YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase

Extremophiles - YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular...     

Radioiodinated clioquinol as a biomarker for beta-amyloid: Zn complexes in Alzheimer's disease

Neocortical beta-amyloid (Abeta) aggregates in Alzheimer's disease (AD) are enriched in transition metals that mediate assembly. Clioquinol (CQ) targets metal interaction with Abeta and inhibits amyloid pathology in transgenic mice. Here, we investigated the binding properties of radioiodinated CQ ([(125)I]CQ) to different in vitro and in vivo Alzheimer models. We observed saturable binding of [(125)I]CQ to synthetic Abeta precipitated by Zn(2+) (K(d)=0.45 and 1.40 nm for Abeta(1-42) and Abeta(1-40), respectively), which was fully displaced by free Zn(2+), Cu(2+), the chelator DTPA (diethylene triamine pentaacetic acid) and partially by Congo red. Sucrose density gradient of post-mortem AD brain indicated that [(125)I]CQ concentrated in a fraction enriched for both Abeta and Zn, which was modulated by exogenous addition of Zn(2+) or DTPA. APP transgenic (Tg2576) mice injected with [(125)I]CQ exhibited higher brain retention of tracer compared to non-Tg mice. Autoradiography of brain sections of these animals confirmed selective [(125)I]CQ enrichment in the neocortex. Histologically, both thioflavine-S (ThS)-positive and negative structures were labeled by [(125)I]CQ. A pilot SPECT study of [(123)I]CQ showed limited uptake of the tracer into the brain, which did however, appear to be more rapid in AD patients compared to age-matched controls. These data support metallated Abeta species as the neuropharmacological target of CQ and indicate that this drug class may have potential as in vivo imaging agents for Alzheimer neuropathology.     

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.     

Engineering Triterpene and Methylated Triterpene Production in Plants Provides Biochemical and Physiological Insights into Terpene Metabolism

