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Properties of Phosphorylated Thymidylate Synthase
... Les auteurs suggèrent qu'il est nécessaire que l'extrémité N-ter soit libre pour l'entrée de la TS dans la chambre protéasomale. Récemment, Frączyk et collaborateurs (2015) ont localisé par nano-chromatographie en phase liquide couplée à la spectrométrie de masse en tandem NanoLC-MS/MS (Nano-Liquid Chromatography tandem-Mass Spectrometry) la N α -acétylation sur l'initiateur de la traduction Met 1 de la TS dans les cellules leucémiques murines L1210 (Frączyk et al., 2015). La caractérisation de différents mutants a révélé que la Pro 2 protège l'extrémité N-ter de la d'activation, des enzymes E2 de conjugaison, des ligases E3 et des DUB (DésUBiquitinases). ...
... Plus ARNm hétérologues (codant la DHFR, la DCD (dCMP Désaminase), la SHMT ou encore la TK). Les auteurs suggèrent ainsi que la phosphorylation pourrait augmenter les interactions protéine-ARN ou les interactions protéine-protéine entre, par exemple, la TS et le ribosome (Frączyk et al., 2015). ...
... Comme dit précédemment, une seule des deux sous-unités enzymatiques de la TS lie ses substrats, or, une seule des deux sous-unités est phosphorylée suggérant un lien entre ces deux évènements (Frączyk et al., 2015). In vitro, CK2α catalyse la phosphorylation de la TS humaine sur des résidus Ser, notamment la Ser 124 , entrainant une diminution de son activité catalytique (Fraczyk et al., 2010). ...
La O-GlcNAcylation (O-N-acétylglucosaminylation) est une MPT (modification post-traductionnelle) dynamique et réversible catalysée par un unique couple d’enzymes antagonistes : l’OGT (O-GlcNAc transférase) et l’OGA (O GlcNAcase). Elle est considérée comme un véritable senseur nutritionnel et régule un grand nombre de mécanismes cellulaires fondamentaux. En ciblant des oncoprotéines et des suppresseurs de tumeur, sa dérégulation est associée à la cancérogenèse et la progression tumorale. En revanche, son rôle dans la réponse aux thérapies anti-cancéreuses est très peu étudié. Il a été néanmoins montré récemment que l’hyper-O-GlcNAcylation impacte la réponse de certains cancers à des drogues telles que le tamoxifène, le cisplatine, le bortézomib et le 5-FU (5-fluorouracile). Le 5-FU est la chimiothérapie de référence du CCR (cancer colorectal) et la TS (Thymidylate Synthase) sa cible principale. La surexpression de la TS est un biomarqueur de résistance au 5-FU utilisé en clinique. La TS a été montrée comme étant O-GlcNAcylée mais le rôle de cette MPT n’a pas été élucidé. Il nous est donc paru intéressant d’analyser le « cross-talk » entre O-GlcNAcylation et réponse au 5-FU dans le CCR dans l’hypothèse que la O-GlcNAcylation pourrait impacter la sensibilité au 5-FU en régulant sa cible TS. Un modèle murin in vivo de CCR humains et des cellules coliques non cancéreuses et cancéreuses ont été utilisés pour analyser l’effet du 5-FU sur la O-GlcNAcylation globale des protéines et réciproquement l’impact de la O-GlcNAcylation sur le niveau et l’activité de la TS, et la réponse au 5-FU. Nos données in vitro corroborent nos résultats in vivo et soutiennent que le 5-FU diminue la O-GlcNAcylation globale et que, réciproquement, la O-GlcNAcylation augmente le niveau de TS et sensibilise le CCR au 5-FU. Nous avons déchiffré le mécanisme moléculaire sous-jacent mettant en lumière le rôle de la O-GlcNAcylation dans la stabilisation de la TS et sa protection contre la dégradation protéasomale. Deux sites de O-GlcNAcylation de la TS ont été identifiés : la Thr251 à l’interface de dimérisation de l’enzyme et la Thr306 dans la séquence dégron carboxy-terminale connue pour contrôler sa dégradation. Ensemble nos résultats proposent une nouvelle stratégie thérapeutique combinant le 5-FU à un inhibiteur de l’OGA afin d’améliorer la réponse du CCR à la chimiothérapie à base de 5-FU.
... Mouse HisTag-free (mTS) recombinant thymidylate synthase protein was over-expressed and purified as previously described [12,13]. Phosphatase inhibitors (50 mM NaF, 5 mM Na-pyrophosphate, 0.2 mM EGTA, 0.2 mM EDTA and 2 mM Na3VO4) were present in the purification buffers. ...
... The results showed the extent of binding, reflected by the amount of filter paperbound radioactivity, to be dependent on FdUMP, tetrahydrofolate and thymidylate synthase protein concentrations ( Fig. 2A-C). In addition, the amount of filter paper-bound F[2-14 C]dUMP increased with time, and comparison of the reaction run with the phosphorylated and nonphosphorylated thymidylate synthase fractions, differing in the apparent specific activity [13], showed the binding rate to depend on the latter (Fig. 3). ...
... Note: The apparent specific activity of mTS, compared to PimTS, is six-fold higher [cf. Frączyk et al., 2015]. With mTS-catalyzed reaction the linear fit is almost perpendicular to the x-axis, pointing to a very rapid THF → DHF transformation and suggesting that the linear stage is not observable, thus allowing only rough approximation of the "specific activity" value that is presumably underestimated. ...
In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.
... In humans, the TSase system represents the first reported instance of such translational autoregulation [10]. Control over expression and function of TSase may also be achieved by posttranslational modifications [14]. In contrast to the bacterial one, mammalian TSases are reportedly known to undergo certain posttranslational modifications in certain cell lines under certain conditions [14]. ...
... Control over expression and function of TSase may also be achieved by posttranslational modifications [14]. In contrast to the bacterial one, mammalian TSases are reportedly known to undergo certain posttranslational modifications in certain cell lines under certain conditions [14]. Those modifications could include methylation, phosphorylation [14] and/or acetylation of the N-terminal methionine [15]. ...
