Role of N-terminal residues in the ubiquitin-independent degradation of human thymidylate synthase

Thymidylate synthase (TS) catalyses the reductive methylation of dUMP to form dTMP, a reaction that is essential for maintenance of nucleotide pools during cell growth. Because the enzyme is indispensable for DNA replication in actively dividing cells, it is an important target for cytotoxic drugs used in cancer chemotherapy, including fluoropyrimidines (e.g. 5-fluorouracil and 5-fluoro-2′-deoxyuridine) and anti-folates (e.g. raltitrexed, LY231514, ZD9331 and BW1843U89). These drugs generate metabolites that bind to the enzyme's active site and inhibit catalytic activity, leading to thymidylate deprivation and cellular apoptosis. Ligand binding to TS results in stabilization of the enzyme and an increase in its intracellular concentration. Previously, we showed that degradation of the TS polypeptide is carried out by the 26 S proteasome in a ubiquitin-independent manner. Such degradation is directed by the disordered N-terminal region of the TS polypeptide, and is abrogated by ligand binding. In the present study, we have verified the ubiquitin-independent nature of TS proteolysis by showing that a ‘lysine-less’ polypeptide, in which all lysine residues were replaced by arginine, is still subject to proteasome-mediated degradation. In addition, we have mapped the structural determinants of intracellular TS degradation in more detail and show that residues at the N-terminal end of the molecule, particularly the penultimate amino acid Pro², play an important role in governing the half-life of the enzyme. This region is capable on its own of destabilizing an evolutionarily distinct TS molecule that normally lacks this domain, indicating that it functions as a degradation signal. Interestingly, degradation of an intrinsically unstable mutant form of TS, containing a Pro→Leu substitution at residue 303, is directed by C-terminal, rather than N-terminal, sequences. The implications of these findings for the control of TS expression, and for the regulation of protein degradation in general, are discussed.