PubMed Health⌕ Search

Biomedical subjects

Antoni R Slabas

Publications and source records attributed to Antoni R Slabas.

At least 19 recordsLinked to original sources
PubMed2007

Evaluation of an in vitro model of androgen ablation and identification of the androgen responsive proteome in LNCaP cells.

Proteins responsive to androgen and anti-androgen may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-ablation therapy. These proteins represent potential diagnostic and therapeutic targets for improved management of prostate cancer. We have investigated the effect of androgen (R1881) and anti-androgen (bicalutamide) on the androgen-responsive prostate cancer LNCaP cell line using a quantitative gel-based proteomic approach. Prior to analysis, the in vitro system was evaluated for reproducibility and validated by appropriate molecular responses to treatment. Six replicate samples were independently generated and analysed by 2-D DIGE. According to strict statistical criteria, 197 spots were differentially expressed, of which we have successfully identified 165 spots corresponding to 125 distinct proteins. Following androgen supplementation, 108 spots (68 proteins) were increased and 57 spots (39 proteins) were decreased. Essentially no difference was observed between control and anti-androgen-treated samples, confirming the absence of "off-target" effects of bicalutamide. Identified proteins were involved in diverse processes including the stress response and intracellular signalling. The potential contribution to disease of these processes and identified constituent proteins are discussed. This rigorous, statistically supported study of androgen responses has provided a number of potential candidates for development as diagnostic/prognostic markers and drug targets.

Androgens↗
PubMed2006

Identification and functional expression of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals.

Seed oil from castor bean (Ricinus communis) contains high amounts of hydroxy fatty acid rich triacylglycerols (TAGs) that can serve as raw material for production of bio-based products such as nylon, cosmetics, lubricants, foams, and surfactants. Diacylglycerol acyltransferase (DGAT) catalyses the terminal reaction in the acyl-CoA dependent Kennedy pathway of triglyceride biosynthesis. There is still some debate whether there are three or four enzymes in yeast that have DGAT activity and catalyse the synthesis of TAG but of these the DGAT2 homologue Dga1 contributes in a major way to TAG biosynthesis. Here we report on the cloning of a cDNA for DGAT2 from castor bean and prove its biological activity following expression in yeast and enzymatic assays using diricinolein as the acceptor and ricinoleoyl-CoA as the donor. Previous reports of DGAT in castor have focussed on DGAT1 which has little amino acid sequence homology to DGAT2. Expressional studies demonstrate that DGAT2 is 18-fold more highly expressed in seeds than in leaves and shows temporal specific expression during seed development. In contrast, DGAT1 shows little difference in expression in seeds versus leaves. We conclude that in castor bean DGAT2 is more likely to play a major role in seed TAG biosynthesis than DGAT1.

Amino Acid Sequence↗
PubMed2006

Structural studies of fatty acyl-(acyl carrier protein) thioesters reveal a hydrophobic binding cavity that can expand to fit longer substrates.

A knowledge of the structures of acyl chain loaded species of the acyl carrier protein (ACP) as used in fatty acid biosynthesis and a range of other metabolic events, is essential for a full understanding of the molecular recognition at the heart of these processes. To date the only crystal structure of an acylated species of ACP is that of a butyryl derivative of Escherichia coli ACP. We have now determined the structures of a family of acylated E. coli ACPs of varying acyl chain length. The acyl moiety is attached via a thioester bond to a phosphopantetheine linker that is in turn bound to a serine residue in ACP. The growing acyl chain can be accommodated within a central cavity in the ACP for transport during the elongation stages of lipid synthesis through changes in the conformation of a four alpha-helix bundle. The results not only clarify the means by which a substrate of varying size and complexity is transported in the cell but also suggest a mechanism by which interacting enzymes can recognize the loaded ACP through recognition of surface features including the conformation of the phosphopantetheine linker.

Acyl Carrier Protein↗
PubMed2006

The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics.

When cells of the cyanobacterium Synechocystis sp. PCC 6803 are exposed to high temperature they perceive changes in the growth conditions and regulate the expression of genes and synthesize heat-inducible proteins as a response to the heat stress. DNA microarray analysis revealed that genes for chaperonins and proteases, such as groESL1, groEL2, htpG, hspA, and clpB1 were transiently induced after incubation of the cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that the levels of these chaperonins and proteases were elevated after incubation of cells at 44 degrees C for 60 min. These findings indicated that levels of the mRNAs and proteins of chaperonins were well correlated in the cells of Synechocystis. However, the level of elongation factors are mainly regulated at the protein level. These results indicated that acclimation to the heat-shock conditions might be governed by transcriptional and translational regulation in Synechocystis.

Acclimatization↗
PubMed2006

Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana.

De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.

Arabidopsis↗
PubMed2006

Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures.

