Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Abstract

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.

Keywords:

• thymidylate synthesis
• protein–protein interaction
• protein–protein complex model
• protein–protein interface
• molecular dynamics

Acknowledgements

Part of the computations was performed at the Interdisciplinary Center for Mathematical and Computational Modelling (ICM) of the Warsaw University, Poland. Flow cytometry experiments and confocal microscopy observations were performed at the Laboratory of Cytometry and Laboratory of Imaging Tissue Structure and Function, respectively, at the Nencki Institute of Experimental Biology, Poland. We would like to extend our sincere gratitude to Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD) for his kind gift of HCT 116 cell line.