... In contrast to the bacterial one, mammalian TSases are reportedly known to undergo certain posttranslational modifications in certain cell lines under certain conditions [14]. Those modifications could include methylation, phosphorylation [14] and/or acetylation of the N-terminal methionine [15]. It was reported that hsTSase could under particular circumstances have methylated glutamates and phosphorylation at serine. ...
Thymidylate Synthase (TSase) is a highly conserved enzyme that catalyzes the production of the DNA building block thymidylate. Structurally, functionally and mechanistically, bacterial and mammalian TSases share remarkable similarities. Because of this closeness, bacterial enzymes have long been used as model systems for human TSase. Furthermore, while TSase inhibitors have long served as chemotherapeutic drugs, no TSase inhibitor serves as an antibiotic. Despite their high resemblance, the mammalian TSases are distinct in a few known aspects, such as having a N-terminal tail and two insertions in the primary sequence and active/inactive conformations. Here, we aim to comprehensively characterize human (hs) TSase and delineate its contrasts and the similarities to the well-studied Escherichia coli (ec) TSase. We found that, in contrast to ecTSase, Mg²⁺ does not enhance reaction rates for hsTSase. The temperature dependence of intrinsic kinetic isotope effects (KIEs), on the other hand, suggests that Mg²⁺ has little or no impact on the transition state of hydride transfer in either enzyme, and that the transition state for the hydride transfer in hsTSase is looser than in ecTSase. Additionally, the substrates’ binding order is strictly ordered for ecTSase but slightly less ordered for hsTSase. The observed kinetic and functional differences between bacterial and human enzymes may aid in the development of antibiotic drugs with reduced toxicity.
... lanes 2 and 5; note: of the two protein bands stained in lane 5, corresponding to the two subunits (cf. ref. 16), only the upper one shows the presence of a phosphate group, as detected in lane 2, causing lower electrophoretic mobility of the phosphorylated subunit). Assuming the presence of a fraction of TS molecules phosphorylated on both subunits, it was too small to be detected. ...
... Alternatively, rare enzyme molecules that underwent phosphorylation in bacterial cells on both subunits could be lost in the process of MOAC, the loss also remaining undetected. The two TS fractions have been previously characterized by comparing their 31 P NMR spectra and found to contain unmodified and histidine-phosphorylated enzymes, 16 respectively. The latter was evidenced by the phosphorylated fraction spectrum containing a resonance at 2.14 ppm, belonging to inorganic phosphate, and two upfield shifted singlet peaks at À7.39 ppm and À9.87 ppm, with the singlet resonances in the negative spectrum region assigned to reflect N-phosphorylated histidine species (3-pHis and 1-pHis, respectively). ...
... The latter was confirmed by the acidlabile character of both resonances. 16 Different lines of evidence pointed to modification of His 298 (affinity chromatography of a phosphate-containing peptide, followed by sequencing) and His 33 (MS). In addition, by MS analysis phospho-Ser 118 was determined. ...
Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependences of the inactivation rate on time and accompanied by differing inhibition parameters. Considering importance for chemotherapeutic drug resistance, a possibility was tested of thymidylate synthase inhibition to be affected by post-translational modification, e.g. phosphorylation, by comparing sensitivities to inhibition by each of two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparation, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered with each inhibitor studied both the pattern of the dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy -dCMP was found strongly dependent on [Mg2+], the cations demonstrated previously to influence also activity of endogenous mouse TS isolated from tumour cells.
... Among those substrates are also three enzymes, namely, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT), involved in the metabolic cycle resulting in de novo synthesis of thymidylate, a crucial substrate for DNA synthesis and repair. The in vitro CK2mediated phosphorylation of TS was shown by Frączyk et al. Frączyk et al., 2015) and Ludwiczak et al., 2016. The phosphorylation lowered TS catalytic activity and affected binding of the TS inhibitor, 5-fluorodeoxyuridylate (FdUMP). ...
... There are reports indicating the occurrence of CK2-mediated phosphorylation of TS, DHFR, and SHMT Frączyk et al., 2015;Ludwiczak et al., 2016;Rusin et al., 2017;Skierka et al., 2019); however, to this date, there have been no studies showing the physiological role of CK2-mediated phosphorylation of thymidylate synthesis cycle enzymes. Our previous and present qPCR results indicate that inhibition of CK2 in CCRF-CEM cells treated with a specific CX-4945 inhibitor does not significantly influence the mRNA levels. ...
Thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT) constitute the thymidylate synthesis cycle providing thymidylate for DNA synthesis and repair. Our previous studies indicated that TS and DHFR are the substrates of protein kinase CK2. This work has been aimed at the elucidation of the effect of CK2 activity on cell cycle progression, thymidylate synthesis enzyme expression and localization, and the role of CK2-mediated TS phosphorylation in in vitro di- and trimolecular complex formation. The results were obtained by means of western blot, confocal microscopy, flow cytometry, quantitative polymerase chain reaction (QPCR), quartz crystal microbalance with dissipation monitoring (QCM-D), and microthermophoresis (MST). Our research indicates that CK2 inhibition does not change the levels of the transcripts; however, it affects the protein levels of DHFR and TS in both tested cell lines, i.e., A549 and CCRF-CEM, and the level of SHMT1 in CCRF-CEM cells. Moreover, we show that CK2-mediated phosphorylation of TS enables the protein (pTS) interaction with SHMT1 and leads to the stability of the tri-complex containing SHMT1, DHFR, and pTS. Our results suggest an important regulatory role of CK2-mediated phosphorylation for inter- and intracellular protein level of enzymes involved in the thymidylate biosynthesis cycle.
... Mouse (mTS), C. elegans (CeTS), and T. spiralis (TspTS) thymidylate synthase recombinant proteins were overexpressed and purified as previously described [22,[35][36][37][38], with phosphatase inhibitors (50 mM NaF, 5 mM Na-pyrophosphate, 0.2 mM EGTA, 0.2 mM EDTA and 2 mM Na 3 VO 4 ) present in the purification buffers. TS activity was measured either spectrophotometrically [39] or with the use of the tritium release assay [40]. ...