Slow progress has been made in discovering plant genes governing the interaction of plant pathogens and their hosts using classical genetic approaches. Extensive studies employing DNA microarray techniques to identify global changes in gene expression during pathogen-host interaction have greatly enhanced discovery of genetic components regulating the plant defence response to pathogen attack. In this study, a complementary approach was used to identify changes in protein abundance during interaction of Arabidopsis cell cultures with a pathogen-derived elicitor. The soluble protein fractions were analysed by two-dimensional difference gel electrophoresis and proteins differentially expressed in response to treatment with fungal elicitor were identified via matrix-assisted laser desorption ionization-time of flight mass spectrometry. Elicitor responsive proteins included molecular chaperones, oxidative stress defence proteins, mitochondrial proteins, and enzymes of a diverse number of metabolic pathways. The findings, in combination with currently available microarray data, will form the basis of a filter to identify pivotal genes whose role in pathogen defence systems will require confirmation using gene knockout mutants.

Antioxidants↗
PubMed2006

Proteomic analysis of the heat shock response in Synechocystis PCC6803 and a thermally tolerant knockout strain lacking the histidine kinase 34 gene.

Proteomic analysis of the heat shock response of wild type and a mutant of the histidine kinase 34 gene (Deltahik34), which shows increased thermal tolerance, has been performed in the cyanobacterium Synechocystis sp. PCC6803. In vivo radioactive labelling demonstrates that major proteomic changes occur within 1 h of heat shock. 2-D DIGE and MS have been used to quantify changes in specific proteins following heat shock in the wild type and the mutant. Over 100 spots, corresponding to 65 different proteins alter following heat shock. Changes occur not only in the classical heat shock proteins but also in the protein biosynthetic machinery, amino acid biosynthetic enzymes, components of the light and dark acts of photosynthesis and energy metabolism. The Deltahik34 cells have elevated levels of heat shock proteins under both non-heat shock and heat shock conditions, in comparison to the wild type, consistent with Hik34, or a down stream component, being a negative regulator of heat shock-responsive genes.

Bacterial Proteins↗
PubMed2005

Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability.

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with beta,gamma-methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

Adenosine Triphosphate↗
PubMed2005

Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein and plays an important role in long chain fatty acid metabolism.

The industrial yeasts Candida tropicalis or Candida cloacae are able to grow on a variety of long chain alkanes and fatty acids as the sole carbon source. The complete oxidation of these substrates involves two sequential oxidative pathways: omega-oxidation, comprising the P450 alkane oxidase, a flavin-dependent membrane-bound long chain fatty alcohol oxidase [FAO] and a possible separate aldehyde oxidase [F.M. Dickinson, C. Wadforth, Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis, Biochem. J. 282 (1992) 325-331], and the beta-oxidation pathway, which utilises acylCoA substrates. We recently purified the membrane-bound long chain fatty alcohol oxidase FAO1 and confirmed it is also a c-type haemoprotein. Multiple isoforms may exist for many of these long chain fatty alcohol oxidases and the in vivo requirements for individual genes with respect to specific substrates are still being elucidated. In vitro reconstitution experiments have demonstrated that in Candida maltosa, the cytochrome P450 52A3 gene product can completely oxidise alkanes to dicarboxylic acids [U. Scheller, T. Zimmer, D. Becher, F. Schauer, W. Schunck, Oxygenation Cascade in Conversion of n-Alkanes to, -Dioic Acids Catalyzed by Cytochrome P450 52A3, J. Biol. Chem. 273 (1998) 32528-32534], potentially obviating requirements for a long chain alcohol oxidase. Here, we directly determine in vivo the role of the long chain alcohol oxidase (FAOT) in C. tropicalis, grown on a variety of substrates, followed by gene deletion. The faot double knockout has no detectable faot activity and is incapable of growth on octadecane, but it grows well on oleic acid, palmitic acid and shorter chain alkanes/fatty acids. A spontaneous mutation[s] may have occurred in the faot double gene knockout of C. tropicalis resulting in its inability to grow on oleic acid and hexadecane. The mutations demonstrate that different pathways of octadecane, hexadecane, oleic acid and palmitic acid utilisation exist in C. tropicalis.

Alcohol Oxidoreductases↗
PubMed2005

The histidine kinase Hik34 is involved in thermotolerance by regulating the expression of heat shock genes in synechocystis.

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (DeltaHik34 cells) survived incubation at 48 degrees C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in DeltaHik34 cells after incubation of cells at 42 degrees C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44 degrees C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

Bacterial Proteins↗
PubMed2005

Identification of Arabidopsis salt and osmotic stress responsive proteins using two-dimensional difference gel electrophoresis and mass spectrometry.

