Detection of methyltransferase activities which modify Gppp G TO m7GpppGm in embryonic chick lens

GpppG was modified to m⁷GpppGm by a cytoplasmic extract, prepared from embryonic lens cells, in a reaction mixture which contained S-adenosyl-methionine as methyl group donor. The appearance of m⁷GpppGm was a function of time and lens extract concentration. S-adenosyl-homocysteine inhibited both the m⁷G and Gm modification reactions. Analogues of GpppG, pG, ppG and pppG were relatively ineffective as substrates.     

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

N⁷-methylguanine at position 46 (m⁷G46) in tRNA is produced by tRNA (m⁷G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m⁷G46 modification in tRNA^Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m⁷G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m¹G37, suggesting that the m⁷G46 positively affects their formations. Although the lack of the m⁷G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA^Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA^Phe and tRNA^Ile. ³⁵S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m⁷G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m⁷G46 modification supports introduction of other modifications.     

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5′ cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn⁷GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N⁷-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5′-phosphorothioate (m⁷GMPS), which is a translation inhibitor with in vitro potency higher than bn⁷GMP.     

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5′-mRNA cap, i.e., m⁷GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m⁷GMP (or m^2,7GMP) as one of the reaction products. Interestingly, m⁷Gpppm₃^{N6, N6, 2′O}A was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m⁷Gpppm₃^{N6, N6, 2′O}Apm^2′OApm^2′OCpm₂^{N3, 2′O}U, that occurs in these organisms.     

Structural Insights into Parasite eIF4E Binding Specificity for m7G and m2,2,7G mRNA Caps

The eukaryotic translation initiation factor eIF4E recognizes the mRNA cap, a key step in translation initiation. Here we have characterized eIF4E from the human parasite Schistosoma mansoni. Schistosome mRNAs have either the typical monomethylguanosine (m⁷G) or a trimethylguanosine (m^2,2,7G) cap derived from spliced leader trans-splicing. Quantitative fluorescence titration analyses demonstrated that schistosome eIF4E has similar binding specificity for both caps. We present the first crystal structure of an eIF4E with similar binding specificity for m⁷G and m^2,2,7G caps. The eIF4E·m⁷GpppG structure demonstrates that the schistosome protein binds monomethyl cap in a manner similar to that of single specificity eIF4Es and exhibits a structure similar to other known eIF4Es. The structure suggests an alternate orientation of a conserved, key Glu-90 in the cap-binding pocket that may contribute to dual binding specificity and a position for mRNA bound to eIF4E consistent with biochemical data. Comparison of NMR chemical shift perturbations in schistosome eIF4E on binding m⁷GpppG and m^2,2,7GpppG identified key differences between the two complexes. Isothermal titration calorimetry demonstrated significant thermodynamics differences for the binding process with the two caps (m⁷G versus m^2,2,7G). Overall the NMR and isothermal titration calorimetry data suggest the importance of intrinsic conformational flexibility in the schistosome eIF4E that enables binding to m^2,2,7G cap.     

Virus-Specific mRNA Capping Enzyme Encoded by Hepatitis E Virus

Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m⁷GTP or m⁷GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m⁷GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m⁷GMP, in the presence of AdoMet and GTP, because radioactivity from both [α-³²P]GTP and [³H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m⁷GTP, m⁷GDP, et²m⁷GMP, and m²et⁷GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.     

Phosphorothioate analogs of m7GTP are enzymatically stable inhibitors of cap-dependent translation

We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5′-(1-thiotriphosphate). These new analogs of mRNA 5′cap (referred to as m⁷GTPαS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m⁷GTPαS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate ～8-fold and ～15-fold more efficiently than m⁷GTP and m⁷GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.     

The predominant contribution of platelets to baseline and ascorbate-stimulated increments in cyclic GMP in human mononuclear leukocyte preparations.

