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.     

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.     

γH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products

Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as γH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that γH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring γH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that γH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that γH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.     

Methylene analogues of adenosine 5'-tetraphosphate. Their chemical synthesis and recognition by human and plant mononucleoside tetraphosphatases and dinucleoside tetraphosphatases

Adenosine 5'-polyphosphates have been identified in vitro, as products of certain enzymatic reactions, and in vivo. Although the biological role of these compounds is not known, there exist highly specific hydrolases that degrade nucleoside 5'-polyphosphates into the corresponding nucleoside 5'-triphosphates. One approach to understanding the mechanism and function of these enzymes is through the use of specifically designed phosphonate analogues. We synthesized novel nucleotides: alpha,beta-methylene-adenosine 5'-tetraphosphate (pppCH2pA), beta,gamma-methylene-adenosine 5'-tetraphosphate (ppCH2ppA), gamma,delta-methylene-adenosine 5'-tetraphosphate (pCH2pppA), alphabeta,gammadelta-bismethylene-adenosine 5'-tetraphosphate (pCH2ppCH2pA), alphabeta, betagamma-bismethylene-adenosine 5'-tetraphosphate (ppCH2pCH2pA) and betagamma, gammadelta-bis(dichloro)methylene-adenosine 5'-tetraphosphate (pCCl2pCCl2ppA), and tested them as potential substrates and/or inhibitors of three specific nucleoside tetraphosphatases. In addition, we employed these p4A analogues with two asymmetrically and one symmetrically acting dinucleoside tetraphosphatases. Of the six analogues, only pppCH2pA is a substrate of the two nucleoside tetraphosphatases (EC 3.6.1.14), from yellow lupin seeds and human placenta, and also of the yeast exopolyphosphatase (EC 3.6.1.11). Surprisingly, none of the six analogues inhibited these p4A-hydrolysing enzymes. By contrast, the analogues strongly inhibit the (asymmetrical) dinucleoside tetraphosphatases (EC 3.6.1.17) from human and the narrow-leafed lupin. ppCH2ppA and pCH2pppA, inhibited the human enzyme with Ki values of 1.6 and 2.3 nm, respectively, and the lupin enzyme with Ki values of 30 and 34 nm, respectively. They are thereby identified as being the strongest inhibitors ever reported for the (asymmetrical) dinucleoside tetraphosphatases. The three analogues having two halo/methylene bridges are much less potent inhibitors for these enzymes. These novel nucleotides should prove valuable tools for further studies on the cellular functions of mono- and dinucleoside polyphosphates and on the enzymes involved in their metabolism.     

Polo-like kinase 3 is Golgi localized and involved in regulating Golgi fragmentation during the cell cycle

The Golgi apparatus undergoes extensive fragmentation during mitosis in animal cells. Protein kinases play a critical role during fragmentation of the Golgi apparatus. We reported here that Polo-like kinase 3 (Plk3) may be an important mediator during Golgi breakdown. Specifically, Plk3 was concentrated at the Golgi apparatus in HeLa and A549 cells during interphase. At the onset of mitosis, Plk3 signals disintegrated and redistributed in a manner similar to those of Golgi stacks. Nocodazole activated Plk3 kinase activity, correlating with redistribution of Plk3 signals and Golgi fragmentation. In addition, treatment with brefeldin A (BFA), a Golgi-specific poison, also resulted in disappearance of concentrated Plk3 signals. Plk3 interacted with giantin, a Golgi-specific protein. Expression of Plk3, but not the kinase-defective Plk3 (Plk3(K52R)), resulted in significant Golgi breakdown. Given its role in cell cycle progression, Plk3 may be a protein kinase involved in regulation of Golgi fragmentation during the cell cycle.     

Thermodynamics of mRNA 5' cap binding by eukaryotic translation initiation factor eIF4E

Translation of mRNA in eukaryotes begins with specific recognition of the 5' cap structure by the highly conserved protein, eIF4E. The thermodynamics of eIF4E interaction with nine chemical cap analogues has been studied by means of emission spectroscopy. High-sensitivity measurements of intrinsic protein fluorescence quenching upon cap binding provided equilibrium association constants in the temperature range of 279 to 314 K. A van't Hoff analysis yielded the negative binding enthalpies for the entire cap analogue series, -16.6 to -81 kJ mol(-1), and the entropies covering the range of +40.3 to -136 J mol(-1) K(-1) at 293 K. The main enthalpic contributions come from interactions of the phosphate chains and positively charged amino acids and the cation-pi stacking of 7-methylguanine with tryptophans. A nontrivial, statistically important isothermal enthalpy-entropy compensation has been detected (T(c) = 399 +/- 24 K), which points to significant fluctuations of apo-eIF4E and indicates that the cap-binding microstate lies 9.66 +/- 1.7 kJ mol(-1) below the mean energy of all available conformational states. For five cap analogues, large and positive heat capacity changes have been found. The values of DeltaC(p) degrees correlate with the free energies of eIF4E binding due to stiffening of the protein upon interaction with cap analogues. At biological temperatures, binding of the natural caps has both favorable enthalpy and favorable entropy. Thermodynamic coupling of cap-eIF4E association to intramolecular self-stacking of dinucleotide cap analogues strongly influences the enthalpies and entropies of the binding, but has a negligible effect on the resultant DeltaG degrees and DeltaC(p) degrees values.     

Charge distribution in 7-methylguanine regarding cation-pi interaction with protein factor eIF4E

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.