suggested as a therapeutic approach against difficult to cure blood cancers. Herein, we investigated for the first time the combined effect of exogenous nitric oxide (NO) donor and recombinant human arginase (rhARG) as arginine-depleting agent on viability of several human leukemic cell lines and normal peripheral blood lymphocytes (PBL). We found that exogenous NO donor, sodium nitroprusside (SNP), at physiologically compatible dose did not counteract but augmented rhARG-mediated pro-apoptotic effect of arginine depletion in leukemic cells but not in resting lymphocytes. Thus, we hypothesize that NO deficiency resulting from arginine deprivation is not the primary cause of high leukemic cells sensitivity to the action of rhARG. The results of this study further support the notion that not arginine catabolism but other cell response mechanisms must be involved in determining cell fate upon arginine restriction. SNP or alternative NO donors can be proposed as components of metabolic anti-leukemia therapy based on arginine deprivation.
Background: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting ye... more Background: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve hightemperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Results: Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30-40 times more ethanol than is produced by the wild-type strain.
Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia l... more Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.
Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient... more Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We dir...
Escherichia coli strain BL21(DE3)/pET42a/ASS-an efficient producer of recombinant human argininos... more 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, a-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.
In this study we analyzed how exogenous glucose levels affect enzymatic and non-enzymatic antioxi... more In this study we analyzed how exogenous glucose levels affect enzymatic and non-enzymatic antioxidant defense systems and markers of oxidative stress in cells of the methylotrophic yeast Ogataea polymorpha producing recombinant human α-synuclein, implicated in pathogenesis of neurodegenerative Parkinson's disease (PD). We found that glucose depletion up-induced activity of antioxidant enzymes superoxide dismutase, and catalase, and increased content of reduced and oxidized glutathione in the cells cultivated in the medium with 0,1% glucose, as compared to physiological growth condition (1% glucose-containing medium). In addition, low glucose concentration in the medium upregulated content of proteins carbonyl groups and of products of lipid peroxidation. Notably, the shift in the equilibrium toward pro-oxidant changes was similar for recombinant α-synuclein producer and parental wild-type strain. Thus, glucose limitation leads to the overproduction of reactive oxygen species in the methylotrophic yeast cells independently of the recombinant human α-synuclein production.
Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic po... more Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-ad...
Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of P... more Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of Parkinson’s disease. The most important factor influencing the process of α-synuclein aggregation is the level of this protein in neurons which depends on the balance between its synthesis, degradation and secretion. Under certain conditions, when α-synuclein is synthesized at a high level, monomers of this protein can aggregate on the lipid membrane, which leads to the formation of amyloids, fibrils and protofibrils unable to perform their physiological functions. Since it is virtually impossible to study the properties of α-synuclein in vivo, researchers are actively using model biological systems (single-celled microorganisms, human cell lines, animal models etc.). The aim of this study was to construct a recombinant strain of Saccharomyces cerevisiae with controlled expression of human α-synuclein to study the regulation and properties of this protein and for screening for new low mol...
Results. In this study, we analyzed the effects of exogenous spermidine in different concentratio... more Results. In this study, we analyzed the effects of exogenous spermidine in different concentrations on the enzymatic (superoxide dismutase and catalase activity) and nonenzymatic (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.
Abnormal oligomerisation and aggregation of the protein called alpha-synuclein (α-syn) are the ke... more 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.
It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arg... more It was previously demonstrated in in vitro experiments that canavanine (Cav), a natural toxic arginine analogue of plant origin, is a promising candidate for augmenting the antineoplastic effects of arginine starvation. We demonstrated herein that recombinant human arginase, an arginine degrading enzyme, abrogated growth and significantly increased Cav cytotoxicity toward cultured L1210 murine leukemic cells. Cav co-treatment further reduced cells viability in a time-dependent manner and significantly promoted apoptosis induction. In the pilot study we also evaluated for the first time the potential toxicity of the combined arginine deprivation and Cav treatment in healthy mice. administration of Cav alone or in combination with pegylated cobalt-containing human arginase (Co-hARG) did not evoke any apparent toxic effects in these animals, with no significant behavioural and survival changes after several weeks of the treatment. The therapeutic effects of the combination of Co-hARG and Cav were provisionally evaluated on the highly aggressive murine L1210 leukemia, which is semi-sensitive to arginine deprivation as a monotreatment. Combination of two drugs did not result in significant prolongation of the survival of leukemia-bearing mice. thus, we have shown that the proposed combinational treatment is rather non-toxic for the animals. It has to be further evaluated in animal studies with alternative tumor models and/or drug doses and treatment modalities. k e y w o r d s: arginase, canavanine, murine leukemia, animal model.
A cold sensitive (cs) suppressor mutant was isolated from the H. polymorpha рехб strain defective... more A cold sensitive (cs) suppressor mutant was isolated from the H. polymorpha рехб strain defective in peroxisome biogenesis. The restored cs рехб 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 рехб strain. The isolated mutation has no effect on the peroxisome degradation in H. polymorpha.
