1,486 research outputs found

    Altered expression of an ankyrin-repeat protein results in leaf abnormalities, necrotic lesions, and the elaboration of a systemic signal

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    Summary: The PR-like proteins, class I β-1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated G\underline{G} lucanohydrolase B\underline{B} inding P\underline{P} roteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved P\underline{P} lant-specific Ank\underline{Ank} yrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolis

    Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target

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    The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells

    抗赤痢アメーバ新規薬剤開発を目的とした補酵素A生合成経路の解明

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    筑波大学 (University of Tsukuba)201

    Pantothenate Kinase 1 Is Required to Support the Metabolic Transition from the Fed to the Fasted State

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    Coenzyme A (CoA) biosynthesis is regulated by the pantothenate kinases (PanK), of which there are four active isoforms. The PanK1 isoform is selectively expressed in liver and accounted for 40% of the total PanK activity in this organ. CoA synthesis was limited using a Pank1−/− knockout mouse model to determine whether the regulation of CoA levels was critical to liver function. The elimination of PanK1 reduced hepatic CoA levels, and fasting triggered a substantial increase in total hepatic CoA in both Pank1−/− and wild-type mice. The increase in hepatic CoA during fasting was blunted in the Pank1−/− mouse, and resulted in reduced fatty acid oxidation as evidenced by abnormally high accumulation of long-chain acyl-CoAs, acyl-carnitines, and triglycerides in the form of lipid droplets. The Pank1−/− mice became hypoglycemic during a fast due to impaired gluconeogenesis, although ketogenesis was normal. These data illustrate the importance of PanK1 and elevated liver CoA levels during fasting to support the metabolic transition from glucose utilization and fatty acid synthesis to gluconeogenesis and fatty acid oxidation. The findings also suggest that PanK1 may be a suitable target for therapeutic intervention in metabolic disorders that feature hyperglycemia and hypertriglyceridemia

    Energizing miRNA Research: A Review of the Role of miRNAs in Lipid Metabolism, with a Prediction that miR-103/107 Regulates Human Metabolic Pathways

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    MicroRNAs (miRNAs) are powerful regulators of gene expression. Although first discovered in worm larvae, miRNAs play fundamental biological roles-including in humans-well beyond development. MiRNAs participate in the regulation of metabolism (including lipid metabolism) for all animal species studied. A review of the fascinating and fast-growing literature on miRNA regulation of metabolism can be parsed into three main categories: (1) adipocyte biochemistry and cell fate determination; (2) regulation of metabolic biochemistry in invertebrates; and (3) regulation of metabolic biochemistry in mammals. Most research into the \u27function\u27 of a given miRNA in metabolic pathways has concentrated on a given miRNA acting upon a particular \u27target\u27 mRNA. Whereas in some biological contexts the effects of a given miRNA:mRNA pair may predominate, this might not be the case generally. In order to provide an example of how a single miRNA could regulate multiple \u27target\u27 mRNAs or even entire human metabolic pathways, we include a discussion of metabolic pathways that are predicted to be regulated by the miRNA paralogs, miR-103 and miR-107. These miRNAs, which exist in vertebrate genomes within introns of the pantothenate kinase (PANK) genes, are predicted by bioinformatics to affect multiple mRNA targets in pathways that involve cellular Acetyl-CoA and lipid levels. Significantly, PANK enzymes also affect these pathways, so the miRNA and \u27host\u27 gene may act synergistically. These predictions require experimental verification. In conclusion, a review of the literature on miRNA regulation of metabolism leads us believe that the future will provide researchers with many additional energizing revelations