... TS activity was measured either spectrophotometrically [39] or with the use of the tritium release assay [40]. Purified TS preparation was separated into phosphorylated and non-phosphorylated fractions using metal oxide/hydroxide affinity chromatography (MOAC) on Al(OH) 3 beads [38,41]. Protein contents of TS preparations was determined spectrophotometrically at λ = 280 nm [42]. ...
Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.
... It pointed to the high TS level as a result of an unusual cell cycle regulation, leading to a long-term cell cycle arrest, in the developmentally arrested larvae (discussed in Reference [17,18]). Although TS protein in those larvae is probably catalytically irrelevant (no DNA synthesis), it may play a regulatory role in view of the enzyme's certain non-catalytic activities, including capacity to bind mRNA (its own and some others) and inhibit translation, with potential regulation of several cellular genes [19,20], as well as an oncogene-like activity [21]. Thus, in view of the latter, a possibility to selectively interfere with nematode TS catalytic/non-catalytic activities could be applied not only in an attempt to kill a parasite but also to study the physiological significance of the high expression of TS in nematodes' cells, particularly in their developmentally arrested larvae. ...
... The recombinant human, rat, mouse, T. spiralis and C. elegans TS proteins were produced, purified and separated into phosphorylated and non-phosphorylated fraction, as previously described [20]. TS activity was monitored, following tritium release from [5-3 H]dUMP, and its inhibition studied as earlier reported [76]. ...
With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.
... Considering that CX-4945 at 3 μM affected the expression of DHFR at the protein but not mRNA level, the possible regulatory role of CK2-mediated phosphorylation in translation/degradation of DHFR protein should be taken into account. Recently, it was demonstrated that phosphorylation of TYMS affected its catalytic and non-catalytic properties, including translation and binding of its own mRNA (39)(40)(41). CK2-mediated phosphorylation of DHFR might have a similar effect on enzyme properties. It should also be noted that phosphorylated TYMS bound and inhibited translation not only of its own mRNA, but also of other mRNAs encoding DHFR, SHMT, thymidine kinase and deoxycitidylate deaminase proteins (41). ...
... CK2-mediated phosphorylation of DHFR might have a similar effect on enzyme properties. It should also be noted that phosphorylated TYMS bound and inhibited translation not only of its own mRNA, but also of other mRNAs encoding DHFR, SHMT, thymidine kinase and deoxycitidylate deaminase proteins (41). If we assume that in vivo lack of or diminished phosphorylation of TYMS due to inhibition of CK2 prevents the binding of TYMS protein to DHFR mRNA, this mechanism certainly might contribute to an increase of DHFR protein after treatment of cells with CK2 inhibitor. ...
Background/aim:
Recently, we demonstrated the ability of inhibitors of protein kinase 2 (casein kinase II; CK2) to enhance the efficacy of 5-fluorouracil, a thymidylate synthase (TYMS)-directed drug for anticancer treatment. The present study aimed to investigate the antileukemic effect of simultaneous inhibition of dihydrofolate reductase (DHFR), another enzyme involved in the thymidylate biosynthesis cycle, and CK2 in CCRF-CEM acute lymphoblastic leukemia cells.
Materials and methods:
The influence of combined treatment on apoptosis and cell-cycle progression, as well as the endocellular level of DHFR protein and inhibition of CK2 were determined using flow cytometry and western blot analysis, respectively. Real-time quantitative polymerase chain reaction was used to examine the influence of silmitasertib (CX-4945), a selective inhibitor of CK2 on the expression of DHFR and TYMS genes.
Results:
The synergistic effect was correlated with the increase of annexin V-binding cell fraction, caspase 3/7 activation and a significant reduce in the activity of CK2. An increase of DHFR protein level was observed in CCRF-CEM cells after CX-4945 treatment, with the mRNA level remaining relatively constant.
Conclusion:
The obtained results demonstrate a possibility to improve methotrexate-based anti-leukemia therapy by simultaneous inhibition of CK2. The effect of CK2 inhibition on DHFR expression suggests the important regulatory role of CK2-mediated phosphorylation of DHFR inside cells.
... In view of the foregoing conclusion of obvious interest is potential functional consequence of this mechanism, as well as possibility to find structural features enabling the inactive conformation stabilization (arginine residue in a position homologous to hTS 163) in other TS structures. Considering strong influences of posttranslational modifications on kinetics and inhibition of TS-catalyzed reaction [9,26,27] and resulting wide variability of properties of the enzyme preparations of the same specific origin [2,9,27], a simple comparison of mTS and hTS properties would not answer the former question. However, Gibson et al. [28] obtained a mutant R163K of the human enzyme and found its catalytic activity to be higher than that of the parental hTS. ...
... In view of the foregoing conclusion of obvious interest is potential functional consequence of this mechanism, as well as possibility to find structural features enabling the inactive conformation stabilization (arginine residue in a position homologous to hTS 163) in other TS structures. Considering strong influences of posttranslational modifications on kinetics and inhibition of TS-catalyzed reaction [9,26,27] and resulting wide variability of properties of the enzyme preparations of the same specific origin [2,9,27], a simple comparison of mTS and hTS properties would not answer the former question. However, Gibson et al. [28] obtained a mutant R163K of the human enzyme and found its catalytic activity to be higher than that of the parental hTS. ...
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.
... Ufosforylowane formy enzymu cechowała przynajmniej 3-krotnie niższa (w porównaniu z formami nieufosforylowanymi) aktywność cząsteczkowa (liczba obrotów) oraz wyżej wspomniane aktywności niekatalityczne. Porównawcze analizy 31 PNMR dowiodły, że w czterech badanych rekombinowanych białkach ST fosforylacja dotyczy reszt histydynowych [67]. ...
... slow-binding) dynowych. Z tych dwóch form tylko ufosforylowana posiadała wspomniane aktywności niekatalityczne, dysponując jednocześnie znacząco obniżoną aktywnością katalityczną [67]. ...