Для генно-інженерного конструювання надпродуцента глутатіонзалежної формальдегіддегідро генази (Ф... more Для генно-інженерного конструювання надпродуцента глутатіонзалежної формальдегіддегідро генази (ФдДГ) обрано термотолерантні метилотрофні дріжджі Н. polymorpha NCYC 495 (leul-l). Ген FLD1 з власним промотором введено у плазміду інтегративного типу pYTl, яка містила ген LEU2 Saccharomyces cerevisiae, з подальшим включенням цього гена у геном штаму-реципіента leul-J. Здійснено селекцію інтегративних трансформантів за ознаками прототрофності по лейцину та резистентності до підвищених концентрацій формальдегіду (до 15 мМ) у ростовому середовищі. Визначено оптимальні умови культивування штамів для досягнення максимального рівня синтезу ФдДГ. Відібрано кращий трансформант як перспективний продуцент ФдДГ з активністю до 4 мкмольхв' 1 мг' 1 білка у безклітинному екстракті.
Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane... more Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAs), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. however, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35Δ mutant was not affected in macropexophagy, cytoplasm-tovacuole targeting or general autophagy. however, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MiPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral eR to the nuclear envelope was required to organize a peri-nuclear structure (PNs), the site of Atg35 colocalization during micropexophagy. in summary, Atg35 is a new Atg protein that relocates to the PNs and specifically regulates MiPA formation during micropexophagy.
Arginine deprivation has been recently suggested as a therapeutic approach against difficult to c... more Arginine deprivation has been recently suggested as a therapeutic approach against difficult to cure blood cancers. Herein, we investigated for the first time the combined effect of exogenous nitric oxide (NO) donor and recombinant human arginase (rhARG) as arginine-depleting agent on viability of several human leukemic cell lines and normal peripheral blood lymphocytes (PBL). We found that exogenous NO donor, sodium nitroprusside (SNP), at physiologically compatible dose did not counteract but augmented rhARG-mediated pro-apoptotic effect of arginine depletion in leukemic cells but not in resting lymphocytes. Thus, we hypothesize that NO deficiency resulting from arginine deprivation is not the primary cause of high leukemic cells sensitivity to the action of rhARG. The results of this study further support the notion that not arginine catabolism but other cell response mechanisms must be involved in determining cell fate upon arginine restriction. SNP or alternative NO donors can be proposed as components of metabolic anti-leukemia therapy based on arginine deprivation.
Background: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting ye... more Background: Ogataea (Hansenula) polymorpha is one of the most thermotolerant xylose-fermenting yeast species reported to date. Several metabolic engineering approaches have been successfully demonstrated to improve hightemperature alcoholic fermentation by O. polymorpha. Further improvement of ethanol production from xylose in O. polymorpha depends on the identification of bottlenecks in the xylose conversion pathway to ethanol. Results: Involvement of peroxisomal enzymes in xylose metabolism has not been described to date. Here, we found that peroxisomal transketolase (known also as dihydroxyacetone synthase) and peroxisomal transaldolase (enzyme with unknown function) in the thermotolerant methylotrophic yeast, Ogataea (Hansenula) polymorpha, are required for xylose alcoholic fermentation, but not for growth on this pentose sugar. Mutants with knockout of DAS1 and TAL2 coding for peroxisomal transketolase and peroxisomal transaldolase, respectively, normally grow on xylose. However, these mutants were found to be unable to support ethanol production. The O. polymorpha mutant with the TAL1 knockout (coding for cytosolic transaldolase) normally grew on glucose and did not grow on xylose; this defect was rescued by overexpression of TAL2. The conditional mutant, pYNR1-TKL1, that expresses the cytosolic transketolase gene under control of the ammonium repressible nitrate reductase promoter did not grow on xylose and grew poorly on glucose media supplemented with ammonium. Overexpression of DAS1 only partially restored the defects displayed by the pYNR1-TKL1 mutant. The mutants defective in peroxisome biogenesis, pex3Δ and pex6Δ, showed normal growth on xylose, but were unable to ferment this sugar. Moreover, the pex3Δ mutant of the non-methylotrophic yeast, Scheffersomyces (Pichia) stipitis, normally grows on and ferments xylose. Separate overexpression or co-overexpression of DAS1 and TAL2 in the wild-type strain increased ethanol synthesis from xylose 2 to 4 times with no effect on the alcoholic fermentation of glucose. Overexpression of TKL1 and TAL1 also elevated ethanol production from xylose. Finally, co-overexpression of DAS1 and TAL2 in the best previously isolated O. polymorpha xylose to ethanol producer led to increase in ethanol accumulation up to 16.5 g/L at 45 °C; or 30-40 times more ethanol than is produced by the wild-type strain.
Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia l... more Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.
Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient... more Glioblastomas are the most frequent and aggressive form of primary brain tumors with no efficient cure. However, they often exhibit specific metabolic shifts that include deficiency in the biosynthesis of and dependence on certain exogenous amino acids. Here, we evaluated, in vitro, a novel combinatory antiglioblastoma approach based on arginine deprivation and canavanine, an arginine analogue of plant origin, using two human glioblastoma cell models, U251MG and U87MG. The combinatory treatment profoundly affected cell viability, morphology, motility and adhesion, destabilizing the cytoskeleton and mitochondrial network, and induced apoptotic cell death. Importantly, the effects were selective toward glioblastoma cells, as they were not pronounced for primary rat glial cells. At the molecular level, canavanine inhibited prosurvival kinases such as FAK, Akt and AMPK. Its effects on protein synthesis and stress response pathways were more complex and dependent on exposure time. We dir...
Escherichia coli strain BL21(DE3)/pET42a/ASS-an efficient producer of recombinant human argininos... more 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, a-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.
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