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g⁻¹ fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.Terpenes and terpenoids represent a distinct class of natural products (Buckingham, 2003) that are derived from two universal five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotic fungi and animals, IPP and DMAPP are synthesized via the mevalonate (MVA) pathway, whereas in prokaryotes, they are synthesized via the methylerythritol phosphate (MEP) pathway. In higher plants, the pathways are present in separate compartments and are believed to operate independently. The MVA pathway in the cytoplasm is predominantly responsible for sesquiterpene (C15), triterpene (C30), and polyprenol (greater than C45) biosynthesis and associated with the endoplasmic reticulum (ER) system. The MEP pathway resides in plastids and is dedicated to monoterpenes (C10), diterpenes (C20), carotenoids (C40), and long-chain phytol biosynthesis. All these compounds are usually produced by plants for a variety of physiological (i.e. hormones, aliphatic membrane anchors, and maintaining membrane structure) and ecological (i.e. defense compounds and insect/animal attractants) roles (Kempinski et al., 2015). Terpenes are also important for various industrial applications, ranging from flavors and fragrances (Schwab et al., 2008) to medicines (Dewick, 2009; Niehaus et al., 2011; Shelar, 2011).The utility of terpenes as chemical and biofuel feedstocks has also received considerable attention recently. Isoprenoid-derived biofuels include farnesane (Renninger and McPhee, 2008; Rude and Schirmer, 2009), bisabolene (Peralta-Yahya et al., 2011), pinene dimers (Harvey et al., 2010), isopentenal (Withers et al., 2007), and botryococcene (Moldowan and Seifert, 1980; Hillen et al., 1982; Glikson et al., 1989; Mastalerz and Hower, 1996). The richness of branches within these hydrocarbon scaffolds correlate with their high-energy content, which enables them to serve as suitable alternatives to crude petroleum (Peralta-Yahya and Keasling, 2010). Indeed, some of them are already major contributors to current-day petroleum-based fuels. One of the best examples of this is the triterpene oil accumulating in the green alga Botryococcus braunii race B, which is considered a major progenitor to oil and coal shale deposits (Moldowan and Seifert, 1980). This alga has been well studied, and the major constituents of its prodigious hydrocarbon oil are a group of triterpenes including squalene (C30), organism-specific botryococcene (C30), methylated squalene (C31–C34), and methylated botryococcene (C31–C37; Metzger et al., 1988; Huang and Poulter, 1989; Okada et al., 1995), which can be readily converted into all classes of combustible fuels under hydrocracking conditions (Hillen et al., 1982).The unique biosynthetic mechanism for the triterpenes in B. braunii was recently described by Niehaus et al. (2011), and a series of novel squalene synthase-like genes were identified (Fig. 1). In short, squalene synthase-like enzyme, SSL-1, performs a head-to-head condensation of two farnesyl diphosphate (FPP) molecules into presqualene diphosphate, followed by a reductive rearrangement to yield squalene (C30) by the enzyme SSL-2, or is converted by SSL-3 to form botryococcene through a different reductive rearrangement (Niehaus et al., 2011). Methylated derivatives are the dominant triterpene species generated by B. braunii race B (Metzger, 1985; Metzger et al., 1988), and these derivatives are known to yield higher quality fuels due to their high energy content and the hydrocracking products derived by virtue of having more hydrocarbon branches. Triterpene methyltransferases (TMTs) that can methylate squalene and botryococcene have been successfully characterized by Niehaus et al. (2012). TRITERPENE METHYLTRANSFERASE1 (TMT-1) and TMT-2 prefer squalene C30 as their substrate for the production of monomethylated (C31) or dimethylated (C32) squalene, while TMT-3 prefers botryococcene as its substrate for the biosynthesis of monomethylated (C31) or dimethylated (C32) botryococcene (Fig. 1). These TMTs are believed to be insoluble enzymes; they exhibit large hydrophobic areas, and their activities were only observed in vitro using yeast microsomal preparations (no activity was observed when expressed in bacteria; Niehaus et al., 2012).Open in a separate windowFigure 1.Depiction of the catalytic roles of novel SSL and TMT enzymes in B. braunii race B and their putative contributions to the triterpene constituents (Niehaus et al., 2011; Niehaus et al., 2012). SSL-1 catalyzes the condensation of two farnesyl diphosphate (FPP) molecules to presqualene diphosphate (PSPP), which is converted to either squalene or botryococcene by SSL-2 or SSL-3, respectively. Squalene can also be synthesized directly from the condensation of two FPP molecules catalyzed by squalene synthase (SQS). TMT-1 and TMT-2 transfer the methyl donor group from S-adenosylmethionine (SAM) to squalene to form monomethylated and dimethylated squalene, whereas TMT-3 acts on botryococcene to form monomethylated and dimethylated botryococcene (Niehaus et al., 2012).Like the majority of identified methyltransferases, these TMTs utilize the methyl donor S-adenosyl methionine (SAM), which is ubiquitous in prokaryotes and eukaryotes (Scheer et al., 2011; Liscombe et al., 2012). In plants, SAM is one of the most abundant cofactors (Fontecave et al., 2004; Sauter et al., 2013) and is synthesized exclusively in the cytosol (Wallsgrove et al., 1983; Ravanel et al., 1998, 2004; Bouvier et al., 2006). While it is used predominantly as a methyl donor in the methylation reaction (Ravanel et al., 2004), it also serves as the primary precursor for the biosynthesis of ethylene (Wang et al., 2002b), polyamines (Kusano et al., 2008), and nicotianamine (Takahashi et al., 2003), which play a variety of important roles for plant growth and development (Huang et al., 2012; Sauter et al., 2013). The SAM present in organelles, like the chloroplast, appears to be imported from the cytosol by specific SAM/S-adenosylhomocysteine exchange transporters that reside on the envelope membranes of plastids (Ravanel et al., 2004; Bouvier et al., 2006). The imported