Gladys F. Maley

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

ABSTRACT A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.

Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro.

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.

Processing of the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level.

The interrupted T4 phage td gene, which encodes thymidylate synthase, is the first known example of an intron-containing prokaryotic structural gene. Analysis of td-encoded transcripts provides evidence in favor of maturation at the RNA level. Northern blotting with T4 RNA and with region-specific probes revealed three classes of RNA: diffuse premessage (ca. 2.5 kb), a low-abundance mature mRNA (ca. 1.3 kb), and an abundant free intron RNA (ca. 1.0 kb). The existence of covalently joined mature mRNA was suggested by hybridization and S1 protection experiments and was confirmed by primer extension analysis of the splice junction. In analogy to expression of interrupted eukaryotic genes, these results are consistent with an RNA processing model that would account for the direct gene transcript serving as precursor for both free intron RNA and a spliced mRNA that is colinear with the thymidylate synthase product.

Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP.

The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 Å and 2.5 Å resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild‐type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 Å away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten‐fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.

Intracellular Location of Thymidylate Synthase and Its State of Phosphorylation

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50–100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme’s contribution to the cell cycle may be more complex than believed previously.

Tetrahydrodeoxyuridylate: A potent inhibitor of deoxycytidylate deaminase☆

Abstract Deoxycytidylate deaminase is inhibited competitively by tetrahydrodeoxyuridine 5′-monophosphate with a Ki of 1.0–2.0 × 10−8 m . The potential effectiveness of the inhibition as revealed by the K m K i ratio is of the order of 3.0–6.0 × 104. Although the deamination of deoxycytidine and cytidine in a chick embryo mince system is impaired rather drastically by tetrahydrodeoxyuridine and tetrahydrouridine, respectively, DNA synthesis is not greatly affected. Tetrahydrodeoxyuridine appears to impose a sparing effect on deoxycytidine utilization and limits the incorporation of cytidine into the thymidylate of DNA. Tetrahydrouridine severely inhibits cytidine deamination, but does not affect its incorporation into DNA. The most probable site of action of these compounds in vivo is deoxycytidylate deaminase. Of the two known pathways for the formation of deoxyuridylate, the deamination of deoxycytidylate and the ribonucleotide reductase conversion of uridine diphosphate to deoxyuridine diphosphate, the results suggest that most of the DNA thymidylate is provided by the latter. Cytidine and deoxycytidine can still be deaminated in the chick embryo mince system, in spite of sufficient tetrahydrodeoxyuridine and tetrahydrodeoxyuridylate to inhibit deoxycytidine and deoxycytidylate deaminases completely, a result that suggests an alternate pathway for the formation of deoxyuridine nucleotides from deoxycytidine nucleotides.

Studies on identifying the folylpolyglutamate binding sites of Lactobacillus casei thymidylate synthetase.

Abstract Pteroylheptaglutamate labeled with 14 C in its proximal glutamate was activated with a water-soluble carbodiimide and reacted with Lactobacillus casei thymidylate synthetase in the presence and absence of dUMP. The latter nucleotide appeared to enhance the specificity of the reaction as the quantity of folylpolyglutamate fixed plateaued at 1.5 mol/mol of enzyme, but continued to increase above 2.0 in the absence of dUMP. The location of the covalently bound pteroylheptaglutamate was determined by first cleaving the labeled protein with cyanogen bromide and establishing that 80% of the radioactivity was present in the second of the five cyanogen bromide peptides of thymidylate synthetase. This peptide was then digested with chymotrypsin and the resulting peptides were purified by high-performance liquid chromatography. Two labeled peptides were isolated, with one being separated further into two pure peptides on gel exclusion chromatography. The three peptides represented residues 48–54, 52–61, and 55–61 of the synthetase, with the pteroylheptaglutamate fixed covalently to lysines 50 or 51 and 58. From the results presented, it would appear that each of the enzyme subunits is labeled, with the folylpolyglutamate being fixed to lysine 58 of one subunit and 50, 51 of the other.

A Tale of Two Enzymes, Deoxycytidylate Deaminase and Thymidylate Synthase

Publisher Summary This chapter explains the two critical enzymes— namely, deoxycytidylate deaminase and 1-thymidylate synthase, involved in providing substrate precursors for DNA synthesis, with particular emphasis on the contributions to these areas of research. dCMP deaminase and dTMP synthase appear to be intimately associated both in their association with the mitotic process and in maintaining a flux of nucleotides into the pools of dCTP and dTTP. It was perhaps intuitive on a part to study dTMP synthesis in conjunction with the newly discovered dCMP deaminase, since it was felt that, if one enzyme provides substrate for another, particularly, at a rate-limiting step in DNA synthesis, something would come of these studies and it did. In the course of studying the properties of dCMP deaminase, it was found to be an exquisitely regulated enzyme, controlled to a fine degree by the end products of its metabolic pathway, dCTP and dTTP. However, the potential role of these enzymes in cell division took on added significance when it was found that both increased from practically undetectable levels to those that were easily measurable during a time frame that was just prior to mitosis. Since these enzymes appeared to be associated mainly with dividing tissues, it was reasonable to consider them as potential diagnostic tools for various disease states, in which the enzymes are elevated.

The Regulatory Influence of Allosteric Effectors on Deoxycytidylate Deaminases

Publisher Summary The discovery of deoxycytidylate deaminase resulted from independent investigations on the metabolism of deoxycytidine derivatives in sea urchin eggs and in rat embryo extracts. At the time, the deaminase appeared to provide the only direct means of forming deoxyuridine monophosphate (dUMP), a substrate for the critical enzyme thymidylate synthetase, and it was viewed as playing a possible role in the regulation of DNA synthesis or contributing to the efficiency of pyrimidine nucleotide utilization. Potential evidence in support of these assumptions was provided by studies indicating that deaminase activity was elevated in mitotically active normal and neoplastic tissues. The role of the deaminase in most bacterial systems is marginal at best or non-existent, except in those cases where the enzyme is induced on bacteriophage infection, and even in this case, the presence of the enzyme is not essential for survival. In the course of studies, the enzyme to be markedly susceptible to feedback inhibition by deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) has been found, and it has proved to be a convenient model for the study of the mechanisms involved in eliciting allosteric transitions associated with deoxycytidylate deaminase.