In nine consecutive experiments with Ficoll-Hypaque-purified human mononuclear leukocytes containing 2.8 (range 1.1–4.3) platelets per leukocyte, 2–5 mM sodium ascorbate produced a 14-fold (range, 7- to 18-fold) rise in guanosine 3′: 5′-cyclic monophosphate (cyclic GMP) from baseline levels of 0.103 ± 0.056 pmol/10⁷ mononuclear leukocytes. In five experiments with mononuclear leukocytes prepared by the Ficoll-Hypaque method from human blood depleted of platelets by defibrination, 2–5 mM sodium ascorbate produced a twofold (range, one- to fourfold) rise in cyclic GMP from baseline levels of 0.030 ± 0.012 pmol/10⁷ mononuclear leukocytes. Thus, platelets contribute substantially to baseline and ascorbate-stimulated levels of cyclic GMP in standard Ficoll-Hypaque preparations of mononuclear leukocytes. The rise in cyclic GMP concentration in mononuclear leukocyte preparations elicited by ascorbate was independent of a calcium requirement, persisted for up to 3 hr in the presence of ascorbate, and was prevented by the introduction of nonsteroidal anti-inflammatory agents such as aspirin and indomethacin (ID₅₀ = 105 and 23.5 μM, respectively).     

Interaction of Adenosine and Guanine Derivatives in the Rat Hippocampus: Effects on Cyclic AMP Levels and on the Binding of Adenosine Analogues and GMP

Guanine nucleotides (GN) have been implicated in many intracellular mechanisms. Extracellular actions, probably as glutamate receptor antagonists, have also been recently attributed to these compounds. GN may have a neuroprotective role by inhibiting excitotoxic events evoked by glutamate. Effects of extracellular GN on adenosine-evoked cellular responses have also been reported. However, the exact mechanism of such interaction is not known. In the present study, we showed that GN potentiated adenosine-induced cAMP accumulation in slices of hippocampus from young rats. However, neither GMP nor the metabotropic glutamate receptor agonist, 1S,3R-ACPD, inhibited the binding of the adenosine receptor agonist [³H]NECA (when binding to adenosine A2 receptors), or the binding of the adenosine A2a receptor agonist [³H]CGS 21680 in hippocampal membrane preparations. GppNHp, probably by interacting with G-proteins, decreased [³H]CGS 21680 binding. [³H]GMP binding was assayed in order to evaluate the GN sites which are not G-proteins. [³H]GMP binding was inhibited by GMP and GppNHp, but not by 1S,3R-ACPD. The interaction of endogenous adenosine with the GMP-binding sites was determined by incubating membranes in the presence or absence of adenosine deaminase (ADA). NECA, CADO, CGS 21680 and CPA (only at the highest concentration used) increased GMP binding in the presence of ADA. However, in the absence of ADA, the control levels of GMP binding were as high as in the presence of added ADA plus adenosine agonists, indicating that endogenous adenosine modulates the binding of GMP. If this site has a neuroprotective role, adenosine may be increasing its neuromodulator and proposed protective action.     

Cyclic nucleotide binding and protein kinase in the central nervous system of Galleria mellonella (L.)

The binding of cyclic AMP, IMP and GMP by the central nervous system of Galleria mellonella was studied. The K_m for cyclic AMP was 1 · 10^?7 and that for cyclic GMP 1 · 10^?8. The results suggest a different binding protein and cyclic nucleotide-stimulated protein kinase for each of these nucleotides. In addition a cyclic IMP-dependent protein kinase may also be present.     

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode

Poly(A)-specific ribonuclease (PARN) is a processive 3′-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3′ end of the mRNA but also with its 5′ end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m⁷G) cap. The interaction of PARN with the 5′ cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m⁷G triphosphate nucleotide, revealing a novel binding mode for the m⁷G cap. The structure of the m⁷G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m⁷G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two α-helices with an adjacent protein molecule in the crystal lattice.     

Conformational Polymorphism of m7GTP in Crystal Structure of the PB2 Middle Domain from Human Influenza A Virus

