Adenylosuccinate synthetase and adenylosuccinate lyase from plant tissues.
Ontology highlight
ABSTRACT:
SUBMITTER: Hatch MD
PROVIDER: S-EPMC1264815 | biostudies-other | 1966 Jan
REPOSITORIES: biostudies-other
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Project description:Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family (T. brucei, T. cruzi and L. major) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L. major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNASec kinase (PSTK)-Sec-tRNASec synthase (SEPSECS) complex and the tRNASec-specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T. brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T. brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T. brucei selenophosphate synthetase in the regulation of the parasite's ER stress response.
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Project description:BACKGROUND:Isocitrate lyase (ICL) is a key enzyme in the glyoxylate cycle. In a previous study in rice, the expression of the ICL-encoding gene (OsICL) was highly induced by salt stress and its expression was enhanced in transgenic rice lines overexpressing OsCam1-1, a calmodulin (CaM)-encoding gene. CaM has been implicated in salt tolerance mechanisms in plants; however, the cellular mechanisms mediated by CaM are not clearly understood. In this study, the role of OsICL in plant salt tolerance mechanisms and the possible involvement of CaM were investigated using transgenic plants expressing OsICL or OsCam1-1. RESULTS:OsICL was highly expressed in senesced leaf and significantly induced by salt stress in three OsCam1-1 overexpressing transgenic rice lines as well as in wild type (WT). In WT young leaf, although OsICL expression was not affected by salt stress, all three transgenic lines exhibited highly induced expression levels. In Arabidopsis, salt stress had negative effects on germination and seedling growth of the AtICL knockout mutant (Aticl mutant). To examine the roles of OsICL we generated the following transgenic Arabidopsis lines: the Aticl mutant expressing OsICL driven by the native AtICL promoter, the Aticl mutant overexpressing OsICL driven by the 35SCaMV promoter, and WT overexpressing OsICL driven by the 35SCaMV promoter. Under salt stress, the germination rate and seedling fresh and dry weights of the OsICL-expressing lines were higher than those of the Aticl mutant, and the two lines with the icl mutant background were similar to the WT. The Fv/Fm and temperature of rosette leaves in the OsICL-expressing lines were less affected by salt stress than they were in the Aticl mutant. Finally, glucose and fructose contents of the Aticl mutant under salt stress were highest, whereas those of OsICL-expressing lines were similar to or lower than those of the WT. CONCLUSIONS:OsICL, a salt-responsive gene, was characterized in the transgenic Arabidopsis lines, revealing that OsICL expression could revert the salt sensitivity phenotypes of the Aticl knockout mutant. This work provides novel evidence that supports the role of ICL in plant salt tolerance through the glyoxylate cycle and the possible involvement of OsCam1-1 in regulating its transcription.
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Project description:Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.
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Project description:A method for mapping tissue permeability based on time-dependent diffusion measurements is presented. A pulsed field gradient sequence to measure the diffusion encoding time dependence of the diffusion coefficients based on the detection of stimulated spin echoes to enable long diffusion times is combined with a turbo spin echo sequence for fast NMR imaging (MRI). A fitting function is suggested to describe the time dependence of the apparent diffusion constant in porous (bio-)materials, even if the time range of the apparent diffusion coefficient is limited due to relaxation of the magnetization. The method is demonstrated by characterizing anisotropic cell dimensions and permeability on a subpixel level of different tissues of a carrot (Daucus carota) taproot in the radial and axial directions.
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Project description:1. The polysaccharide compositions of the cell walls of sycamore cambium and sycamore callus tissue have been analysed and found to be directly comparable. 2. Electrophoretic analyses of the whole pectins prepared from actively growing callus and cambial tissue have shown that these preparations contain, in addition to the neutral and weakly acidic components present in apple fruit, a strongly acidic polygalacturonic acid component. 3. The weakly acidic component of all the pectins was directly comparable with that of the pectinic acid of apple fruit. 4. The components of the whole pectin of sycamore callus tissue have been partially purified and analysed. The neutral and weakly acidic components also found in apple fruit were isolated. 5. The pattern of the composition of the neutral sugars present in the pectins of actively growing tissues of cambium and callus has been compared with those present in apple-fruit pectinic acid. 6. The presence of rhamnose linked as galacturonosyl-(1-->2)-rhamnose has been found in sycamore whole pectin. 7. The difference in the pectins of callus, cambium and fruit appears not to be that of species difference but is more characteristic of the nature of the growth and growth conditions of the cells. This is discussed in relation to the problems of the control and mechanism of plant-cell growth and differentiation.
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Project description:BACKGROUND:Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals. RESULTS:We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment. CONCLUSIONS:Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass.
| S-EPMC3909318 | biostudies-literature
Project description:3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.
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Project description:1. A phosphatase that hydrolyses sucrose phosphate (phosphorylated at the 6-position of fructose) was isolated from sugar-cane stem and carrot roots. With partially purified preparations fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, glucose 1-phosphate and fructose 1,6-diphosphate are hydrolysed at between 0 and 2% of the rate for sucrose phosphate. 2. The activity of the enzyme is increased fourfold by the addition of Mg(2+) ions and inhibited by EDTA, fluoride, inorganic phosphate, pyrophosphate, Ca(2+) and Mn(2+) ions. Sucrose (50mm) reduces activity by 60%. 3. The enzyme exhibits maximum activity between pH6.4 and 6.7. The Michaelis constant for sucrose phosphate is between 0.13 and 0.17mm. 4. At least some of the specific phosphatase is associated with particles having the sedimentation properties of mitochondria. 5. A similar phosphatase appears to be present in several other plant species.
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Project description:3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG-CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients.
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Project description:Verticillium dahliae is a wide-host-range fungal pathogen that causes soil-borne disease in hundreds of dicotyledonous hosts. In search of V. dahliae Vd991 cell death-inducing proteins, we identified a pectate lyase (VdPEL1) that exhibited pectin hydrolytic activity, which could induce strong cell death in several plants. Purified VdPEL1 triggered defense responses and conferred resistance to Botrytis cinerea and V. dahliae in tobacco and cotton plants. Our results demonstrated that the mutant VdPEL1rec lacking the enzymatic activity lacked functions to induce both cell death and plant resistance, implying that the enzymatic activity was necessary. In addition, VdPEL1 was strongly induced in V. dahliae infected Nicotiana benthamiana and cotton roots, and VdPEL1 deletion strains severely compromised the virulence of V. dahliae. Our data suggested that VdPEL1 contributed to V. dahliae virulence and induced plant defense responses. These findings provide a new insight for the function of pectate lyase in the host-pathogen interaction.
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