Joanna Cieśla

Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.

Crystal structures of thymidylate synthase from nematodes, Trichinella spiralis and Caenorhabditis elegans , as a potential template for species-specific drug design

Abstract Crystal structures were solved of the binary complexes Trichinella spiralis and Caenorhabditis elegans thymidylate synthases with deoxyuridine monophosphate (dUMP), with crystals obtained by the vapor diffusion method in hanging drops. For the T. spiralis thymidylate synthase-dUMP complex, the diffraction data were collected at the BESSY Synchrotron to 1.9 Å resolution. The crystal belongs to the space group P1 with two dimers in the asymmetric unit (ASU). For the C. elegans TS-dUMP complex crystal, the diffraction data were collected at the BESSY Synchrotron to 2.48 Å resolution, and the crystal belongs to the space group P 32 2 1, with two monomers (one dimer) in the ASU. Structural comparisons were made of both structures and each of them with the corresponding mouse thymidylate synthase complex.

Unusual Developmental Pattern of Expression of Enzymes Involved in DNA Biosynthesis in Trichinella spiralis and Trichinella pseudospiralis

All species in the genus Trichinella, between them T. spiralis and T. pseudospiralis, have been successful in colonizing striated sceletal muscle tissue and remain infective in this niche for months to years. Trichinella spiralis causes trichinellosis, a serious disease in man and other mammals. Mating of adult worms (developing from infective larvae, deriving from digested infected meat) occurs in a non membrane-bound portion of columnar epithelium of the host’s small intestine. The fertilized females enter the intestinal wall and release to the bloodstream the newborn larvae. Each of these penetrates host’s skeletal muscle cell and lives in its modified portion, the nurse cell, surrounded by a collagen capsule around which a circulatory rete develops. The nurse cell development, initiated by T. spiralis infection, is associated with a variety of changes, including cell cycle re-entry and induction of DNA synthesis, followed by the apparent G2/M arrest of the infected cell in the cell cycle. Similar changes appear to be caused by T. pseudospiralis infection, albeit the nurse cell complexes are not encapsulated by collageneous fibres and the larvae may move between muscle cells. Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2′-deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of N5,10-methylenetetrahydrofolate, with concomitant production of thymidylate and dihydrofolate. The coenzyme tetrahydrofolate is regenerated via dihydrofolate reduction by the enzyme dihydrofolate reductase (DHFR). One of the sources of TS substrate, dUMP, is dUTP hydrolysis in a pyrophosphatase reaction catalyzed by the enzyme dUTPase. TS and dUTPase induction is known to be associated with cell proliferation. Thymidylate synthesis inhibition by drugs targeted at either TS or DHFR is taken advantage of in chemotherapy. TS, DHFR and dUTPase were found to be persistently expressed at a high and constant level, comparable to that found in regenerating rat liver, in crude extracts from adult worms of Trichinella spiralis, as well as from developmentally arrested muscle larvae of both Trichinella spiralis (isolated 1–24 months after infection) and Trichinella pseudospiralis (isolated 5.5–13 months after infection). The results obtained with Trichinella pseudospiralis muscle larvae isolated with the use of pepsin did not differ from those obtained when pepsin was not used. Moreover, T. spiralis muscle larvae (T. pseudospiralis larvae were not tested) contained also high level, comparable with that found in mouse leukemia L1210 cells, of DNA polymerase α, a key enzyme of the eukaryotic replication complex, its expression also known to be associated with cell proliferation. Immunofluorescent detection of TS protein was done with the use of monoclonal antibodies, developed by in vivo immunization of Balb/c mice with homogeneous recombinant rat hepatoma TS protein as an antigen. The specific anti-rat TS antibodies recognized also T. spiralis TS, as indicated by cross-reactivity on Western blot. Localization of the enzyme was based on analysis of pictures collected by confocal microscopy. Two types of T. spiralis muscle larvae preparations were studied: muscle larvae isolated from mouse muscles by a procedure destroying nurse cells and muscle larvae remaining in nurse cells, isolated as an intact nurse cell preparation. The results revealed reproducible TS localization patterns, reflected by strong fluorescence emitted by cells of both female and male gonad primordium, as well as from the regions around stichocyte nuclei. High expression in Trichnella muscle larva of thymidylate synthase, and certain other enzymes involved in DNA biosynthesis, was found also in Caenorhabditis dauer larva and appears to be connected with their cells being arrested in the cell cycle.

