Versatile 5′ phosphoryl coupling of small and large molecules to an RNA

A Ca2+-requiring catalytic RNA is shown to create 5′ phosphate–phosphate linkages with all nucleotides and coenzymes including CoA, nicotinamide adenine dinucleotide phosphate, thiamine phosphate, thiamine pyrophosphate, and flavin mononucleotide. In addition to these small molecules, macromolecules such as RNAs with 5′-diphosphates, and nonnucleotide molecules like Nɛ-phosphate arginine and 6-phosphate gluconic acid also react. That is, the self-capping RNA isolate 6 is an apparently universal 5′ phosphate-linker, reacting with any nucleophile containing an unblocked phosphate. These RNA reactions demonstrate a unique RNA catalytic capability and imply versatile and specific posttranscriptional RNA modification by RNA catalysis.

A mouse model for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder with impaired β-oxidation of very long chain fatty acids (VLCFAs) and reduced function of peroxisomal very long chain fatty acyl-CoA synthetase (VLCS) that leads to severe and progressive neurological disability. The X-ALD gene, identified by positional cloning, encodes a peroxisomal membrane protein (adrenoleukodystrophy protein; ALDP) that belongs to the ATP binding cassette transporter protein superfamily. Mutational analyses and functional studies of the X-ALD gene confirm that it and not VLCS is the gene responsible for X-ALD. Its role in the β-oxidation of VLCFAs and its effect on the function of VLCS are unclear. The complex pathology of X-ALD and the extreme variability of its clinical phenotypes are also unexplained. To facilitate understanding of X-ALD pathophysiology, we developed an X-ALD mouse model by gene targeting. The X-ALD mouse exhibits reduced β-oxidation of VLCFAs, resulting in significantly elevated levels of saturated VLCFAs in total lipids from all tissues measured and in cholesterol esters from adrenal glands. Lipid cleft inclusions were observed in adrenocortical cells of X-ALD mice under the electron microscope. No neurological involvement has been detected in X-ALD mice up to 6 months. We conclude that X-ALD mice exhibit biochemical defects equivalent to those found in human X-ALD and thus provide an experimental system for testing therapeutic intervention.

Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1

The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH2-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.

Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays

Protein acetylation has been implicated in the regulation of HIV-1 gene transcription. Here, we have exploited the activities of four native histone acetyltransferase (HAT) complexes from yeast to directly test whether acetylation regulates HIV-1 transcription in vitro. HAT activities acetylating either histone H3 (SAGA, Ada, and NuA3) or H4 (NuA4) stimulate HIV-1 transcription from preassembled nucleosomal templates in an acetyl CoA-dependent manner. HIV-1 transcription from histone-free DNA is not affected by the HATs, indicating that these activities function in a chromatin-specific fashion. For Ada and NuA4, we demonstrate that acetylation of only histone proteins mediates enhanced transcription, suggesting that these complexes facilitate transcription at least in part by modifying histones. To address a potential mechanism by which HAT complexes stimulate transcription, we performed a restriction enzyme accessibility analysis. Each of the HATs increases the cutting efficiencies of restriction endonucleases targeting the HIV-1 chromatin templates in a manner not requiring transcription, suggesting that histone acetylation leads to nucleosome remodeling.

Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation

Ocular dominance column formation in visual cortex depends on both the presence of subplate neurons and the endogenous expression of neurotrophins. Here we show that deletion of subplate neurons, which supply glutamatergic inputs to visual cortex, leads to a paradoxical increase in brain-derived neurotrophic factor mRNA in the same region of visual cortex in which ocular dominance columns are absent. Subplate neuron ablation also increases glutamic acid decarboxylase-67 levels, indicating an alteration in cortical inhibition. These observations imply a role for this special class of neurons in modulating activity-dependent competition by regulating levels of neurotrophins and excitability within a developing cortical circuit.

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

Alcaligenes eutrophus genes encoding the enzymes, β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-d-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous β-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, β-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 × 106 to 1.8 × 106 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid β-oxidation

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the β-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the β-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH2-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH2-terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The high-cholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70–90% reduction in mRNAs encoding 3-hydroxy-3-methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells.

Der3p/Hrd1p Is Required for Endoplasmic Reticulum-associated Degradation of Misfolded Lumenal and Integral Membrane Proteins

We have studied components of the endoplasmic reticulum (ER) proofreading and degradation system in the yeast Saccharomyces cerevisiae. Using a der3–1 mutant defective in the degradation of a mutated lumenal protein, carboxypeptidase yscY (CPY*), a gene was cloned which encodes a 64-kDa protein of the ER membrane. Der3p was found to be identical with Hrd1p, a protein identified to be necessary for degradation of HMG-CoA reductase. Der3p contains five putative transmembrane domains and a long hydrophilic C-terminal tail containing a RING-H2 finger domain which is oriented to the ER lumen. Deletion of DER3 leads to an accumulation of CPY* inside the ER due to a complete block of its degradation. In addition, a DER3 null mutant allele suppresses the temperature-dependent growth phenotype of a mutant carrying the sec61–2 allele. This is accompanied by the stabilization of the Sec61–2 mutant protein. In contrast, overproduction of Der3p is lethal in a sec61–2 strain at the permissive temperature of 25°C. A mutant Der3p lacking 114 amino acids of the lumenal tail including the RING-H2 finger domain is unable to mediate degradation of CPY* and Sec61–2p. We propose that Der3p acts prior to retrograde transport of ER membrane and lumenal proteins to the cytoplasm where they are subject to degradation via the ubiquitin-proteasome system. Interestingly, in ubc6-ubc7 double mutants, CPY* accumulates in the ER, indicating the necessity of an intact cytoplasmic proteolysis machinery for retrograde transport of CPY*. Der3p might serve as a component programming the translocon for retrograde transport of ER proteins, or it might be involved in recognition through its lumenal RING-H2 motif of proteins of the ER that are destined for degradation.

Ste6p Mutants Defective in Exit from the Endoplasmic Reticulum (ER) Reveal Aspects of an ER Quality Control Pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6–166p, is highly unstable. We show that its degradation involves the ubiquitin–proteasome system, as indicated by its in vivo stabilization in certain ubiquitin–proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6–13p and Ste6–90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using β-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.

Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the α subunit and the third domain is made up entirely by the β subunit. The N-terminal portion of the α subunit and the majority of the β subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the α subunit. Alignment of all the known sequences for the ETF α subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of αT266, suggesting why the pathogenic mutation, αT266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between αH286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands

The current paper describes a line of cultured rat hepatoma cells (McA-RH7777 cells) that mimics the behavior of rat liver by producing an excess of mRNA for sterol regulatory element-binding protein 1c (SREBP-1c) as opposed to SREBP-1a. These two transcripts are derived from a single gene by use of alternative promoters that are separated by many kilobases in the genome. The high level of SREBP-1c mRNA is abolished when cholesterol synthesis is blocked by compactin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase that inhibits cholesterol synthesis. Levels of SREBP-1c mRNA are restored by mevalonate, the product of the HMG CoA reductase reaction, and by ligands for the nuclear hormone receptor LXR, including 22(R)-hydroxycholesterol and T0901317. These data suggest that transcription of the SREBP-1c gene in hepatocytes requires tonic activation of LXR by an oxysterol intermediate in the cholesterol biosynthetic pathway. Reduction of this intermediate lowers SREBP-1c levels, and this in turn is predicted to lower the rates of fatty acid biosynthesis in liver.