Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Mohsen, Al-Walid A.; Aull, John L.; Payne, DJ; Daron, Harlow H.

doi:10.1021/bi00005a023

Cited by 15 publications

(6 citation statements)

References 37 publications

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“…It is well documented that upon formation of either the catalytic or the inhibitory ternary complex, the enzyme, particularly its Cterminal residues, undergoes a major conformational shift that is stabilized by a hydrogen bonding network involving the folate co-substrate (1). Indeed, this shift alters the susceptibility of TS to proteolytic enzymes in vitro (15). It is therefore not surprising that the conformational change stabilizes the enzyme in vivo.…”

Section: Resultsmentioning

confidence: 99%

Ligand-mediated Induction of Thymidylate Synthase Occurs by Enzyme Stabilization

Kitchens¹,

Forsthoefel²,

Rafique³

et al. 1999

Journal of Biological Chemistry

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Thymidylate synthase (TS) is indispensable in the de novo synthesis of dTMP. As such, it has been an important target at which anti-neoplastic drugs are directed. The fluoropyrimidines 5-fluorouracil and 5-fluoro-2-deoxyuridine are cytotoxic as a consequence of inhibition of TS by the metabolite 5-fluoro-2-deoxyuridine 5-monophosphate (FdUMP). This inhibition occurs through formation of a stable ternary complex among the enzyme, the nucleotide analog, and the co-substrate N 5 ,N 10 -methylenetetrahydrofolate. Numerous studies have shown that cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. An extensive body of in vitro studies has led to the proposal that this induction occurs because of relief of the translational repression brought on by the binding of TS to its own mRNA. In the current study, we have tested several predictions of this autoregulatory translation model. In contrast to expectations, we find that fluoropyrimidines do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations within the mRNA that abolish its ability to bind TS have no effect on the induction. Finally, enzyme turnover measurements show that the induction is associated with an increase in the stability of the TS polypeptide. Our results, in total, indicate that enzyme stabilization, rather than translational derepression, is the primary mechanism of TS induction by fluoropyrimidines and call into question the general applicability of the autoregulatory translation model.Thymidylate synthase (TS, 1 EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH 2 H 4 PteGlu, generating dTMP and dihydrofolate (for a review, see Ref. 1). Because the enzyme is indispensable in the de novo synthesis of dTMP, it plays an important role in DNA replication in actively dividing cells and has been an attractive target at which anti-neoplastic agents are directed. Fluoropyrimidines (e.g. 5-fluorouracil and FdUrd) and, more recently, anti-folates (e.g. AG337, ZD1694, BW1843U89) have been useful in the clinical management of tumors of the breast, colon, stomach, and head and neck (2-5). Fluoropyrimidines exert their effects through formation of the nucleotide analog FdUMP, which inhibits TS via formation of a covalent complex containing the analog CH 2 H 4 PteGlu and the enzyme (1). This complex, which is termed the inhibitory ternary complex, is quite stable and leads to prolonged inhibition of the enzyme, depletion of dTMP pools, and thymineless death.A number of studies with cultured cell lines, tumor models, and clinical specimens have shown that TS inhibitors induce enzyme levels by about 2-4-fold (6 -8). Because response to TS-directed chemotherapy is dependent upon the enzyme concentration, such induction has been viewed as a potential barrier to successful therapeutic outcomes. As a result, there has been a great deal of interest in the mechanism of the induction and in strategies to ameliorate its effects. The increases in TS levels do not involve change...

