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Biomedical subjects

Edward Darzynkiewicz

Publications and source records attributed to Edward Darzynkiewicz.

At least 19 recordsLinked to original sources
PubMed2006

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

3' Untranslated Regions↗
PubMed2006

Stopped-flow kinetic analysis of eIF4E and phosphorylated eIF4E binding to cap analogs and capped oligoribonucleotides: evidence for a one-step binding mechanism.

Recruitment of eukaryotic mRNA to the 48 S initiation complex is rate-limiting for protein synthesis under normal conditions. Binding of the 5' -terminal cap structure of mRNA to eIF4E is a critical event during this process. Mammalian eIF4E is phosphorylated at Ser-209 by Mnk1 and Mnk2 kinases. We investigated the interaction of both eIF4E and phosphorylated eIF4E (eIF4E(P)) with cap analogs and capped oligoribonucleotides by stopped-flow kinetics. For m(7)GpppG, the rate constant of association, k(on), was dependent on ionic strength, decreasing progressively up to 350 mm KCl, but the rate constant of dissociation, k(off), was independent of ionic strength. Phosphorylation of eIF4E decreased k(on) by 2.1-2.3-fold at 50-100 mm KCl but had progressively less effect at higher ionic strengths, being negligible at 350 mm. Contrary to published evidence, eIF4E phosphorylation had no effect on k(off). Several observations supported a simple one-step binding mechanism, in contrast to published reports of a two-step mechanism. The kinetic function that best fit the data changed from single- to double-exponential as the eIF4E concentration was increased. However, measuring k(off) for dissociation of a pre-formed eIF4E.m(7)GpppG complex suggested that the double-exponential kinetics were caused by dissociation of eIF4E dimers, not a two-step mechanism. Addition of a 12-nucleotide chain to the cap structure increased affinity at high ionic strength for both eIF4E (24-fold) and eIF4E(P) (7-fold), primarily due to a decrease in k(off). This suggests that additional stabilizing interactions between capped oligoribonucleotides and eIF4E, which do not occur with cap analogs alone, act to slow dissociation.

Animals↗
PubMed2006

Enzymatically stable 5' mRNA cap analogs: synthesis and binding studies with human DcpS decapping enzyme.

Four novel 5' mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl2. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3' to 5' cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.

Endoribonucleases↗
PubMed2006

Trimethylguanosine nucleoside inhibits cross-linking between Snurportin 1 and m3G-CAPPED U1 snRNA.

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin alpha/beta. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import.

Animals↗
PubMed2006

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.

Acid Anhydride Hydrolases↗
PubMed2005

Differential inhibition of mRNA degradation pathways by novel cap analogs.

mRNA degradation predominantly proceeds through two alternative routes: the 5'-->3' pathway, which requires deadenylation followed by decapping and 5'-->3' hydrolysis; and the 3'-->5' pathway, which involves deadenylation followed by 3'-->5' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp(3)G, m(7)Gp(3)G, or m(2)(7,3'-O) Gp(3)G) and 3' termini (A(31),A(60), or G(16)) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'-end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5'-->3' versus 3'-->5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the alpha- and beta-phosphate moieties, m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m(2)(7,3'-O)Gpp(CH2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2)(7,3'-O)Gpp(CH2)pG, but not m(2)(7,3'-O)Gp(CH2)ppG, were more stable in vivo, indicating that the 5'-->3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2)(7,3'-O)Gpp(CH2)pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.

5' Untranslated Regions↗
PubMed2005

The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro.

The eukaryotic initiation factor eIF4E binds the mRNA 5' cap structure and has a central role during translational initiation. eIF4E and the mechanisms to control its activity have oncogenic properties and thus have become targets for anticancer drug development. A recent study (Kentsis et al. 2004) presented evidence that the antiviral nucleoside ribavirin and its phosphorylated derivatives were structural mimics of the mRNA cap, high-affinity ligands for eIF4E, and potent repressors of eIF4E-mediated cell transformation and tumor growth. Based on these findings, we tested ribavirin, ribavirin triphosphate (RTP), and the dinucleotide RpppG for their ability to inhibit translation in vitro. Surprisingly, the ribavirin-based compounds did not affect translation at concentrations where canonical cap analogs efficiently block cap-dependent translation. Using a set of reporter mRNAs that are translated via either cap-dependent or viral internal ribosome entry sites (IRES)-dependent initiation, we found that these ribavirin-containing compounds did inhibit translation at high (millimolar) concentrations, but there was no correlation of this inhibition with an eIF4E requirement for translation. The addition of a ribavirin-containing cap to mRNA did not stimulate translation. Fluorescence titration experiments with eIF4E and the nuclear cap-binding complex CBC indicated affinities for RTP and RpppG that were two to four orders of magnitude lower than those of m(7)GTP and m(7)GpppG. We conclude that, at least with respect to translation, ribavirin does not act in vitro as a functional mimic of the mRNA cap.

Antiviral Agents↗
PubMed2005

Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.

The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.

Binding Sites↗
PubMed2005

Cross-talk between snurportin1 subdomains.

The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. After formation of an Sm-core and a trimethylguanosine (TMG) cap, the RNPs are transported into the nucleus via the import adaptor snurportin1 (SPN) and the import receptor importin-beta. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin-beta, and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin-beta binding domain (IBB) disrupted the interaction with importin-beta, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are required for TMG binding. However, SPN can be imported into the nucleus without cargo. Interestingly, SPN targets to Cajal bodies when U2 but not U1 snRNPs are imported as cargo. SPN also relocalizes to Cajal bodies upon treatment with leptomycin B. Finally, we uncovered an interaction between the N- and C-terminal domains of SPN, suggesting an autoregulatory function similar to that of importin-alpha.

Active Transport, Cell Nucleus↗
PubMed2005

Synthesis and biochemical properties of novel mRNA 5' cap analogs resistant to enzymatic hydrolysis.

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m7GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m7GpppG.

Animals↗
PubMed2005

Novel dinucleoside 5',5'-triphosphate cap analogues. Synthesis and affinity for murine translation factor eIF4E.

Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin A, 3'-O-methylguanosine, 9-beta-D-arabinofuranosyladenine, and isoguanosine, has been performed using our new methodology. The key reactions of pyrophosphate bonds formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures of the new cap analogues were confirmed by 1H NMR and 31p NMR spectra. The binding affinity of the new cap analogues for murine eIF4E(28-217) were determined spectroscopically showing the highest association constant for the analogue that contains formycin A.

Adenosine↗
PubMed2005

New affinity resin for purification of cap-binding proteins.

Cap binding proteins, which recognize the cap structure present at 5' termini of RNA polymerase II transcripts, have been routinely isolated and purified using affinity resins with mononucleotide cap analogs attached. Here we present a new methodology in which dinucleotide cap analog, m7GpppG, has been linked to the EAH-Sepharose. The method is based on derivatization of 2',3'-cis diol of the second nucleotide within the cap structure, with levulinic acid, and subsequent coupling of resulted acetal through its carboxylic group with aminohexyl-agarose.

Carrier Proteins↗
PubMed2005

Deaggregation of eIF4E induced by mRNA 5' cap binding.

All eukaryotic mRNAs contain a 5' terminal cap structure, which consists of 7-methylguanosine linked by a 5-5' triphosphate bridge to the first transcribed nucleoside (m7GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33-217) was shown to aggregate. After addition of m7G7P, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.

Animals↗
PubMed2005

Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex.

Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.

Dose-Response Relationship, Drug↗
PubMed2005

Synthesis and enzymatic characterization of methylene analogs of adenosine 5'-tetraphosphate (P4A).

A new methodology for synthesis of biologically important nucleoside tri- and tetraphosphates containing a bisphosphonate moiety instead of the terminal pyrophosphate bond is described. The series consists of tri- and tetraphosphate analogs of adenosine, guanosine and 7-methylguanosine (characteristic for mRNA cap). We have adopted a two-step procedure that allowed us to insert a methylene bridge into the phosphate chain. Nucleoside mono- or diphosphates were first activated (as imidazole derivatives) and then used in coupling reactions with organic salts of bisphosphonate. The resulting synthetic method enabled us to obtain the desired compounds with high yields and does not require any protective groups. This makes it very useful for the synthesis of labile compounds such as those containing the 7-methylguanosine ring. The structures of the synthesized compounds were confirmed by NMR spectroscopy. They were tested as potential substrates and inhibitors of several hydrolases.

Adenine Nucleotides↗
PubMed2005

Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5',5'-triphosphate chain.

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the alpha, beta, or gamma position of 5',5'-triphosphate chain [m7Gp(s)ppG, m7Gpp(s)pG, and m7Gppp(s)G]. These are the first examples of the biologically important 5'mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the alpha or gamma position within the dinucleotide cap analog was achieved using PSCl3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in beta position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1H and 31P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.

Animals↗
PubMed2004

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.

Animals↗