[show abstract][hide abstract] ABSTRACT: Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein.
Molecular and Biochemical Parasitology 02/2012; 183(1):63-9. · 2.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.
[show abstract][hide abstract] ABSTRACT: 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.
-Thymidylate synthase-Dihydrofolate reductase, dUTPase-Pyrimidine salvage
[show abstract][hide abstract] ABSTRACT: Thymidylate synthase (TS) may undergo phosphorylation as endogenous protein present in mammalian cells and as recombinant protein (corresponding to human, rat, mouse or Trichinella spiralis TS) expressed in bacterial cells. The phosphorylated, compared to non-phosphorylated, recombinant enzyme forms show decreased (at least by 3-fold) molecular activity, bind their cognate mRNA (tested only with the rat enzyme) and repress their own mRNA translation (tested with human, rat and mouse enzyme). Recombinant human, mouse, rat and Trichinella spiralis TSs, expressed in E. coli, undergo phosphorylation on histidine residues; so do probably L1210 and calf thymus endogenous TS proteins. Endogenous calf thymus and L1210 thymidylate synthase proteins undergo nitration in vivo. Chemical nitration of human, mouse and C. elegans recombinant TS proteins distinctly affects catalytic potency, by lowering the V max app value.