FRANKLIN G. BERGER

Co-localization of human TS and promyelocytic leukemia (PML) bodies. TS-negative cells (top row), wild-type cells (middle row), and cells expressing TS predominantly in the nucleus (bottom row) were stained for nuclei (blue), TS (green) and PML bodies (red), and observed by fluorescence microscopy. For each cell type, red and green fluorescence were merged in the panels to the far right.

Extensive studies both in our laboratory and in others, have shown that both the structure and the concentration of TS are important factors governing cellular response to TS inhibitors. Early studies established that TS gene amplification, leading to mRNA and enzyme overproduction, is an important mechanism of 5-fluoro-2'-deoxyuridine (FdUrd)-resistance in human cells. We have characterized several structural variants of TS that confer changes in the enyzme's expression and function. Alterations in the stability of TS and its interactions with ligands have been identified among these variants.

Ligand-mediated induction of TS concentrations: Numerous investigations have shown that the cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. In vitro studies have led to the proposal that this induction is due to ligand-mediated relief of the translational repression brought on by binding of TS to its own mRNA. We have tested several predictions of this autoregulatory translation model, and find that contrary to expectations, TS ligands do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations that abolish the ability of TS mRNA to bind the enzyme 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. Transfection studies with human cell lines have shown that covalent, inhibitory ternary complex formation among the enzyme and its ligands is not required for the ligand-mediated stabilization.

Mechanisms of intracellular TS degradation: We have embarked on a study of the mechanism by which the TS molecule is degraded within the cell. Our work has shown that degradation of TS is ubiquitin-independent and is mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. This region, in cooperation with an alpha-helix formed by the next 15 residues, forms a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the polypeptide's N-terminal domain is hypervariable among species yet is conserved with regard to its disordered structure, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro2 protects the N-terminus against N-α-acetylation, a post-translational process that inhibits TS degradation. However, although a free, unblocked amino group at the N-terminus is necessary, it is not sufficient for degradation of the polypeptide.

In further studies, we have used targeted in vitro mutagenesis to define additional elements within the disordered domain that regulate TS degradation. We have shown that an Arg-Arg motif at residues 10-11 is required for proteolysis; deletion or substitution of both of these residues leads to profound stabilization of the polypeptide, an observation that was confirmed by functional analysis of the TS N-terminus from a number of mammalian species. The effects of stabilizing mutations on hTS degradation are maintained when the enzyme is fused to the protein PSMD4, which provides an alternate means of proteasome association; this indicates that such mutations perturb one or more post-docking steps in the degradation pathway. Interestingly, deletions that remove large segments of the disordered domain cause degradation to occur by a distinct mechanism, as deduced from assessment of the impact of modifying one or the other terminus of the polypeptide.

Destabilizing the TS polypeptide as a means of improving cytotoxicity of TS inhibitors: The fact that TS is a relatively stable protein (half-life = 5-8 h), along with the observation that active-site inhibitors of the enzyme increase its half-life to > 24 h, means that elevated concentrations of enzyme that are resistant to change will be maintained during drug treatment, thereby decreasing effective inhibition of dTMP synthesis during chemotherapy. In order to combat this constraint to effective TS-directed therapy, we are using chemical genetic strategies to specific destabilize the TS molecule. Novel drugs are being designed that should promote ubiquitin modification of the enzyme, which should make it a more efficient proteasomal substrate; this in turn will render the enzyme more unstable, thereby increasing the efficacy of inhibitors directed at it.

Selected Recent References

Peña, M., Y.Y. Xing, S. Koli, and F.G. Berger. 2006. Role of N-terminal residues in ubiquitin-independent degradation of human thymidylate synthase. Biochem. J. 394:355-363.

Berger, F.G. and S.H. Berger. 2006. Thymidylate synthase as a chemotherapeutic drug target: Where are we after fifty years? Cancer Biol. Therapy 5:1238-1241.

Barbour, K.W. and F.G. Berger. 2008. Cell death in response to antimetabolites directed at thymidylate synthase. Cancer Chemother. Pharmacol. 61:189-201.

Peña, M.M., S. Melo, Y.-Y. Xing, K. White, K.W. Barbour, and F.G. Berger. 2009. The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway. J. Biol. Chem. 284:31597-31607.

Huang, X., L.M. Gibson, B.J. Bell, L.L. Lovelace, M.M.O. Peña, F.G. Berger, S.H. Berger, and L. Lebioda. 2010. Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability. Biochemistry 29:2475-2482.

Melo, S.P., A. Yoshida, and F.G. Berger. 2010. Functional dissection of the N-terminal degron of human thymidylate synthase. Biochem. J. 432:217-226.

Franklin G. Berger

Molecular Genetics of Response to Cancer Chemotherapy

Selected Recent References