Thymidylate synthase ThyA (EC 2.1.1.45; encoded by the Tyms gene), having been for 60 years a molecular target in chemotherapy, catalyses the dUMP pyrimidine ring C(5) methylation reaction, encompassing a transfer of one-carbon group (the methylene one, thus at the formaldehyde oxidation level) from 6R-N5,10-methylenetetrahydrofolate, coupled with a reduction of this group to the methyl one, with concomitant
generation of 7,8-dihydrofolate and thymidylate. New facts are presented, concerning (i) molecular mechanism of the catalyzed reaction, including the substrate selectivity mechanism, (ii) mechanism of inhibition by a particular inhibitor, N4-hydroxy-dCMP, (iii) structural properties of the enzyme, (iv) cellular localization, (v) potential posttranslational modifications of the enzyme protein and their influence on the
catalytic properties and (vi) non-catalytic activities of the enzyme.
... We precised the O-GlcNAc position at the T251 residue and its role on TS stabilization by transfection of the three single threonine mutated TS-FLAG ( Supplementary Fig. S4). Our MS data also pointed out an O-GlcNAc site at the T306 (Fig. 4e) along with the previously described [27] phosphorylation showing a reciprocal phosphorylation/O-GlcNAcylation interplay on this residue. We also mapped two phosphorylated sites at S114 and T170 (Supplementary Table S2 and Fig. S5). ...
Alteration of O-GlcNAcylation, a dynamic posttranslational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O-GlcNAcylation was reported but not investigated yet. We hypothesize that O-GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G (O-GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O-GlcNAcylation and, reciprocally, elevation of O-GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O-GlcNAcylation by decreasing O-GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O-GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O-GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.
... Site-directed mutagenesis was performed using GeneArt Site-Directed Mutagenesis System (Thermofisher Scientific, Waltham, MA, USA). Recombinant thymidylate synthase protein and its mutants (H190A and W103G) were over-expressed and purified as previously described [72][73][74], with some modifications. Phosphatase inhibitors (50 mM NaF, 5 mM Na-pyrophosphate, 0.2 mM EGTA, 0.2 mM EDTA and 2 mM Na 3 VO 4 ) were present in the purification buffers. ...
A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.
... Until recently, phosphorylation of the hydroxyl-containing amino acids serine (Ser), threonine (Thr) and tyrosine (Tyr) was thought to be the primary mode of phosphorylation-mediated signalling in non-plant eukaryotes. However, a growing body of evidence (Besant & Attwood, 2012;Fan et al, 2012;Fraczyk et al, 2015;Fuhs et al, 2015;Shen et al, 2015;Wieland & Attwood, 2015;Panda et al, 2016;Srivastava et al, 2016;Xu et al, 2016a,b;Fuhs & Hunter, 2017) indicates that phosphorylation of other amino acids, termed here "non-canonical" phosphorylation, including His, Asp, Glu, Lys, Arg and Cys [in addition to pyrophosphorylation of Ser and Thr to form ppSer and ppThr (Chanduri et al, 2016;Harmel & Fiedler, 2018), and Lys polyphosphorylation (Bentley-DeSousa & Downey, 2019)], may also regulate protein signalling functions. Of note, the generation of both sitespecific and generic antibodies against phosphohistidine (pHis) (Kee et al, 2013(Kee et al, , 2015Fuhs et al, 2015;Lilley et al, 2015) has recently allowed mammalian cell-type independent roles for this PTM to be elucidated (Fuhs et al, 2015) in processes as diverse as ion channel regulation and T-cell signalling , and during cell proliferation, differentiation and migration. ...
Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.
... Until recently, phosphorylation of the hydroxyl-containing amino acids serine (Ser), threonine (Thr) and tyrosine (Tyr) was thought to be the primary mode of phosphorylation-mediated signalling in non-plant eukaryotes. However, a growing body of evidence [3][4][5][6][7][8][9][10][11][12][13] indicates that phosphorylation on the imidazolecontaining amino acid histidine (His) also plays a significant, but ill-defined, role in human biology. In contrast, and despite occasional reports in the literature 14,15 , the prevalence (or otherwise) of noncanonical amino acid phosphorylation on the five amino acids for which protein phosphorylation is chemically feasible (Asp, Arg, Lys, Glu, Cys) has yet to be revealed in human cells. ...
Protein phosphorylation is a ubiquitous post-translational modification (PTM) that regulates all aspects of life. To date, investigation of human cell signalling has focussed on canonical phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that phosphorylation of histidine also plays a central role in regulating cell biology. Phosphoproteomics workflows rely on acidic conditions for phosphopeptide enrichment, which are incompatible with the analysis of acid-labile phosphorylation such as histidine. Consequently, the extent of non-canonical phosphorylation is likely to be under-estimated.
We report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), which permits enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive and positional phosphorylation patterns on histidine, arginine, lysine, aspartate and glutamate in human cell extracts, including 310 phosphohistidine and >1000 phospholysine sites of protein modification. Remarkably, the extent of phosphorylation on individual non-canonical residues vastly exceeds that of basal phosphotyrosine. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring roles of acid-labile phosphorylation in any proteome using mass spectrometry.
Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (K d below 0.5 μM, k on = 10.31 × 10 ⁴ M ⁻¹ s ⁻¹ and k off = 1.40 × 10 ⁻³ s ⁻¹ ) and calculated Gibbs free energy (−36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level.
Thymidylate is a component of DNA (deoxyribonucleic acid), and is synthesised from the de novo or nucleotide salvage pathways. Many cell types rely on the salvage pathway, but often this is insufficient. Thymidylate is synthesised de novo from deoxyuridylate and methylenetetrahydrofolate by thymidylate synthase (TYMS), with the enzymes dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase (SHMT) or methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) which are required to regenerate methylenetetrahydrofolate. The de novo synthesis pathway forms a nuclear complex at the nuclear lamina at sites of DNA replication. DNA polymerases do not distinguish between deoxyuridylate and thymidylate, and when thymidylate synthesis is insufficient, uracil is misincorporated into DNA. This can lead to futile cycles of DNA repair and subsequent DNA single- and double-strand breaks. Impaired de novo thymidylate synthesis can result in neural tube defects, megaloblastic anaemia and severe combined immunodeficiency (SCID). Inhibitors of TYMS and DHFR impair cell replication and have been used in the treatment of cancer.
