Purification and identification of activating enzymes of CS-0777, a selective sphingosine 1-phosphate receptor 1 modulator, in erythrocytes

CS-0777 is a selective sphingosine 1-phosphate (S1P) receptor 1 modulator with potential benefits in the treatment of autoimmune diseases, including multiple sclerosis. CS-0777 is a prodrug that requires phosphorylation to an active S1P analog, similar to the first-in-class S1P receptor modulator FTY720 (fingolimod). We sought to identify the kinase(s) involved in phosphorylation of CS-0777, anticipating sphingosine kinase (SPHK) 1 or 2 as likely candidates. Unlike kinase activity for FTY720, which is found predominantly in platelets, CS-0777 kinase activity was found mainly in red blood cells (RBCs). N,N-Dimethylsphingosine, an inhibitor of SPHK1 and -2, did not inhibit CS-0777 kinase activity. We purified CS-0777 kinase activity from human RBCs by more than 10,000-fold using ammonium sulfate precipitation and successive chromatography steps, and we identified fructosamine 3-kinase (FN3K) and fructosamine 3-kinase-related protein (FN3K-RP) by mass spectrometry. Incubation of human RBC lysates with 1-deoxy-1-morpholinofructose, a competitive inhibitor of FN3K, inhibited ～10% of the kinase activity, suggesting FN3K-RP is the principal kinase responsible for activation of CS-0777 in blood. Lysates from HEK293 cells overexpressing FN3K or FN3K-RP resulted in phosphorylation of CS-0777 and structurally related molecules but showed little kinase activity for FTY720 and no kinase activity for sphingosine. Substrate preference was highly correlated among FN3K, FN3K-RP, and rat RBC lysates. FN3K and FN3K-RP are known to phosphorylate sugar moieties on glycosylated proteins, but this is the first report that these enzymes can phosphorylate hydrophobic xenobiotics. Identification of the kinases responsible for CS-0777 activation will permit a better understanding of the pharmacokinetics and pharmacodynamics of this promising new drug.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.     

Purification and Characterization of a Novel Galloyltransferase Involved in Catechin Galloylation in the Tea Plant (Camellia sinensis)

Catechins (flavan-3-ols), the most important secondary metabolites in the tea plant, have positive effects on human health and are crucial in defense against pathogens of the tea plant. The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant. The results suggested that galloylated catechins were biosynthesized via 1-O-glucose ester-dependent two-step reactions by acyltransferases, which involved two enzymes, UDP-glucose:galloyl-1-O-β-d-glucosyltransferase (UGGT) and a newly discovered enzyme, epicatechin:1-O-galloyl-β-d-glucose O-galloyltransferase (ECGT). In the first reaction, the galloylated acyl donor β-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose. In the second reaction, galloylated catechins were produced by ECGT catalysis from β-glucogallin and 2,3-cis-flavan-3-ol. 2,3-cis-Flavan-3-ol and 1-O-galloyl-β-d-glucose were appropriate substrates of ECGT rather than 2,3-trans-flavan-3-ol and 1,2,3,4,6-pentagalloylglucose. Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241 to 121 kDa by gel filtration. Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer, trimer, or tetramer of 60- and/or 58-kDa monomers, and these monomers represent a heterodimer consisting of pairs of 36- or 34- of and 28-kDa subunits. MALDI-TOF-TOF MS showed that the protein SCPL1199 was identified. Epigallocatechin and epicatechin exhibited higher substrate affinities than β-glucogallin. ECGT had an optimum temperature of 30 °C and maximal reaction rates between pH 4.0 and 6.0. The enzyme reaction was inhibited dramatically by phenylmethylsulfonyl fluoride, HgCl₂, and sodium deoxycholate.     

Inhibition of Arabidopsis O-acetylserine(thiol)lyase A1 by tyrosine nitration

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.     

一个未知的子宫雌激素反应蛋白ULF-250的鉴定研究

摘要 鉴定一个未知的子宫雌激素反应蛋白（ULF-250）。去卵巢大鼠补充给予雌二醇造成子宫产生大量宫腔液（ULF）,受雌激素调节的蛋白分泌其中。收集ULF分别进行SDS-PAGE和双向电泳（2-DE）分离;通过Western 和2D-Western确认抗ULF-250抗体识别的蛋白成分并与凝胶上的条带或斑点相对应;对目标蛋白分别用MALDI-TOF-MS和HPLC-ESI-MS/MS两种方法进行质谱分析并获得肽段序列数据;与蛋白数据库比对及文献检索鉴定未知蛋白。2D-Western显示抗ULF-250抗体特异识别的蛋白成分。从2D胶上切下对应的蛋白斑点进行MALDI-TOF-MS。 结果提示：ULF-250是ebnerin/DMBT1。另外,经SDS-PAGE分离的250 kD蛋白条带进行液-质联用分析。结果同样提示：ULF-250是ebnerin/DMBT1。与文献报道比较,ULF-250与ebnerin/DMBT1在子宫的组织定位和调节是相同的。因此,我们认为ULF-250是子宫表达的ebnerin/DMBT1。通过2-DE结合质谱分析以及查阅文献确认未知的雌激素反应蛋白ULF-250是ebnerin/DMBT1。此外,我们的研究提示,该蛋白不仅在子宫上皮细胞表达而且分泌到子宫腔液中,其功能值得进一步研究。     