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Section: Resultsmentioning

confidence: 99%

Ligand-mediated Induction of Thymidylate Synthase Occurs by Enzyme Stabilization

Kitchens¹,

Forsthoefel²,

Rafique³

et al. 1999

Journal of Biological Chemistry

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“…One important mechanism is the disruption of the autoregulatory feedback loop for the repression of translation. TS ligands, such as 5-FU, disrupt the binding of the TS enzyme with TYMS mRNA, leading to translational derepression and overproduction of the TS enzyme. ,, In addition to translational derepression, enzyme stabilization has been suggested as the primary mechanism of TS induction by fluoropyrimidines in human CRC and ovarian cancer cell lines. − Furthermore, it is proposed that fluoropyrimidine-mediated increases in TS levels are induced by its effect on TS enzyme stability, with no effect on TYMS mRNA. ,, The amplification of TYMS , leading to the overproduction of TYMS mRNA and TS protein, is another mechanism of resistance to fluoropyrimidines like 5-FU and its derivatives . These observations indicated that an understanding of translational derepression, enzyme stabilization, and gene amplification as the process of TS induction can help to elucidate the mechanism of the acquisition of 5-FU resistance.…”

Section: Discussionmentioning

confidence: 99%

“…27−29 Furthermore, it is proposed that fluoropyrimidine-mediated increases in TS levels are induced by its effect on TS enzyme stability, with no effect on TYMS mRNA. 28,30,31 The amplification of TYMS, leading to the overproduction of TYMS mRNA and TS protein, is another mechanism of resistance to fluoropyrimidines like 5-FU and its derivatives. 12 These observations indicated that an understanding of translational derepression, enzyme stabilization, and gene amplification as the process of TS induction can help to elucidate the mechanism of the acquisition of 5-FU resistance.…”

Section: ■ Discussionmentioning

confidence: 99%

Trapping of 5-Fluorodeoxyuridine Monophosphate by Thymidylate Synthase Confers Resistance to 5-Fluorouracil

et al. 2022

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The major metabolite of the anticancer agent 5-fluorouracil (5-FU) is 5-fluorodeoxyuridine monophosphate (FdUMP), which is a potent inhibitor of thymidylate synthase (TS). Recently, we hypothesized that 5-FU-resistant colorectal cancer (CRC) cells have increased levels of TS protein relative to 5-FU-sensitive CRC cells and use a fraction of their TS to trap FdUMP, which results in resistance to 5-FU. In this study, we analyzed the difference between the regulation of the balance of the free, active form of TS and the inactive FdUMP-TS form in 5-FU-resistant HCT116 cells and parental HCT116 cells. Silencing of TYMS, the gene that encodes TS, resulted in greater enhancement of the anticancer effect of 5-FU in the 5-FU-resistant HCT116RF10 cells than in the parental HCT116 cells. In addition, the trapping of FdUMP by TS was more effective in the 5-FU-resistant HCT116RF10 cells than in the parental HCT116 cells. Our observations suggest that the regulation of the balance between the storage of the active TS form and the accumulation of FdUMP-TS is responsible for direct resistance to 5-FU. The findings provide a better understanding of 5-FU resistance mechanisms and may enable the development of anticancer strategies that reverse the sensitivity of 5-FU resistance in CRC cells.

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“…Furthermore, it is proposed that fluoropyrimidine-mediated increases in TS levels occur through an effect on enzyme stability with no effect on its mRNA [25,27]. It is also suggested that TS stabilization could be the result of conformational changes that may occur upon the formation of a ternary complex, reducing the susceptibility of the TS enzyme to proteolysis [28]. These findings indicated that understanding translational derepression and enzyme stabilization as the process of TS induction has significance for elucidating the mechanism of resistance acquisition.…”

Section: Discussionmentioning

confidence: 99%

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

Kurasaka

Ogino

Sato

2021

IJMS

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5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116RF10. HCT116RF10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116RF10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116RF10 cells. Interestingly, HCT116RF10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.

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Ligand-Induced Conformational Changes of Thymidylate Synthase Detected by Limited Proteolysis

Cited by 15 publications

References 37 publications

Ligand-mediated Induction of Thymidylate Synthase Occurs by Enzyme Stabilization

Ligand-mediated Induction of Thymidylate Synthase Occurs by Enzyme Stabilization

Trapping of 5-Fluorodeoxyuridine Monophosphate by Thymidylate Synthase Confers Resistance to 5-Fluorouracil

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

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