    Chemoenzymatic Study of CoA-Linked RNA in Bacteria

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    The ability of RNA to store genetic information and to catalyze biochemical transformations led to the speculation of the existence of RNA world before the evolution of contemporary ribonucleoprotein (RNP) world. Recent discovery of RNA molecules containing metabolic cofactors including coenzyme A and its various thioesters at their 5’ end further supported the RNA world hypothesis as these CoA-linked RNA molecules could be the molecular fossils with very ancient origin. As both RNA and Coenzyme A are believed to have co-existed since last universal common ancestor (LUCA) or even before, the CoA-RNA conjugates in current biology may reveal fundamental molecular secrets involved in evolution. Furthermore, these CoA-RNA conjugates may not be just remnants of evolution, rather may have an important functional significance in contemporary metabolism and gene regulation. The successful characterization of these conjugates will expand our knowledge in RNA function and CoA function. However, the sequence, metabolism and biological role of these RNA species are still unknown. The aim of this study is to capture CoA-RNA sequences form E. coli total RNA to uncover their sequences and to investigate their biogenesis. The successful characterization of CoA-RNA requires a specific protocol to capture them. The development of such protocol requires an easy access to synthetic CoA-RNA. While our lab previously developed a method to incorporate CoA into RNA co-transcriptionally by using dephospho CoA as a transcription initiator, the limited availability of dephospoCoA restricts an easy access to synthetic CoA-RNA. In the first section, a simple and easy method for the synthesis of dephospho CoA and its oxidized dimer was developed. Two enzymes of CoA biosynthetic pathway, PanK and PPAT were cloned in a single plasmid, and purified in a single enzyme preparation. The synthesis of dephsphoCoA was achieved by the enzyme cocktail and pure product was obtained by a simple reverse phase column chromatography. The method was extended further to synthesize various dephosphoCoA analogs including amino dephosphoCoA and biotin dephosphoCoA. In the second section, two strategies were investigated for their application in CoA-RNA capture. In first strategy, various acyl-CoA ligases including acetyl CoA synthetase (ACS), Malonyl CoA synthetase (MatB), Succinyl CoA synthetase (SucCD), medium chain fatty acyl CoA synthetase (FadK) and Long chain fatty acyl CoA synthetase were cloned, expressed and tested their ability to accept biotinylated fatty acid and CoA-RNA as substrates. In the second strategy, various pantetheine and phosphopantetheine analogs with biotin as a purification handle, [14-C] acetate as a reporter tag, and variable number of positive charges to mediate cellular uptake were synthesized. During the synthesis of such analogs, a novel method for the selective protection of secondary alcohols in presence of their primary counterparts was developed. In the third section of this work, a mechanism of CoA-RNA biogenesis was explored. Synthesized phosphopantetheine analog was used to investigate whether CoA-RNA can be generated post-transcriptionally. We found that a CoA biosynthetic enzyme, PPAT (coaD), can accept ATP-RNA as its substrate and yields CoA-RNA. our results showed that the suitability of ATP initiated RNA to serve as a PPAT substrate is determined by its 5’ structure. An RNA having at least four unstructured nucleotides at the 5’ end can participate in PPAT catalyzed phosphopantetheine transfer reaction. Furthermore, the rate of the reaction was independent to the number of 5’ unstructured nucleotides at least for the range of 4-10. These findings established the post-transcriptional transfer of 4’-phosphopantetheine to ATP-RNA as another mode of CoA-RNA biogenesis besides previously characterized RNAP mediated co-transcriptional incorporation. Collectively, we have developed an easy and simple method to prepare dephospho CoA and its analogs, investigated chemo-enzymatic strategies for CoA-RNA capture and, discovered a post-transcriptional mechanism of CoA-RNA biogenesis. Our results suggest that the existence of CoA-RNA in extant biology may be evolutionary. Future studies may lead to development of specific CoA-RNA capture and will expand our understandings on how ribozyme catalysis of the RNA world was transformed into protein enzyme-based catalysis of contemporary world

    The Coenzyme A Level Modulator Hopantenate (HoPan) Inhibits Phosphopantotenoylcysteine Synthetase Activity

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    The pantothenate analogue hopantenate (HoPan) is widely used as a modulator of coenzyme A (CoA) levels in cell biology and disease models-especially for pantothenate kinase associated neurodegeneration (PKAN), a genetic disease rooted in impaired CoA metabolism. This use of HoPan was based on reports that it inhibits pantothenate kinase (PanK), the first enzyme of CoA biosynthesis. Using a combination of in vitro enzyme kinetic studies, crystal structure analysis, and experiments in a typical PKAN cell biology model, we demonstrate that instead of inhibiting PanK, HoPan relies on it for metabolic activation. Once phosphorylated, HoPan inhibits the next enzyme in the CoA pathway-phosphopantothenoylcysteine synthetase (PPCS)-through formation of a nonproductive substrate complex. Moreover, the obtained structure of the human PPCS in complex with the inhibitor and activating nucleotide analogue provides new insights into the catalytic mechanism of PPCS enzymes-including the elusive binding mode for cysteine-and reveals the functional implications of mutations in the human PPCS that have been linked to severe dilated cardiomyopathy. Taken together, this study demonstrates that the molecular mechanism of action of HoPan is more complex than previously thought, suggesting that the results of studies in which it is used as a tool compound must be interpreted with care. Moreover, our findings provide a clear framework for evaluating the various factors that contribute to the potency of CoA-directed inhibitors, one that will prove useful in the future rational development of potential therapies of both human genetic and infectious diseases
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