Influenza pandemics with human-to-human transmission of the virus are of great public concern. It is now recognized that a number of factors are necessary for human transmission and virulence, including several key mutations within the PB2 subunit of RNA-dependent RNA polymerase. The structure of the middle domain in PB2 has been revealed with or without m⁷GTP, thus the middle domain is considered to be novel target for structure-based drug design. Here we report the crystal structure of the middle domain of H1N1 PB2 with or without m⁷GTP at 1.9Å and 2.0Å resolution, respectively, which has two mutations (P453H, I471T) to increase electrostatic potential and solubility. Here we report the m⁷GTP has unique conformation differ from the reported structure. 7-methyl-guanine is fixed in the pocket, but particularly significant change is seen in ribose and triphosphate region: the buried 7-methyl-guanine indeed binds in the pocket forming by H357, F404, E361 and K376 but the triphosphate continues directly to the outer domain. The presented conformation of m⁷GTP may be a clue for the anti-influenza drug-design.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Dinucleoside 5′,5-P,P-Triphosphate Hydrolase from Yellow Lupin (Lupinus luteus) Seeds: Purification to Homogeneity and Hydrolysis of mRNA 5′-Cap Analogs

Three separable forms of diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15MKCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS–polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain ofM_r41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5′-cap structure, have been tested as potential substrates of the lupin “Ap₃A hydrolase.” All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m⁷Gp₃G, et⁷Gp₃G, and bz⁷Gp₃G were hydrolyzed randomly, whereas m⁷Gp₃A, m⁷Gp₃C, and m⁷Gp₃U yielded at least 80% m⁷GMP plus corresponding NDP and 20% or less NMP plus m⁷GDP. Hydrolysis of the guanosine-containing hybrids, Ap₃G, Cp₃G, and Up₃G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m⁷G- moiety were hydrolyzed 2- to 4.5-fold faster than Ap₃A. Thus a general name, “dinucleoside triphosphate hydrolase,” is more appropriate for the enzymatic activity described.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

The blocked and methylated 5′ terminal sequence of a specific cellular messenger: the mRNA for silk fibroin of bombyx mori

The messenger RNA for silk fibroin, labeled with ³²PO₄ and methyl-³H L-methionine, was purified to near homogeneity from the posterior silk gland of the silkworm Bombyx mori, and the sequence of a methylated, RNAase T2-resistant structure was determined. This sequence is similar structurally to 5′ terminal blocked and methylated sequences found on the total populations of polyadenylated eucaryotic cellular and certain viral mRNAs. The RNAase T2-resistant oligomer from fibroin mRNA was cleaved by nuclease P1 into three components: a blocked and methylated sequence containing three phosphates; a 2′-0-methyl UMP residue (pU^m), and an unmethylated CMP (pC). The blocked and methylated sequence comigrated in three chromatographic systems with the blocked and methylated terminus of silkworm cytoplasmic polyhedrosis virus mRNA, which has the structure m⁷GpppA^m. The fibroin mRNA cap was cleaved by nucleotide pyrophosphatase to yield 7-methyl GMP (pm⁷G) and 2′-0-methyl AMP (pA^m). This sequence also appeared to be terminally located, with the m⁷G joined by a 5′-5′ pyrophosphate linkage to the A^m. It was concluded that the 5′ terminal sequence of fibroin mRNA molecules is m⁷G(5′)ppp(5′)A^mpU^mpCp. The regulation of expression of the highly specialized gene for fibroin is discussed in light of this finding.     

Technetium-99m in the Diagnosis of Thyrotoxicosis

The thyroid uptake at 20 minutes of intravenously administered Technetium-99^m (^99mTc) was measured in 117 patients with a standard scintillation counter. Patients were divided into three groups on the basis of clinical assessment, four-hour ¹³¹I uptake, triiodothyronine (T-3) resin uptake, and protein-bound iodine measurements.In 31 patients with no evidence of thyroid disease the mean ^99m Tc uptake was 1·8% ±S.D. 1·1%. In 32 patients with thyroid enlargement who were euthyroid the mean uptake was 2·5% ±S.D. 2·2%. In 54 thyrotoxic patients the mean uptake was 17·7% with a range of 4·1 to 44%, all cases having an uptake above the upper limit of normal (4·0%). These results agree closely with reported uptake studies using scanning techniques. In seven patients the extrathyroidal neck activity was measured by using a scanner, and the mean was 6·3% of the extrathyroidal total body radioactivity comparing favourably with an assumed 6% used in our calculations.We have shown that the measurement of the thyroid uptake of ^99mTc with a scintillation counter is of value, and that it is not necessary to use scanning techniques in the diagnosis of thyrotoxicosis. Advantages of ^99m Tc are minimal radiation, reduction in patient and laboratory time, and low cost.