Ribonucleoprotein complexes involved in pre-mRNA splicing and mRNA decay are often regulated by phosphorylation of RNA-binding proteins. Cells use phosphorylation-dependent signaling pathways to turn on and off gene expression. Not much is known about how phosphorylation-dependent signals transmitted by exogenous factors or cell cycle checkpoints regulate RNA-mediated gene expression at the atomic level. Several human diseases are linked to an altered phosphorylation state of an RNA binding protein. Understanding the structural response to the phosphorylation “signal” and its effect on ribonucleoprotein assembly provides mechanistic understanding, as well as new information for the design of novel drugs. In this review, I highlight recent structural studies that reveal the mechanisms by which phosphorylation can regulate protein-protein and protein-RNA interactions in ribonucleoprotein complexes.
The hydrogen bonds and electrostatic interactions that form between the protonated side chain of a basic residue and the negatively charged phosphate of a phosphopeptide can play crucial roles in governing their dissociation pathways under low-energy collision-induced dissociation (CID). Understanding how phosphoramidate (i.e. phosphohistidine, phospholysine and phosphoarginine), rather than phosphomonoester-containing peptides behave during CID is paramount in investigation of these problematic species by tandem mass spectrometry. To this end, a synthetic peptide containing either phosphohistidine (pHis) or phospholysine (pLys) was analysed by ESI-MS using a Paul-type ion trap (AmaZon, Bruker) and by travelling wave ion mobility-mass spectrometry (Synapt G2-Si, Waters). Analysis of the products of low-energy CID demonstrated formation of a doubly ‘phosphorylated’ product ion arising from intermolecular gas-phase phosphate transfer within a phosphopeptide dimer. The results are explained by the formation of a homodimeric phosphohistidine (pHis) peptide non-covalent complex (NCX), likely stabilized by the electrostatic interaction between the pHis phosphate group and the protonated C-terminal lysine residue of the peptide. To the best of our knowledge this is the first report of intermolecular gas-phase phosphate transfer from one phosphopeptide to another, leading to a doubly phosphorylated peptide product ion.
A significant number of proteins in both eukaryotes and prokaryotes are known to be post-translationally modified by the addition of phosphate, serving as a means of rapidly regulating protein function. Phosphorylation of the amino acids serine, threonine and tyrosine are the focus of the vast majority of studies aimed at elucidating the extent and roles of such modification, yet other amino acids, including histidine and aspartate, are also phosphorylated. Although histidine phosphorylation is known to play extensive roles in signalling in eukaryotes, plants and fungi, roles for phosphohistidine are poorly defined in higher eukaryotes. Characterization of histidine phosphorylation aimed at elucidating such information is problematic due to the acid-labile nature of the phosphoramidate bond, essential for many of its biological functions. Although MS-based strategies have proven extremely useful in the analysis of other types of phosphorylated peptides, the chromatographic procedures essential for such approaches promote rapid hydrolysis of phosphohistidine-containing peptides. Phosphate transfer to non-biologically relevant aspartate residues during MS analysis further complicates the scenario.
Tyrosine nitration is a widespread post-translational modification capable of affecting both the function and structure of the host protein molecule. Enzyme thymidylate synthase (TS), a homodimer, is a molecular target for anticancer therapy. Recently purified TS preparations, isolated from mammalian tissues, were found to be nitrated, suggesting this modification to appear endogenously in normal and tumor tissues. Moreover, human TS (hTS) nitration in vitro led to a by twofold lowered catalytic activity following nitration in average of 1 tyrosine residue per monomer (Dąbrowska-Maś et al. in Org Biomol Chem 10:323-331, 2012), with the modification identified by mass spectrometry at seven different sites (Y33, Y65, Y135, Y213, Y230, Y258 and Y301). In the present paper, combined computational approach, including molecular and essential dynamics and free energy computations, was used to predict the influence on the activity of hTS of nitration of each of the seven tyrosine residues. The simulations were based on the crystal structure of hTS ternary complex with dUMP and Tomudex (PDB code: 1I00), with the Tomudex molecule replaced by the molecule of TS cofactor analogue, tetrahydrofolate. The present results indicate that while with nitration of five out of seven residues (Y33, Y135, Y230, Y258 and Y301), single residue modification appears to have a strong reducing effect on the activity, with the remaining two, Y65 and Y213, no or a weaker influence is apparent. Taken together, these results demonstrate that tyrosine nitrations in the hTS enzyme show clear tendency to influence the structure and dynamics and, in turn, catalytic properties of the host enzyme. These effects are overall distance-dependent.
The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.
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.
Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.
Phosphorylation of proteins is an essential signalling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In the present study, two MS fragmentation methods, ECD (electron-capture dissociation) and CID (collision-induced dissociation), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE and PLSRTLSVAAKK. The peptides were phosphorylated by reaction with monopotassium phosphoramidate. The results were confirmed by 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to the phosphoramidate moiety. Moreover, the results indicate that the ECD-MS may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.
Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.
Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.
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.
Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N(5,10)-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, 'wild')(dUMP)) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.
The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.
Crude extract specific activities of thymidylate synthase, dUTPase, thymidine kinase and dihydrofolate reductase were high during the development of Caenorhabditis elegans, the dauer larva activities being similar to those previously determined in Trichinella spiralis and T. pseudospiralis muscle larvae (with the exception of thymidine kinase, not detected in Trichinella). High thymidylate synthase expression in developmentally arrested larvae, demonstrated also at the mRNA and protein levels, is in agreement with a global cell cycle arrest of dauer larvae and indicates this unusual cell cycle regulation pattern can be shared by developmentally arrested larvae of C. elegans and the two Trichnella species. Hence, the phenomenon may be characteristic for developmentally arrested larvae of different nematodes, rather than specific for the parasitic Trichinella muscle larvae. Endogenous C. elegans thymidylate synthase was purified and its molecular properties compared with those of the recombinant protein, expression of the latter in E. coli cells confirming the NCBI database sequence identity.
Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.
A new method for enrichment of phosphopeptides in complex mixtures derived by proteolytic digestion of biological samples has been developed. The method is based on calcium phosphate precipitation of the phosphopeptides prior to further enrichment with established affinity enrichment methods. Calcium phosphate precipitation combined with phosphopeptide enrichment using Fe(III) IMAC provided highly selective enrichment of phosphopeptides. Application of the method to a complex peptide sample derived from rice embryo resulted in more than 90% phosphopeptides in the enriched sample as determined by mass spectrometry. Introduction of a two-step IMAC enrichment procedure after calcium phosphate precipitation resulted in observation of an increased number of phosphopeptides.
Over the past 60 years, chemotherapeutic agents that target thymidylate biosynthesis and the enzyme thymidylate synthase (TS) have remained among the most-successful drugs used in the treatment of cancer. Fluoropyrimidines, such as 5-fluorouracil and capecitabine, and antifolates, such as methotrexate and pemetrexed, induce a state of thymidylate deficiency and imbalances in the nucleotide pool that impair DNA replication and repair. TS-targeted agents are used to treat numerous solid and haematological malignancies, either alone or as foundational therapeutics in combination treatment regimens. We overview the pivotal discoveries that led to the rational development of thymidylate biosynthesis as a chemotherapeutic target, and highlight the crucial contribution of these advances to driving and accelerating drug development in the earliest era of cancer chemotherapy. The function of TS as well as the mechanisms and consequences of inhibition of this enzyme by structurally diverse classes of drugs with distinct mechanisms of action are also discussed. In addition, breakthroughs relating to TS-targeted therapies that transformed the clinical landscape in some of the most-difficult-to-treat cancers, such as pancreatic, colorectal and non-small-cell lung cancer, are highlighted. Finally, new therapeutic agents and novel mechanism-based strategies that promise to further exploit the vulnerabilities and target resistance mechanisms within the thymidylate biosynthesis pathway are reviewed.
Crystal structure is presented of the binary complex between potassium phosphoramidate-phosphorylated recombinant C. elegans thymidylate synthase and dUMP. On each monomer a single phosphoserine residue (Ser127) was identified, instead of expected phosphohistidine. As 31P NMR studies of both the phosphorylated protein and of potassium phosphoramidate potential to phosphorylate different amino acids, point to histidine as the only possible site of the modification, thermodynamically favored intermolecular phosphotransfer from histidine to serine is suggested.
Phospho-proteomic studies opened a broad view onto the main mechanisms of regulating cellular processes. Our recent discovery of a protein arginine kinase and its target in bacteria added a previously undescribed type of phosphorylation to control protein activity. Several challenges arise from large in vivo studies of this and other types of phosphorylations. The main factors impeding correct localization are low spectral quality, neutral loss of phosphoric acid and gas-phase rearrangements, which have recently been described for phospho-serine, -threonine and -tyrosine. Studies on histidine-phosphorylated peptides, a nitrogen-bound phosphorylation, also reported loss of phosphoric acid upon collision-induced dissociation. We were interested in studying the behaviour of arginine phosphorylation under different fragmentation conditions and their influence on site localization. First, we determined the percentage of false localizations obtained by three different search engines and a software tool dedicated for phospho-site determination. Next, we demonstrate that application of collisional activation for analysis of arginine-phosphorylated peptides leads to extensive elimination of phosphoric acid and increases the numbers of false localizations, while the modification is maintained on the arginine side chain upon electron-transfer dissociation. Furthermore, we also observed a rearrangement of the phosphorylation onto serine and glutamic acid side chains upon collisional activation.
The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate analogues.
1.1. Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells.2.2. The enzyme is a dimer composed of 35,000 mol. wt monomers.3.3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP.4.4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34† C, reflecting a change of rate-limiting step in dTMP formation.5.5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine ring of the substrate, dUMP, and the pterine ring of the cofactor analogue, THF, in the active site of the phosphorylated enzyme. The ED analysis indicates changes in the behavior of collective motions in the phosphorylated enzyme, suggesting that the formation of the closed ternary complex is hindered. Computed free energies, in agreement with structural analysis, predict that the binding of dUMP and THF to hTS is favored in the native compared to phosphorylated state of the enzyme. The paper describes at the structural level how phosphorylation at the distant site influences the ligand binding. We propose that the 'phosphorylation effect' is transmitted from the outside loop of Ser 124 into the active site via a subtle mechanism initiated by the long-range electrostatic repulsion between the phosphate groups of dUMP and Ser124. The mechanism can be described in terms of the interplay between the two groups of amino acids: the link (residues 125-134) and the patch (residues 189-192), resulting in the change of orientation of the pyrimidine ring of dUMP, which, in turn, prevents the correct alignment between the latter ring and the pterin ring of THF.
Thymidylate synthase (TS) was found to be a substrate for both catalytic subunits of human CK2, with phosphorylation by CK2alpha and CK2alpha' characterized by similar K(m) values, 4.6microM and 4.2microM, respectively, but different efficiencies, the apparent turnover number with CK2alpha being 10-fold higher. With both catalytic subunits, phosphorylation of human TS, like calmodulin and BID, was strongly inhibited in the presence of the regulatory subunit CK2beta, the holoenzyme being activated by polylysine. Phosphorylation of recombinant human, rat, mouse and Trichinella spiralis TSs proteins was compared, with the human enzyme being apparently a much better substrate than the others. Following hydrolysis and TLC, phosphoserine was detected in human and rat, and phosphotyrosine in T. spiralis, TS, used as substrates for CK2alpha. MALDI-TOF MS analysis led to identification of phosphorylated Ser(124) in human TS, within a sequence LGFS(124)TREEGD, atypical for a CK2 substrate recognition site. The phosphorylation site is located in a region considered important for the catalytic mechanism or regulation of human TS, corresponding to the loop 107-128. Following phosphorylation by CK2alpha, resulting in incorporation of 0.4mol of phosphate per mol of dimeric TS, human TS exhibits unaltered K(m) values for dUMP and N(5,10)-methylenetetrahydrofolate, but a 50% lower turnover number, pointing to a strong influence of Ser(124) phosphorylation on its catalytic efficiency.
