PT - JOURNAL ARTICLE AU - Sharon Spizzichino AU - Dalila Boi AU - Giovanna Boumis AU - Roberta Lucchi AU - Francesca Romana Liberati AU - Davide Capelli AU - Roberta Montanari AU - Giorgio Pochetti AU - Alessio Paone AU - Serena Rinaldo AU - Roberto Contestabile AU - Alessandro Paiardini AU - Angela Tramonti AU - Giorgio Giardina AU - Francesca Cutruzzolà TI - Cytosolic localization and <em>in vitro</em> assembly of human <em>de novo</em> thymidylate synthesis complex AID - 10.1101/2020.12.23.423904 DP - 2021 Jan 01 TA - bioRxiv PG - 2020.12.23.423904 4099 - http://biorxiv.org/content/early/2021/01/31/2020.12.23.423904.short 4100 - http://biorxiv.org/content/early/2021/01/31/2020.12.23.423904.full AB - 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 StatementThe authors have declared no competing interest.dTMPDeoxythymidine monophosphatedUMPdeoxyuridine monophosphateTYMSthymidylate synthaseSHMTserine hydroxymethyltransferaseDHFRdihydrofolate reductaseCH2-THF5,10-methylene-tetrahydrofolateTHFtetrahydrofolateDHFdihydrofolatedTMP-SCthymidylate synthesis complexIFImmunofluorescenceis-PLAin situ proximity ligation assayIPTGisopropil-β-D-1-tiogalattopiranoside5-FU5-fluorouracilMTXmethotrexatePTXpemetrexedFdUMPfluorodeoxyuridine-monophosphatecryo-EMcryogenic electron microscopyIMACimmobilized metal affinity chromatographyDFSDifferential scanning fluorimetrySPRsurface plasmon resonanceo.n.overnightR.T.room temperature.