ABSTRACT
De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), the latter two targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result strongly indicates that the role of the dTMP-SC assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, may provide a useful tool to develop drugs targeting the entire complex instead of the individual components.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Addition of Co-localization experiments. Figures update to include all supplementary data that are now no longer needed.
ABBREVIATIONS
- dTMP
- Deoxythymidine monophosphate
- dUMP
- deoxyuridine monophosphate
- TYMS
- thymidylate synthase
- SHMT
- serine hydroxymethyltransferase
- DHFR
- dihydrofolate reductase
- CH2-THF
- 5,10-methylene-tetrahydrofolate
- THF
- tetrahydrofolate
- DHF
- dihydrofolate
- dTMP-SC
- thymidylate synthesis complex
- IF
- Immunofluorescence
- is-PLA
- in situ proximity ligation assay
- IPTG
- isopropil-β-D-1-tiogalattopiranoside
- 5-FU
- 5-fluorouracil
- MTX
- methotrexate
- PTX
- pemetrexed
- FdUMP
- fluorodeoxyuridine-monophosphate
- cryo-EM
- cryogenic electron microscopy
- IMAC
- immobilized metal affinity chromatography
- DFS
- Differential scanning fluorimetry
- SPR
- surface plasmon resonance
- o.n.
- overnight
- R.T.
- room temperature.