J. Neurochem. (2010) 113, 848–859.
The present study uses a proteomic approach to examine possible alterations of protein expression in the hippocampus of rats that are subjected to chronic mild stress (CMS). These rats serve as an animal model that was developed to mimic anhedonia, which is one of the core symptoms of depression. As antidepressant treatment is effective after a few weeks of administration, we also aimed to identify changes that were linked to chronic (once daily for 4 weeks) and ‘pulse’ (once a week) administration of imipramine. Fifteen differential proteins were identified with 2D electrophoresis followed by mass spectrometry. Although both methods of imipramine administration restored normal sucrose consumption in rats that were subjected to CMS, the molecular mechanisms of these two therapies were different. CMS-induced changes in the levels of dynactin 2, Ash 2, non-neuronal SNAP25 and alpha-enolase were reversed by chronic imipramine, but ‘pulse’ treatment was not that effective.
Thymidylate synthase (TS) is a homodimeric enzyme with two equivalent active sites composed of residues from both subunits. Despite the structural symmetry of the enzyme, certain experimental results are consistent with half-the-sites activity, suggesting negative cooperativity between the active sites. To gain insight into the mechanism behind this phenomenon, we explore segmental motions of rat TS in the absence of ligands, with normal mode analysis as a tool. Using solvent accessible surface area of the active site pocket as a monitor of the degree of opening of the active sites, we classified the first 25 nontrivial normal modes, obtained from the web server of the program ElNémo, according to the behavior of the active sites. We found seven modes that open and close both sites symmetrically and nine that do so in an anticorrelated fashion. We characterized the motions of these modes by visual inspection and through measurement of distances between selected atoms lining the active site pockets. The segments that regulate access to the active site correspond to the loop containing R44, helix K, and a long loop containing residues 103–125, in agreement with a large body of crystallographic studies. These elements can be activated together or in isolation. There are more asymmetric modes than symmetric ones in the set we analyzed, probably accounting for the half-the-sites behavior of the enzyme. Three of the asymmetric modes result in changes at the dimer interface and indicate the endpoints of possible communication pathways between the active sites. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 549–559, 2010.
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of (31)P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of (31)P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His(26)) or lysine (Lys(33)) side-chain.
The analysis of PTMs such as phosphorylation has become an important field in MS because they can directly indicate protein states and interactions. Whereas the characterization of singly and doubly phosphorylated peptides has almost become routine, identifying phosphorylation events at multiple residues within a small region of a protein is still problematic. The identification of multiple modifications can be further hampered by low sequence information due to multiple neutral losses from phosphorylated side chains. Here we present a strategy for the analysis of complex phosphopeptides that combines peptide enrichment by titanium dioxide, separation by RP separation on monolithic columns and MS using high energy HE-CAD in a MALDI TOF/TOF analyser. Using synthetic phosphopeptides our approach is compared to multistage activation (MSA) MS/MS and the recently described electron transfer dissociation (ETD) method using an ESI-LTQ mass spectrometer.
This chapter discusses the use of 31p nuclear magnetic resonance (NMR) spectroscopy and, in particular, 31p chemical shifts to probe the structure and dynamics of phosphate ester enzyme complexes. This chapter discusses various phosphorus-31 chemical shifts, such as the interaction of the electron cloud surrounding the phosphorus nucleus with an external applied magnetic field B0 gives rise to a local magnetic field. This induced field shields the nucleus, with a magnitude proportional to the field B0. Measured 31p chemical shifts of a sample relative to a standard in a solvent of different diamagnetic susceptibility can differ by up to 1 ppm. To minimize this problem, it has been suggested that 31p chemical shifts in aqueous samples be measured relative to 85% phosphoric acid in a spherical microcell inside the solution. Several attempts have been made to develop a unified theoretical foundation for 31p chemical shifts of phosphorus compounds. This chapter also discusses the stereoelectronic effects on 31p chemical shifts as probe of DNA and DNA-protein structures.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
1-Phosphohistidine and 3-phosphohistidine have been synthesized by reaction of histidine with phosphoramidate. The two isomers have been separated by paper electrophoresis and anion-exchange chromatography. The synthetic phosphohistidine isomer which is identical with the compound isolated from mitochondria and from a succinate thiokinase preparation has been identified as 3-phosphohistidine. 3-Phosphohistidine has been isolated as the solid lithium salt and has been characterized by composition, ultraviolet and nuclear magnetic resonance spectra, titration curve, and rate of hydrolysis over a wide range of pH values. α-N-Acetyl-3-phosphohistidine, 1-methyl-3-phosphohistidine, α-N-acetyl-l-methyl-S-phosphohistidine, and the isomers of phosphohistidine methyl ester have been prepared and some properties reported.
Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.
Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.
A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli. By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells. The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product. Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus. By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein. Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities. To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system. Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.
Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.
The preparation of a novel phosphorus species, thiophosphoramidate, has enabled the specific thiophosphorylation of histidine at its 3-position. The rates of phosphorylation and thiophosphorylation of histidine are reported, as well as the spectroscopic properties of both thiophosphoramidate and 3-thiophosphohistidine. Structural assignment of the latter was made by analogy to the NMR properties of the known 3-phosphohistidine. The alkylation of 3-thiophosphohistidine by phenacyl bromide serves as a model for the introduction of labeling or probe reagents into histidine phosphorothioate-containing proteins.
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 A and 2.5 A 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 A 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.
In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.
Studies from our laboratory have shown that the folate-dependent enzyme, thymidylate synthase (TS), functions as an RNA binding protein. There is evidence that TS, in addition to interacting with its own TS mRNA, forms a ribonucleoprotein complex with a number of other cellular mRNAs, including those corresponding to the p53 tumor suppressor gene and the myc family of transcription factors. Using both in vitro and in vivo model systems, we have demonstrated that the functional consequence of binding of TS protein to its own cognate mRNA, as well as binding of TS to the p53 mRNA, is translational repression. Herein, we review current work on the translational autoregulatory control of TS expression and discuss the molecular elements that are required for the TS protein-TS mRNA interaction. TS may play a critical role in regulating the cell cycle and the process of apoptosis through its regulatory effects on expression of p53 and perhaps other cell cycle related proteins. Finally, the ability of TS to function as a translational regulator may have important consequences with regard to the development of cellular resistance to various anticancer drugs.
