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Structural determinants for the intracellular
degradation of human thymidylate synthase.
Forsthoefel AM, Pena MM, Xing YY, Rafique Z, Berger FG.
Department of Biological Sciences, University of South Carolina,
Columbia, South Carolina 29208, USA.
Thymidylate
synthase (EC 2.1.1.45) (TS) catalyzes the conversion of dUMP to dTMP
and is therefore indispensable for DNA replication in actively dividing
cells. The enzyme is a critical target at which chemotherapeutic agents
such as fluoropyrimidines (e.g., 5-fluorouracil and
5-fluoro-2'-deoxyuridine) and folic acid analogues (e.g., raltitrexed,
LY231514, ZD9331, and BW1843U89) are directed. These agents exert their
effects through the generation of metabolites that bind the active site
of TS and inhibit catalytic activity. The binding of ligands to the TS
molecule leads to dramatic changes in the conformation of the enzyme,
particularly within the C-terminal domain. Stabilization of the enzyme
and an increase in its intracellular level are associated with ligand
binding and may be important in cellular response to TS-directed drugs.
In the present study, we have examined molecular features of the TS
molecule that control its degradation. We find that the C-terminal
conformational shift is not required for ligand-mediated stabilization
of the enzyme. In addition, we demonstrate that the N-terminus of the
TS polypeptide, which is extended in the mammalian enzyme and is
disordered in crystal structures, is a primary determinant of the
enzyme's half-life. Finally, we show that TS turnover is carried out by
the 26S proteasome in a ubiquitin-independent manner. These findings
provide the basis for a mechanistic understanding of TS degradation and
its regulation by antimetabolites.
PubMed
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