SAM is involved in the biogenesis of Asp-derived amino acids (Curien et al., 1998; Jander and Joshi, 2009; Sauter et al., 2013) and serves as the methyl donor for the methylation of macromolecules, such as plastid DNA (Nishiyama et al., 2002; Ahlert et al., 2009) and proteins (Houtz et al., 1989; Niemi et al., 1990; Ying et al., 1999; Trievel et al., 2003; Alban et al., 2014), and small molecule metabolites, such as prenylipids (e.g. plastoquinone, tocopherol, chlorophylls, and phylloquinone; Bouvier et al., 2005, 2006; DellaPenna, 2005).Although plants and microbes are the natural sources for useful terpenes, most of them are produced in very small amounts and often as complex mixtures. In contrast, B. braunii produces large quantities of triterpenes, but its slow growth makes it undesirable as a viable production platform (Niehaus et al., 2011). Nevertheless, metabolic engineering and synthetic biology offer many strategies to manipulate terpene metabolism in various biological systems to achieve high-value terpene production with high yield and high fidelity for particular practical applications (Nielsen and Keasling, 2011). Many successes have been achieved in engineering valuable terpenes in heterotrophic microbes, such as Escherichia coli (Nishiyama et al., 2002; Martin et al., 2003; Ajikumar et al., 2010) and Saccharomyces cerevisiae (Ro et al., 2006; Takahashi et al., 2007; Westfall et al., 2012; Zhuang and Chappell, 2015). The strategies developed in these efforts usually take advantage of specific microbe strains whose innate biosynthetic machinery is genetically modified to accumulate certain prenyldiphosphate precursors (e.g. IPP or FPP), which can be utilized by other introduced terpene synthase(s) for the production of the desired terpene(s). For example, greater than 900 mg L⁻¹ bisabolene was produced when bisabolene synthase genes from plants were introduced into FPP-overproducing E. coli or S. cerevisiae strains (Peralta-Yahya et al., 2011). High levels of farnesane production for diesel fuels were also achieved by reductive hydrogenation of its precursor farnesene, which was generated from a genetically engineered yeast (e.g. Saccharomyces cerevisiae) strain using plant farnesene synthases (Renninger and McPhee, 2008; Ubersax and Platt, 2010). However, terpene production using microbial platforms is still dependent on exogenous feedstocks (i.e. sugars) and elaborate production facilities, both of which add significantly to their production costs.Compared with microbial systems, engineering terpene production in plant systems seems like an attractive target as well. This is because plants can take advantage of photosynthesis by using atmospheric CO₂ as their carbon resource instead of relying on exogenous carbon feedstocks. Moreover, crop plants such as tobacco (Nicotiana tabacum) can generate a large amount of green tissues efficiently when grown for biomass production (Schillberg et al., 2003; Andrianov et al., 2010), making them a robust, sustainable, and scalable platform for large-scale terpene production. Nonetheless, compared with microbial platforms, there are only a few examples of elevating terpene production in bioengineered plants. This is due partly to higher plants being complex multicellular organisms, in which terpene metabolism generally utilizes more complex innate machinery that can be compartmentalized intracellularly and to cell/tissue specificities (Lange and Ahkami, 2013; Kempinski et al., 2015). Significant efforts have been made to overcome these obstacles to improve the production of valuable terpenes in plants, including monoterpenes (Lücker et al., 2004; Ohara et al., 2010; Lange et al., 2011), sesquiterpenes (Aharoni et al., 2003; Kappers et al., 2005; Wu et al., 2006; Davidovich-Rikanati et al., 2008), diterpenes (Besumbes et al., 2004; Anterola et al., 2009), and triterpenes (Inagaki et al., 2011; Wu et al., 2012). Among these, engineering terpene metabolism into a subcellular organelle, where the engineered enzymes/pathways can utilize unlimited/unregulated precursors as substrates, appears most successful. For example, Wu et al. (2006, 2012) expressed an avian farnesyl diphosphate synthase (FPS) with foreign sesquiterpene/triterpene synthases targeted to the plastid to divert the IPP/DMAPP pool from the plastidic MEP pathway to synthesize high levels of the novel sesquiterpenes patchoulol and amorpha-4,11-diene up to 30 µg g⁻¹ fresh weight and the triterpene squalene up to 1,000 µg g⁻¹ fresh weight. This strategy appears to be particularly robust because it avoids possible endogenous regulation of sesquiterpene and triterpene biosynthesis, which occurs normally in the cytoplasm, and relies upon more plastic precursor pools of IPP/DMAPP inherent in the plastid, which are primarily derived from the local CO₂ fixation (Wright et al., 2014).The goal of this study was to evaluate the prospects for engineering advanced features of triterpene metabolism from B. braunii into tobacco and, thus, to probe the innate intricacies of isoprenoid metabolism in plants. In order to achieve this, we first introduced the key steps of botryococcene biosynthesis into specific subcellular compartments of tobacco cells under the direction of constitutive or trichome-specific promoters. The transgenic lines expressing the enzymes in the chloroplast were found to accumulate the highest levels of botryococcene. Triterpene methyltransferases were next introduced into the same intracellular compartments of selected high-triterpene-accumulating lines. A high yield of methylated triterpenes was also achieved in transgenic lines when the TMTs were targeted to the chloroplast. Through careful comparison of the levels of triterpenes and the methylated triterpene products in the various transgenic lines, we have also gained a deeper insight into the subcellular distribution of the triterpene products in these transgenic lines as well as a better understanding of methylation metabolism for specialized metabolites in particular compartments. These findings all contribute to our understanding of the regulatory elements that control carbon flux through the innate terpene biosynthetic pathways operating in plants.     