Thymidylate synthase (TS) is a critical chemotherapeutic target and intracellular levels of TS are an important determinant of sensitivity to TS inhibitors. Translational autoregulation represents one cellular mechanism for controlling the level of expression of TS. This mechanism involves the binding of TS protein to its own messenger RNA (mRNA), thus, repressing translational efficiency. The presence of excess substrate or inhibitors of TS leads to derepression of protein binding to mRNA, resulting in increased translational efficiency and ultimately increased levels of TS protein. TS protein has been shown to bind to two distinct areas on its mRNA. The goal of the present work is to define the TS domains responsible for this interaction. Using a separate series of overlapping 17-mer peptides spanning the length of both the human and Escherichia coli TS sequences, we have identified six potential domains located in the interface region of the TS protein that bind TS mRNA. The identified domains that bind TS mRNA include three concordant regions in both the human and E. coli peptide series. Five of the six binding peptides contain at least one invariant arginine residue, which has been shown to be critical in other well-defined protein-RNA interactions. These data suggest that the identified highly conserved protein domains, which occur at the homodimeric interface of TS, represent potential participating sites for binding of TS protein to its mRNA.
Thymidylate synthase (TS) is an E2F1-regulated enzyme that is essential for DNA synthesis and repair. TS protein and mRNA levels are elevated in many human cancers, and high TS levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and non-small cell lung cancers. In this study, we show that ectopic expression of catalytically active TS is sufficient to induce a transformed phenotype in mammalian cells as manifested by foci formation, anchorage independent growth, and tumor formation in nude mice. In contrast, comparable levels of two TS mutants carrying single point mutations within the catalytic domain had no transforming activity. In addition, we show that overexpression of TS results in apoptotic cell death following serum removal. These data demonstrate that TS exhibits oncogene-like activity and suggest a link between TS-regulated DNA synthesis and the induction of a neoplastic phenotype.
Thymidylate synthase (TS) is an E2F1-regulated enzyme that is essential for DNA synthesis and repair. TS protein and mRNA levels are elevated in many human cancers, and high TS levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and non-small cell lung cancers. In this study, we show that ectopic expression of catalytically active TS is sufficient to induce a transformed phenotype in mammalian cells as manifested by foci formation, anchorage independent growth, and tumor formation in nude mice. In contrast, comparable levels of two TS mutants carrying single point mutations within the catalytic domain had no transforming activity. In addition, we show that overexpression of TS results in apoptotic cell death following serum removal. These data demonstrate that TS exhibits oncogene-like activity and suggest a link between TS-regulated DNA synthesis and the induction of a neoplastic phenotype.
A novel method termed metal oxide affinity chromatography (MOAC) of enriching for phosphorylated proteins and peptides based on the affinity of the phosphate group for Al(OH)(3) is presented here. When compared to commercial phosphoprotein-enrichment kits, this method is more selective, more cost effective and easily applicable to method optimization. The use of glutamic and aspartic acid in the loading buffer significantly enhances selectivity. Standard protein mixtures and complex Arabidopsis thaliana leaf protein extracts were tested for efficacy of enrichment. The method can be applied to proteins extracted using either mild or denaturing conditions. The same Al(OH)(3) material is suitable for the enrichment of phosphopeptides out of a tryptic digest of alpha-casein. Peptide phosphorylation was revealed by beta-elimination of phosphate groups. Enrichment and in vivo phosphorylation of A. thaliana leaf proteins were confirmed with Pro-Q diamond stain. Several of the phosphoprotein candidates that were identified by MS are known to be phosphorylated in vivo in other plant species.
Phosphorylation of proteins is essential in intracellular signal transduction pathways in eukaryotic and prokaryotic cells. Histidine phosphorylation plays an important role in two-component signal transduction in bacteria. In this study, we describe the characterization of a synthetic histidine-phosphorylated peptide with four different mass spectrometric (MS) fragmentation techniques: Collision-induced dissociation (CID), electron capture dissociation, electron-transfer dissociation, and electron detachment dissociation. Furthermore, LC-MS methods were developed to detect histidine-phosphorylated peptides, which are acid-labile, in more complex samples. From these results, we concluded that nonacidic solvent systems or fast LC methods provide the best conditions for separation of histidine-phosphorylated peptides prior to electrospray ionization mass spectrometry analysis. Electron-based fragmentation methods should be used for determination of histidine phosphorylation sites, since CID results in very facile phosphate-related neutral losses. The developed LC-MS/MS methods were successfully applied to a tryptic digest of the cytoplasmic part of the histidine kinase EnvZ, which was in vitro autophosphorylated. Finally, a new method is described for nonretentive solid-phase extraction of histidine-phosphorylated peptides using polymeric Strata-X microcolumns.
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The effect of Arg 209 to Lys mutation in mouse thymidylate synthase
- J Cieśla
- B Gołos
- E Wałajtys-Rode
- E Jagielska
- A Płócienniczak
- W Rode
J. Cieśla, B. Gołos, E. Wałajtys-Rode, E. Jagielska, A. Płócienniczak, W. Rode, The effect
of Arg 209 to Lys mutation in mouse thymidylate synthase, Acta Biochim. Pol. 49
(2002) 651–658.
Phosphorylation of thymidylate synthase from various sources by human protein kinase CK2 and its catalytic subunits
- T Frączyk
- K Kubiński
- M Mastyk
- J Cieśla
- U Hellman
- D Shugar
- W Rode
T. Frączyk, K. Kubiński, M. Mastyk, J. Cieśla, U. Hellman, D. Shugar, W. Rode, Phosphorylation of thymidylate synthase from various sources by human protein kinase
CK2 and its catalytic subunits, Bioorg. Chem. 38 (2010) 124-131.