Effect of Simultaneous Inhibition of Protein Kinase CK2 and Thymidylate Synthase in Leukemia and Breast Cancer Cells

Background/Aim: Protein kinase CK2 was recently identified as a promising therapeutic target for combination therapy. Our study aims to investigate the anticancer effect of a simultaneous inhibition of thymidylate synthase (TS) and CK2 in MCF-7 breast cancer and CCRF-CEM leukemia cells. Materials and Methods: The type of interaction between CK2 inhibitors: CX-4945, 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi), or recently obtained 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazol-1-yl)acetonitrile (2b) and TS-directed anticancer drug, 5-fluorouracil (5-FU) was determined using the MTT assay and a combination index method. The influence of the combined treatment on apoptosis in leukemia cells, as well as on cell-cycle progression and the levels of TS, CK2α and P-Ser529-p65 were determined in both cell lines, using flow cytometry and western blot analysis, respectively. Results: The best synergistic effect was observed in CCRF-CEM cell line with the combination of 5-FU and 2b which correlated with a decrease in the endocellular CK2 activity and enhancement of the pro-apoptotic effect. Conclusion: The obtained results demonstrate the ability of CK2 inhibitors to enhance the efficacy of 5-FU in anticancer treatment, indicating a different molecular mechanism of the studied CK2 inhibitors interaction with 5-FU.

Uncommon and parallel developmental patterns of thymidylate synthase expression and localization in Trichinella spiralis and Caenorhabditis elegans

Abstract Trichinella spiralis is a parasitic nematode causing trichinellosis, a serious disease, and Caenorhabditis elegans is a free-living nematode, which is used as a model in parasitological studies. High levels of thymidylate synthase (EC 2.1.1.45; ThyA) and certain other enzymes involved in thymidylate biosynthesis were found throughout T. spiralis and C. elegans developmental cycles, including developmentally arrested forms, that is, T. spiralis muscle larva and C. elegans dauer larva. As ThyA activity is characteristic for cells that left the G0 phase of the cell cycle, an exceptional regulation of the cell cycle in nematodes is suggested, manifested by a global cell cycle arrest in developmentally arrested larvae of the two species. ThyA immunolocalization during development of T. spiralis and C. elegans revealed the presence of high enzyme levels not only in the developing embryos, where it was expected, but also in gonad primordia, egg and sperm cells, nerve ring and secretory cells, opening to T. spiralis esophagus and C. elegans pharynx, where it may point to those cell populations remaining cell cycle arrested.

Interaction with 2(4)-Thio-5-Fluoro-dUMP of Thymidylate Synthases with Differing Sensitivities to 5-Fluoro-dUMP

To determine how modifications of 5-fluoro-dUMP (FdUMP), a thymidylate synthase (EC 2.1.1.45) chemotherapeutically active inhibitor, may affect its specificity, a study was conducted on inhibition of the enzyme from parental (L1210P) and 5-fluoro-dUrd-resistant (L1210R) mouse leukemia L1210 cells, the tapeworm Hymenolepis diminuta (H.d.) and regenerating rat liver (RRL) by FdUMP and its 2-thio-and 4-thio-congeners. The enzymes differed in sensitivity to time-and methylenetetrahydrofolate-dependent inactivation by FdUMP, with Ki values ranging from 10-9 M for the L1210P and 10-8 M for the L1210R1 and RRL enzymes,2 to 10-7 M for the H.d. enzyme.2