Traumatin- and Dinortraumatin-containing Galactolipids in Arabidopsis: THEIR FORMATION IN TISSUE-DISRUPTED LEAVES AS COUNTERPARTS OF GREEN LEAF VOLATILES*

Green leaf volatiles (GLVs) consisting of six-carbon aldehydes, alcohols, and their esters, are biosynthesized through the action of fatty acid hydroperoxide lyase (HPL), which uses fatty acid hydroperoxides as substrates. GLVs form immediately after disruption of plant leaf tissues by herbivore attacks and mechanical wounding and play a role in defense against attackers that attempt to invade through the wounds. The fates and the physiological significance of the counterparts of the HPL reaction, the 12/10-carbon oxoacids that are formed from 18/16-carbon fatty acid 13-/11-hydroperoxides, respectively, are largely unknown. In this study, we detected monogalactosyl diacylglycerols (MGDGs) containing the 12/10-carbon HPL products in disrupted leaf tissues of Arabidopsis, cabbage, tobacco, tomato, and common bean. They were identified as an MGDG containing 12-oxo-9-hydroxy-(E)-10-dodecenoic acid and 10-oxo-7-hydroxy-(E)-8-decenoic acid and an MGDG containing two 12-oxo-9-hydroxy-(E)-10-dodecenoic acids as their acyl groups. Analyses of Arabidopsis mutants lacking HPL indicated that these MGDGs were formed enzymatically through an active HPL reaction. Thus, our results suggested that in disrupted leaf tissues, MGDG-hydroperoxides were cleaved by HPL to form volatile six-carbon aldehydes and non-volatile 12/10-carbon aldehyde-containing galactolipids. Based on these results, we propose a novel oxylipin pathway that does not require the lipase reaction to form GLVs.     

Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.     

SUMOylation of Pancreatic Glucokinase Regulates Its Cellular Stability and Activity

Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 β-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic β-cells.     

Intracellular localization of prenyltransferases of isoflavonoid phytoalexin biosynthesis in bean and soybean

The intracellular localization of prenyltransferases involved in the biosynthesis of the phytoalexins glyceollin in soybean (Glycine max L.) and phaseollin in French bean (Phaseolus vulgaris L.) has been investigated. By sucrose- and Percoll-gradient centrifugation of microsomes of an elicitor-challenged soybean cell culture, the membranes containing prenyltransferase were separated from the endoplasmic reticulum and shown to be lighter in density. In a continuous Percoll gradient the peak of prenyltransferase activity coincided with the peak of galactolipid synthesis, as determined by incorporation of uridine 5′-diphospho-[¹⁴C]galactose (UDP-[¹⁴C]galactose). Intact chloroplasts isolated from cupricchloride-treated bean leaves contained both prenyltransferase and UDP-galactose transferase activity. Both activities increased during chloroplast isolation. Fractionation of swollen chloroplasts on a discontinuous sucrose gradient showed prenyltransferase and UDP-galactose transferase activity in the envelope membrane subfraction. It is concluded that in both plants prenyltransferase is located in the envelope membrane of plastids. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

A major portion of DNA gyrase inhibitor microcin B17 undergoes an N,O-peptidyl shift during synthesis

Microcin B17 (McB) is a 43-amino acid antibacterial peptide targeting the DNA gyrase. The McB precursor is ribosomally produced and then post-translationally modified by the McbBCD synthase. Active mature McB contains eight oxazole and thiazole heterocycles. Here, we show that a major portion of mature McB contains an additional unusual modification, a backbone ester bond connecting McB residues 51 and 52. The modification results from an N → O shift of the Ser(52) residue located immediately downstream of one of McB thiazole heterocycles. We speculate that the N,O-peptidyl shift undergone by Ser(52) is an intermediate of post-translational modification reactions catalyzed by the McbBCD synthase that normally lead to formation of McB heterocycles.     

Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis

The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4-reductase (DFR) catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins, two flavonoid classes of importance to plant survival and human nutrition. This enzyme has been widely investigated in many plant species, but little is known about its structural and biochemical properties. To provide a basis for detailed structure-function studies, the crystal structure of Vitis vinifera DFR, heterologously expressed in Escherichia coli, has been determined at 1.8 Å resolution. The 3D structure of the ternary complex obtained with the oxidized form of nicotinamide adenine dinucleotide phosphate and dihydroquercetin, one of the DFR substrates, presents common features with the short-chain dehydrogenase/reductase family, i.e., an N-terminal domain adopting a Rossmann fold and a variable C-terminal domain, which participates in substrate binding. The structure confirms the importance of the 131-156 region, which lines the substrate binding site and enlightens the role of a specific residue at position 133 (Asn or Asp), assumed to control substrate recognition. The activity of the wild-type enzyme and its variant N133D has been quantified in vitro, using dihydroquercetin or dihydrokaempferol. Our results demonstrate that position 133 cannot be solely responsible for the recognition of the B-ring hydroxylation pattern of dihydroflavonols.     

Spectrin Domain of Eukaryotic Initiation Factor 3a Is the Docking Site for Formation of the a:b:i:g Subcomplex

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10–15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.     

Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana: ENZYMES CATALYZING UP TO 10 REARRANGEMENT STEPS YIELDING FRIEDELIN AND OTHER TRITERPENOIDS*

The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C₃₀H₅₀O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761–779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.