Arabidopsis thaliana cell suspension cultures have been used to investigate the effects of salinity and hyperosmotic stress on plant cellular proteins. We show that 200 mM NaCl and 400 mM sorbitol treatments induce extracellular medium acidification in Arabidopsis cell cultures, a typical response of plant cells to salt and hyperosmotic stress. Using (35)S-labelled amino acids, we demonstrated that NaCl causes a transient suppression of de novo protein synthesis, from which the cells recover within 4 h. Changes in the abundance of cellular proteins 6 h post NaCl and sorbitol treatments were analysed by 2-DE. Of a total of 2,949 protein spots detected on the gels, 266 showed significant changes in abundance across five independent experiments. Using MALDI-TOF MS, we identified 75 salt and sorbitol responsive spots. These fall into 10 functional categories that include H(+) transporting ATPases, signal transduction related proteins, transcription/translation related proteins, detoxifying enzymes, amino acid and purine biosynthesis related proteins, proteolytic enzymes, heat-shock proteins, carbohydrate metabolism-associated proteins and proteins with no known biological functions.

Arabidopsis↗
PubMed2005

Pathogen elicitor-induced changes in the maize extracellular matrix proteome.

The extracellular matrix is a vital compartment in plants with a prominent role in defence against pathogen attack. Using a maize cell suspension culture system and pathogen elicitors, responses to pathogen attack that are localised to the extracellular matrix were examined by a proteomic approach. Elicitor treatment of cell cultures induced a rapid change in the phosphorylation status of extracellular peroxidases, the apparent disappearance of a putative extracellular beta-N-acetylglucosamonidase, and accumulation of a secreted putative xylanase inhibitor protein. Onset of the defence response was attended by an accumulation of glyceraldehyde-3-phosphate dehydrogenase and a fragment of a putative heat shock protein. Several distinct spots of both proteins, which preferentially accumulated in cell wall protein fractions, were identified. These three novel observations, viz. (i) secretion of a new class of putative enzyme inhibitor, (ii) the apparent recruitment of classical cytosolic proteins into the cell wall and (ii) the change in phosphorylation status of extracellular matrix proteins, suggest that the extracellular matrix plays a complex role in defence. We discuss the role of the extracellular matrix in signal modulation during pathogen-induced defence responses.

Cell Wall↗
PubMed2004

Functional identification of AtFao3, a membrane bound long chain alcohol oxidase in Arabidopsis thaliana.

The Arabidopsis thalina genome database was searched for homologues of the Candida cloacae fao1 gene which encodes a membrane bound, flavin-containing, hydrogen peroxide generating, long chain alcohol oxidase. This gene has not been isolated from plants or animals. Four putative candidates were found in the database but their function has not been proven. The cDNAs for two of them were cloned by RT-PCR from Arabidopis suspension culture and one of them [AtFAO3] was overexpressed in Escherichia coli and shown to functionally express long chain alcohol oxidase activity. The protein has been solubilised and retains biological activity thereby preparing the way for crystallographic studies. This is the first functional proof identifying a long chain alcohol oxidase in higher plants.

Alcohol Oxidoreductases↗
PubMed2003

Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization.

A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH 5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis.

Arabidopsis↗
PubMed2003

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors.

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.

Arabidopsis↗
PubMed2002

Squash glycerol-3-phosphate (1)-acyltransferase. Alteration of substrate selectivity and identification of arginine and lysine residues important in catalytic activity.

Glycerol-3-phosphate 1-acyltransferase is a soluble chloroplast enzyme involved in glycerol-lipid biosynthesis associated with chilling resistance in plants (). Resistance is associated with higher selectivity for unsaturated acyl substrates over saturated ones. In vitro substrate selectivity assays performed under physiologically relevant conditions have been established that discriminate between selective and non-selective forms of the enzyme. A mutation, L261F, in the squash protein converts it from a non-selective enzyme into a selective one. The mutation lies within 10 A of the predicted acyl binding site and results in a higher K(m) for 16:0 acyl carrier protein (ACP). Site-directed mutagenesis was used to determine the importance of four residues, Arg(235), Arg(237), Lys(193), and His(194), implicated to be involved in binding of the phosphate group of glycerol 3-phosphate to the enzyme. All the proteins were highly homologous in structure to the wild type enzyme. Mutations in Arg(235), Arg(237), and Lys(193) resulted in inactive enzyme, while His(194) had reduced catalytic activity. The mutant proteins retained the ability to bind stoichiometric quantities of acyl-ACPs supporting the potential role of these residues in glycerol 3-phosphate binding.

Amino Acid Sequence↗
PubMed2002

Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.

Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and <or=12.5 pg/microg of membrane protein in developing embryos. The amount of LAT2 reaches a peak at 305 pg/microg of membrane protein 25 daf and is not expressed 20 daf or before. This is the first study to quantify these membrane-bound proteins in a plant tissue. The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds. Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different. Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E. coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot. This is probably due to differences in substrate utilization.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