Effects of 1,25-dihydroxyvitamin D3 and its analogs on butyrate-induced differentiation of HT-29 human colonic carcinoma cells and on the reversal of the differentiated phenotype

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) greatly enhances sodium butyrate (NaB)-induced enterocyte differentiation of HT-29 human colonic carcinoma cells while 1,25-(OH)2D3 alone induces growth restriction without associated differentiation. In the present study, the efficacies of various analogs of 1,25-(OH)2D3 to enhance NaB-induced HT-29 differentiation and to prolong the reversal of the differentiated phenotype under NaB-free growth conditions were subsequently examined. Extent of HT-29 differentiation was assessed by measurement of alkaline phosphatase (AP) activity, appearance of mucin-producing cells, changes in morphological characteristics, and expression of differentiation-associated cytokeratin proteins. Among active analogs of 1,25-(OH)2D3, 26,26,26,27,27,27-hexafluoro-1,25-(OH)2D3 (F6-1,25-(OH)2D3), 24,24-difluoro-24-homo-1,25-(OH)2D3, and 26,27-dimethyl-1,25-(OH)2D3 were 100-, 10-, and 5-fold, respectively, more effective than 1,25-(OH)2D3 in enhancing NaB-induced mucin production. Combined use of NaB and F6-1,25-(OH)2D3 (10(-9) M) also induced HT-29 cells to form highly differentiated goblet-like enterocytes, and increased both cellular AP enzymatic activity and tissue-type cytokeratin content. This differentiated state was qualitatively more advanced than that achieved by a combination of NaB and 10(-7) M 1,25-(OH)2D3. NaB-mediated HT-29 differentiation (in short-term inductions) was found to be reversible following a return to NaB-free medium. HT-29 cells differentiated by combined use of NaB and 1,25-(OH)2D3 or its analogs exhibited a significant prolonged reversal time relative to cells differentiated with NaB alone. The most prominent effect was achieved using cells differentiated with NaB and 10(-9) M F6-1,25-(OH)2D3 which exhibited a 7-fold prolonged reversal time over colonocytes differentiated by NaB alone. Our data suggest that a combined use of NaB and 1,25-(OH)2D3 or its derivatives may provide a convenient in vitro model system to probe molecular events associated with steroid-target tissue interactions in a differentiating cell system as commonly occurs in vivo. Such an analysis might lend itself to design of a rational combination differentiation-based therapy for the clinical management of colon cancer.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.     

Structure of a -D-glucan from the mycelial wall of Basidiomycete QM 806

A water-soluble β-D-glucan has been isolated from the mycelial wall of Basidiomycete QM 806. The structure of this glucan was investigated by methylation, periodate, and enzymic studies. Hydrolysis of the methylated glucan gave 2,3,4,6-tetra-, 2,4,6-, 2,3,4- and 2,3,6-tri-, and 2,4-di-O-methyl-D-glucose in the following molar proportions: 1.0:1.0:O.8:1.2:1.0. Periodate oxidation of the glucan followed by reduction and mild acid hydrolysis gave glycerol, erythritol, and D-glucose in the molar proportions, 2.1, 1.0, and 2.0, respectively. The glucan was degraded to the extent of 38% by an exo-β-(1→3)-glucanase isolated from the same organism, though the branch points (joined through O-1, O-3, and O-6) appeared to be resistant to the enzyme whereas the (1→4) linkages were not. On the basis of these findings, the structure of the glucan and the possible role of the glucanase are discussed.     

RanBP2 modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 causes deficits in glucose metabolism

The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2^−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2^+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies.