Protein S3 in the human 80S ribosome adjoins mRNA 3′ of the A-site codon

The protein environment of mRNA 3′ of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5′-end and a perfluoroarylazide group at one of the nucleotide residues 3′ of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNA^Phe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNA^Phe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in ternary complexes and in the absence of tRNA. Within ternary complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed; it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3′ of the codon in the decoding site.     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

The absence of the CpNpG methylation at the 5′-terminal region of the human calcitonin gene in norm and leukemias

The inner cytosine methylation was analyzed in the CCWGG sequences of the 5′-terminal region of the human calcitonin gene from peripheral blood and bone marrow cells in various forms of leukemia. Since these sequences remain nonmethylated both in the norm and in various leukemia forms, the CpG dinucleotide hypermethylation of the 5′-end of the human calcitonin gene, characteristic for the development of leukemias, does not spread to adjacent CpNpG sequences.     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

Interaction of elongation factor EF-Tu with γ-amides of GTP and β-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate

New types of azidoaryl analogs of GTP: γ-(4-azido)anilide of GTP (I), γ-(N-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: β-(4-azido)anilide of GDP (III), β-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N′-β-(4-methylmorpholinium)- ethylcarbodiimidep-toluene sulfonate and the respective amine. The analog of GTP bearing at the γ-phosphate an alkylating 2-chloroethylamino group: γ-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A. and Samukov, V.V. (1976) FEBS Lett. 70, 105–108). Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu·GDP with the respective K_i values 3.9·10^?7 M (I), 2.9·10^?8 M (II), 6.9·10^?7 M (III), 5.0·10^?7 M (IV) and 3.8·10^?8 M (V) relative to GDP. constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5·10^?6 M, 3.4·10^?7 M and 4.6·10^?8 M, respectively, or approx. 1740-, 70- and 9-times greater than that of GDP. GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

Influence of the Length of the Phosphate Chain in mRNA 5′ Cap Analogues on Their Interaction with Eukaryotic Initiation Factor 4E

Abstract

The recognition of the 5′mRNA cap structure m⁷G(5′)ppp(5′)N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

Inhibition of hexose transport in the human erythrocyte by 5, 5′-dithiobis(2-nitrobenzoic acid): Role of an exofacial carrier sulfhydryl group

Summary The sulfhydryl reagent 5, 5-dithiobis (2-nitrobenzoic acid) (DTNB) was used to study the functional role of an exofacial sulfhydryl group on the human erythrocyte hexose carrier. Above 1mm DTNB rapidly inhibited erythrocyte 3-O-methylglucose influx, but only to about half of control rates. Efflux was also inhibited, but to a lesser extent. Uptake inhibition was completely reversed by incubation and washing with 10mm cysteine, whereas it was only partially reduced by washing in buffer alone, suggesting both covalent and noncovalent interactions. The covalent thiol-reversible reaction of DTNB occurred on the exofacial carrier, since (i) penetration of DTNB into cells was minimal, (ii) blockade of potential uptake via the anion transporter did not affect DTNB-induced hexose transport inhibition, and (iii) DTNB protected from transport inhibition by the impermeant sulfhydryl reagent glutathione-maleimide-I. Maltose at 120mm accelerated the covalent transport inhibition induced by DTNB, whereas 6.5 m cytochalasin B had the opposite effect, indicating under the one-site carrier model that the reactive sulfhydryl is on the outward-facing carrier but not in the substrate-binding site. In contrast to glutathione-maleimide-I, however, DTNB did not restrict the ability of the carrier to reorient inwardly, since it did not affect equilibrium cytochalasin B binding. Thus, carrier conformation determines exposure of the exofacial carrier sulfydryl, but reaction of this group may not always lock the carrier in an outward-facing conformation.     

Interactions of cellular histidine triad nucleotide binding protein 1 with nucleosides 5′-O-monophosphorothioate and their derivatives — Implication for desulfuration process in the cell

BackgroundOne of the activities of histidine triad nucleotide-binding protein 1 (Hint1) under in vitro conditions is the conversion of nucleoside 5′-O-phosphorothioate (NMPS) to its 5′-O-phosphate (NMP), which is accompanied by the release of hydrogen sulfide.MethodsNon-hydrolyzable derivatives of AMPS and dCMPS, each containing the residue able to form a covalent bond in nucleic acid–protein complexes via photocrosslinking (at 308 nm), were applied at the complexing experiments with recombinant and cellular Hint1.The cellular lysates prepared after RNAi-mediated knockdown of Hint1 were incubated with AMPS and the level of desulfuration was measured.ResultsRecombinant Hint1 and Hint1 present in the cellular lysate of A549 cells, formed complexes with the used substrate analogs.Computer modeling experiments, in which the ligand was docked at the binding pocket, confirmed that direct interactions between Hint1 and the screened analogs are possible.Using RNAi technology, we demonstrated lowered levels of AMPS substrate desulfuration in reactions that employed the cell lysates with a reduced Hint1 level.ConclusionsThe enzymatic conversion of AMPS to AMP occurred with the participation of cellular Hint1, the protein, which is present in all organisms.General significanceThe intracellular Hint1 could be responsible for the in vivo desulfuration of nucleosides-5′-monophosphorothioate, thus it can contribute to the phosphorothioate oligonucleotides metabolism. H₂S released during this process may participate in several physiological processes, thus NMPSs can be precursors/donors of H₂S in vivo and can be used to study the effects of this gas in biological systems. Moreover, the controlled delivery of (d)NMPSs into cells may be of medicinal utility.     

Properties and reaction with iodoacetamide of adenosine 5′-triphosphate–creatinine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP-Mg(2+) plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP-Mg(2+) plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP-Mg(2+) plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.     

C to T and/or G to A transitions are responsible for loss of a MspI restriction site at the 5′-end of the human apolipoprotein AI gene

We detected the loss of a MspI restriction site by a C to T transition at +83 bp and a G to A transition at +84 bp of the 5-end non-coding region of the human apolipoprotein AI gene. This base change occurred at the hot spot (CCGG) for methylation, which may be important in the regulation of gene expression. The population frequency for the loss of the MspI site is 6.1%.     

Changes of primary sequence and secondary structure proximal to the 5′ end of the stop codon substantially increases the expression of the variable region of an antibody in <Emphasis Type="Italic">E. coli</Emphasis>

The sequence context at the 5 end of the stop codon may influence the efficiency of termination and translation. To increase the expression of a designed variable region of an antibody (named as VH5) against tumor necrosis factor (TNF), two nucleotides (TC) at 25 and 26 nucleotides (nt) upstream of termination codon were substituted with AG, respectively. The free energy of 70 nt (arbitrarily defined from the 32 nt upstream of termination codon to 38 nt downstream) was changed from –13.5 kcal mol^-1 to –17.3 kcal mol^-1. The expression level was increased from 1 ± 0.3% to 10 ± 1.2% of total cellular protein. Although the precise mechanism of this phenomenon remains to be elucidated, this report provides an alternative means to increase the expression of a foreign gene in E. coli. Revisions requested 18 October 2004; Revisions received 26 November 2004