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“Targeting Thymidylate Synthase in Cancer Therapy” F.G. Berger Department of Biological Sciences Center for Colon Cancer Research University of South Carolina. Thymidylate synthase (TS). Catalyzes the conversion of dUMP into dTMP. Figure adapted from Lehninger. N. C.
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“Targeting Thymidylate Synthase in Cancer Therapy” F.G. Berger Department of Biological Sciences Center for Colon Cancer Research University of South Carolina
Thymidylate synthase (TS) Catalyzes the conversion of dUMP into dTMP Figure adapted from Lehninger
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Thymidylate Synthase Inhibitors: 5-Fluoro-2’-deoxyuridine (FdUrd) 5-Fluorouracil (FUra)
Control TNFα FUra FUra FdUrd FdUrd RTX
Role of post-translational processes in TS function: ● Degradation of the TS polypeptide ● SUMO modification of TS ● Nuclear localization
Intracellular stability of human TS HCT15 HCT15/200
Polysome profiles for TS mRNA Kitchens, M. E. et al. 1999. J. Biol. Chem. 274:12544-12547
● TS is degraded intracellularly by the 26S proteasome. ●TS is not ubiquitinylated. ●Abrogation of the ubiquitin ligation pathway does not alter TS degradation. ● Conversion of all Lys residues to Arg does not stabilize the TS polypeptide. Therefore, the TS polypeptide is degraded in a ubiquitin-independent manner.
Role of the penultimate residue in TS degradation. t1/2≤ 12 h t1/2≥ 36 h
N-terminal processing of the TS polypeptide: ● Methionine excision ● N-α-acetylation * From Meinnel et al. 2005.Biochimie 87, 701-712.
Human Pro2 - Val3 - Ala4 - Gly5---- Mouse Ac-Met1 - Leu2 - Val3 - Val4 - Gly5---- Rat Ac-Met1 - Leu2 - Val3 - Glu4 - Gly5----
210 PVAGSELPR
Yeast two-hybrid screen Bait: hTS-(1-313) Gal4-BD Prey Human Placental c-DNA library fused to the C-terminus of Gal4-AD
● UBC9 is a conjugating enzyme required for protein sumoylation, and has been implicated in regulating several critical cellular pathways. ●SUMO is activated for conjugation by the E1 enzyme AOS/UBA2, transferred to the E2 conjugation enzyme UBC9, and finally conjugated to target proteins.
●Known target proteins of sumoylation include p53, MDM2, PML, RanGAP1, IκB, androgen receptor, and c-Jun. ●Modification of these proteins by sumoylation changes their subcellular localization, function, and/or stability. ●Sumoylation is reversible, and there are at least seven mammalian SUMO-specific proteases, which are designated the SENP family proteins.
In vitro TS sumoylation occurs, and requires all components of sumoylation pathway TS + mSUMO-1 TS + SUMO-1 No Protein TS + E1 TS + E2 TS only TS-SUMO1 WB: Anti-hTS Ab TS
Formation of Inhibitory Ternary Complex blocks TS Sumoylation _____TS + SUMO1_____ Sumoylated TS + CH2THF + FdUMP TS + CH2THF + FdUMP + CH2THF + FdUMP + CH2THF only + FdUMP only TS only - TS-SUMO1 * * * TS * - inhibitory ternary complex
- Ψ is bulky hydrophobic residue, X is any residue Ψ-K-X-E Consensus SUMO ligation sites in TS Site A (K284) Site C (K308) Site B (K292)
281 313 ● ● Human TS: …ILRKVEKIDDFKAEDFQIEGYNPHPTIKMEMAV Mouse TS: T V Rat TS: T V
K308 is the site of TS sumoylation TnT-Coupled in vitro Translated human TS K284R K292R K308R Site B K292A Site C K308R K284R K292R No plasmid Site A K284R WT 1 2 1 2 1 2 1 2 1 2 1 2 1 2 hTS-SUMO1 hTS 1 – in vitro synthesized TS 2 – in vitro synthesized TS + SUMO1
Fluorescence complementation assay Fang, Deyu and Kerppola (2004) Proc. Natl. Acad. Sci. USA 101, 14782-14787
CC YN SUMO1 TS K308 SUMO1 + TS
Confocal microscopy of HeLa-55 cells stained by immunofluorescence for thymidylate synthase (TS) Bissoon-Haqqani, S. et al. J. Histochem. Cytochem. 2006;54:19-29
Such ligand-mediated inhibition of nuclear TS accumulation is not a consequence of DNA damage
TS localization to the nucleus is also modulated by residue K308, but does not require sumoylation at this site
“Working” Model for nuclear import of TS: (1.) C-terminal end (including K308) of TS is required for its binding to the nuclear periphery. (2.) This is abrogated by either ligands or the K308A mutation. (3.) TS goes through a sumoylation/desumoylation cycle as it enters the nucleus. (4.) This determines the enzyme’s intra-nuclear locale. Ligands affect exposure of C-terminal region; Sumoylation affects intra-nuclear locale, but not entry.
High nuclear TS levels in colorectal tumors are associated with poorer survival: M. Gustavson et al. AACR Conference on Molecular Diagnostics in Cancer Therapeutic Development (Chicago, IL., Sept. 2006)
Could the cytotoxicity of TS inhibitors derive from abrogation of the enzyme’s nuclear accumulation, and subsequent effects on DNA repair?
Marj Peña Kenn White Karen Barbour Sandra Melo Yang Yang Xing