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Herbert Tabor - One of the best experts on this subject based on the ideXlab platform.
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Microarray studies on the genes responsive to the addition of Spermidine or spermine to a Saccharomyces cerevisiae Spermidine synthase mutant.
Yeast, 2009Co-Authors: Manas K Chattopadhyay, Weiping Chen, David Stiles, Herbert TaborAbstract:The naturally occurring polyamines putrescine, Spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well-established in vivo. In this work we have performed microarray experiments with a Spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with Spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess Spermidine (10−5M) or spermine (10−5M) compared to a control culture containing 10−8M Spermidine. 247 genes were upregulated > two-fold and 11 genes were upregulated >10-fold after Spermidine addition. Functional categorization of the genes showed induction of transport-related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two-fold, and six genes were downregulated > eight-fold after Spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for Spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.
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hypusine modification for growth is the major function of Spermidine in saccharomyces cerevisiae polyamine auxotrophs grown in limiting Spermidine
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Manas K Chattopadhyay, Myung Hee Park, Herbert TaborAbstract:Level of hypusinated eIF5A (A) and total eIF5A (B) in wild-type and different Spermidine-supplemented spe2Δ cultures. The yeast spe2Δ culture was grown in 10−8 M Spermidine and then diluted to an OD600 of 0.001 in media containing 10−6, 10−7, 10−8, and 10−9 M [3H]-Spermidine, as indicated. The wild-type culture was also grown in the presence of 5 μCi/ml [3H]-Spermidine. Cultures were harvested at an OD600 of 1 and subjected to analysis by SDS/PAGE. The migration of [3H]-Spermidine incorporated as hypusine in eIF5A, and standard yeast eIF5A were determined by fluorography and staining. Note that the [3H]-Spermidine level in wild-type cells is diluted by intracellular biosynthesis of cold Spermidine (A). Total eIF5A (hypusinated and precursor) was determined in the above cultures by Western blot analysis against rabbit antiyeast eIF5A antibody, kindly provided by Sandro Valentini (Sao Paulo State University, Universidade Estadual Paulista, Araraquara, SP, Brazil) (B). Equal loading of proteins in the gel lanes in A and B was confirmed by staining with Coomassie and ponceau-S, respectively [see supporting information (SI) Fig. 4].
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Spermidine but not spermine is essential for hypusine biosynthesis and growth in saccharomyces cerevisiae spermine is converted to Spermidine in vivo by the fms1 amine oxidase
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Manas K Chattopadhyay, Celia White Tabor, Herbert TaborAbstract:In our earlier work we showed that either Spermidine or spermine could support the growth of spe2Δ or spe3Δ polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines. In the current work, we demonstrate that Spermidine is specifically required for the growth of Saccharomyces cerevisiae. We were able to show this specificity by using a spe3Δ fms1Δ mutant that lacked both Spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to Spermidine. The polyamine requirement for the growth of this double mutant could only be satisfied by Spermidine; i.e., spermine was not effective because it cannot be oxidized to Spermidine in the absence of the FMS1 gene. We also showed that at least one of the reasons for the absolute requirement for Spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. Spermine itself cannot be used for the hypusine modification, unless it is oxidized to Spermidine by the Fms1 amine oxidase. We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein. We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.
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Spermidine biosynthesis in Saccharomyces cerevisiae : Polyaminerequirement of a null mutant of the SPE3 gene (Spermidine synthase)
Gene, 1997Co-Authors: Nobuko Hamasaki-katagiri, Celia White Tabor, Herbert TaborAbstract:Abstract The Saccharomyces cerevisiae SPE3 gene, coding for Spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian Spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3Δ, which has no Spermidine synthase activity and has an absolute requirement for Spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of Spermidine (10−8 M) or a higher concentration of spermine (10−6 M).
Anthony J Michael - One of the best experts on this subject based on the ideXlab platform.
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different polyamine pathways from bacteria have replaced eukaryotic Spermidine biosynthesis in ciliates tetrahymena thermophila and paramecium tetaurelia
Molecular Microbiology, 2015Co-Authors: Bin Li, Colin Hanfrey, Y Zhang, Katherine A Elliott, S E Ealick, Anthony J MichaelAbstract:Abstract The polyamine Spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding Spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and Spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homoSpermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and Spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homoSpermidine, suggesting it replaces Spermidine for growth. To test this concept, a budding yeast Spermidine auxotrophic strain was found to grow almost normally with homoSpermidine instead of Spermidine. Biosynthesis of Spermidine analogue aminopropylcadaverine, but not exogenously provided norSpermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the Spermidine biosynthetic pathway but retain deoxyhypusine synthase.
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alternative Spermidine biosynthetic route is critical for growth of campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota
Journal of Biological Chemistry, 2011Co-Authors: Colin Hanfrey, Bruce M Pearson, Stuart T Hazeldine, Duncan J H Gaskin, Patrick M Woster, Margaret A Phillips, Anthony J MichaelAbstract:The availability of fully sequenced bacterial genomes has revealed that many species known to synthesize the polyamine Spermidine lack the Spermidine biosynthetic enzymes S-adenosylmethionine decarboxylase and Spermidine synthase. We found that such species possess orthologues of the sym-norSpermidine biosynthetic enzymes carboxynorSpermidine dehydrogenase and carboxynorSpermidine decarboxylase. By deleting these genes in the food-borne pathogen Campylobacter jejuni, we found that the carboxynorSpermidine decarboxylase orthologue is responsible for synthesizing Spermidine and not sym-norSpermidine in vivo. In polyamine auxotrophic gene deletion strains of C. jejuni, growth is highly compromised but can be restored by exogenous sym-homoSpermidine and to a lesser extent by sym-norSpermidine. The alternative Spermidine biosynthetic pathway is present in many bacterial phyla and is the dominant Spermidine route in the human gut, stomach, and oral microbiomes, and it appears to have supplanted the S-adenosylmethionine decarboxylase/Spermidine synthase pathway in the gut microbiota. Approximately half of the gut Firmicutes species appear to be polyamine auxotrophs, but all encode the potABCD Spermidine/putrescine transporter. Orthologues encoding carboxySpermidine dehydrogenase and carboxySpermidine decarboxylase are found clustered with an array of diverse putrescine biosynthetic genes in different bacterial genomes, consistent with a role in Spermidine, rather than sym-norSpermidine biosynthesis. Due to the pervasiveness of ϵ-proteobacteria in deep sea hydrothermal vents and to the ubiquity of the alternative Spermidine biosynthetic pathway in that phylum, the carboxySpermidine route is also dominant in deep sea hydrothermal vents. The carboxySpermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative Spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds.
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a novel polyamine acyltransferase responsible for the accumulation of Spermidine conjugates in arabidopsis seed
The Plant Cell, 2009Co-Authors: Christine Fuell, Katherine A Elliott, Adrian J Parr, Lionel Hill, Paul Bailey, Shirley A Fairhurst, Cathie Martin, Anthony J MichaelAbstract:Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl Spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylSpermidine and N,N′-disinapoylSpermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding Spermidine disinapoyl transferase (SDT) and Spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a Spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl Spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has Spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.
M De Agazio - One of the best experts on this subject based on the ideXlab platform.
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dimethylthiourea a hydrogen peroxide trap partially prevents stress effects and ascorbate peroxidase increase in Spermidine treated maize roots
Plant Cell and Environment, 2001Co-Authors: Massimo Zacchini, M De AgazioAbstract:Inhibition of root growth and accumulation of putrescine caused by exogenous Spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from Spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with Spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing Spermidine-generated H2O2 by DMTU. Over-expression of APX was associated with increased APX activity in Spermidine-treated seedlings whereas the addition of DMTU to Spermidine completely prevented Spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous Spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a Spermidine catabolic product.
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Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in Spermidine‐treated maize roots
Plant Cell and Environment, 2001Co-Authors: Massimo Zacchini, M De AgazioAbstract:Inhibition of root growth and accumulation of putrescine caused by exogenous Spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from Spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with Spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing Spermidine-generated H2O2 by DMTU. Over-expression of APX was associated with increased APX activity in Spermidine-treated seedlings whereas the addition of DMTU to Spermidine completely prevented Spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous Spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a Spermidine catabolic product.
Manas K Chattopadhyay - One of the best experts on this subject based on the ideXlab platform.
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Microarray studies on the genes responsive to the addition of Spermidine or spermine to a Saccharomyces cerevisiae Spermidine synthase mutant.
Yeast, 2009Co-Authors: Manas K Chattopadhyay, Weiping Chen, David Stiles, Herbert TaborAbstract:The naturally occurring polyamines putrescine, Spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well-established in vivo. In this work we have performed microarray experiments with a Spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with Spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess Spermidine (10−5M) or spermine (10−5M) compared to a control culture containing 10−8M Spermidine. 247 genes were upregulated > two-fold and 11 genes were upregulated >10-fold after Spermidine addition. Functional categorization of the genes showed induction of transport-related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two-fold, and six genes were downregulated > eight-fold after Spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for Spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.
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hypusine modification for growth is the major function of Spermidine in saccharomyces cerevisiae polyamine auxotrophs grown in limiting Spermidine
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Manas K Chattopadhyay, Myung Hee Park, Herbert TaborAbstract:Level of hypusinated eIF5A (A) and total eIF5A (B) in wild-type and different Spermidine-supplemented spe2Δ cultures. The yeast spe2Δ culture was grown in 10−8 M Spermidine and then diluted to an OD600 of 0.001 in media containing 10−6, 10−7, 10−8, and 10−9 M [3H]-Spermidine, as indicated. The wild-type culture was also grown in the presence of 5 μCi/ml [3H]-Spermidine. Cultures were harvested at an OD600 of 1 and subjected to analysis by SDS/PAGE. The migration of [3H]-Spermidine incorporated as hypusine in eIF5A, and standard yeast eIF5A were determined by fluorography and staining. Note that the [3H]-Spermidine level in wild-type cells is diluted by intracellular biosynthesis of cold Spermidine (A). Total eIF5A (hypusinated and precursor) was determined in the above cultures by Western blot analysis against rabbit antiyeast eIF5A antibody, kindly provided by Sandro Valentini (Sao Paulo State University, Universidade Estadual Paulista, Araraquara, SP, Brazil) (B). Equal loading of proteins in the gel lanes in A and B was confirmed by staining with Coomassie and ponceau-S, respectively [see supporting information (SI) Fig. 4].
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Spermidine but not spermine is essential for hypusine biosynthesis and growth in saccharomyces cerevisiae spermine is converted to Spermidine in vivo by the fms1 amine oxidase
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Manas K Chattopadhyay, Celia White Tabor, Herbert TaborAbstract:In our earlier work we showed that either Spermidine or spermine could support the growth of spe2Δ or spe3Δ polyamine-requiring mutants, but it was unclear whether the cells had a specific requirement for either of these amines. In the current work, we demonstrate that Spermidine is specifically required for the growth of Saccharomyces cerevisiae. We were able to show this specificity by using a spe3Δ fms1Δ mutant that lacked both Spermidine synthase and the FMS1-encoded amine oxidase that oxidizes spermine to Spermidine. The polyamine requirement for the growth of this double mutant could only be satisfied by Spermidine; i.e., spermine was not effective because it cannot be oxidized to Spermidine in the absence of the FMS1 gene. We also showed that at least one of the reasons for the absolute requirement for Spermidine for growth is the specificity of its function as a necessary substrate for the hypusine modification of eIF5A. Spermine itself cannot be used for the hypusine modification, unless it is oxidized to Spermidine by the Fms1 amine oxidase. We have quantified the conversion of spermine in vivo and have shown that this conversion is markedly increased in a strain overexpressing the Fms1 protein. We have also shown this conversion in enzymatic studies by using the purified amine oxidase from yeast.
Massimo Zacchini - One of the best experts on this subject based on the ideXlab platform.
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dimethylthiourea a hydrogen peroxide trap partially prevents stress effects and ascorbate peroxidase increase in Spermidine treated maize roots
Plant Cell and Environment, 2001Co-Authors: Massimo Zacchini, M De AgazioAbstract:Inhibition of root growth and accumulation of putrescine caused by exogenous Spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from Spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with Spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing Spermidine-generated H2O2 by DMTU. Over-expression of APX was associated with increased APX activity in Spermidine-treated seedlings whereas the addition of DMTU to Spermidine completely prevented Spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous Spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a Spermidine catabolic product.
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Dimethylthiourea, a hydrogen peroxide trap, partially prevents stress effects and ascorbate peroxidase increase in Spermidine‐treated maize roots
Plant Cell and Environment, 2001Co-Authors: Massimo Zacchini, M De AgazioAbstract:Inhibition of root growth and accumulation of putrescine caused by exogenous Spermidine in roots of maize seedlings (Zea mays L., cv Samodek) were partially prevented by a concomitant treatment with dimethylthiourea (DMTU), that traps H2O2 produced from Spermidine by the activity of polyamine oxidase (PAO) in the apoplast. Treatment with Spermidine caused a strong increase of ascorbate peroxidase (APX) gene expression, that was induced to a lesser extent by removing Spermidine-generated H2O2 by DMTU. Over-expression of APX was associated with increased APX activity in Spermidine-treated seedlings whereas the addition of DMTU to Spermidine completely prevented Spermidine-induced increase of APX activity. Thus, DMTU permitted the demonstration that exogenous Spermidine supplied to maize seedlings causes an oxidative stress and induces APX, a key enzyme of the antioxidant defence mechanism, through H2O2, a Spermidine catabolic product.