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

Abstract

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.

The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway

“…First, no ubiquitinconjugated forms of TS are detectable by biochemical assay such as Western blotting or coimmunoprecipitation (16). Second, genetic abrogation of the ubiquitin conjugation pathway has little or no effect on the intracellular half-life of the TS polypeptide (17). Finally, conversion of the enzyme's 15 Lys residues to Arg, which removes sites of potential ubiquitin conjugation, fails to stabilize the protein (17).…”

“…Second, genetic abrogation of the ubiquitin conjugation pathway has little or no effect on the intracellular half-life of the TS polypeptide (17). Finally, conversion of the enzyme's 15 Lys residues to Arg, which removes sites of potential ubiquitin conjugation, fails to stabilize the protein (17). Thus, intracellular degradation of TS does not appear to require either ubiquitinylation or the ubiquitinylation pathway.…”

“…However, in the case of TS, the disordered domain is located at the N-rather than the C-terminal end and is formed by the first 27 amino acids (16,17). Deletion of just the first few residues can result in a very stable enzyme (16,17).…”

Cooperation between an Intrinsically Disordered Region and a Helical Segment Is Required for Ubiquitin-independent Degradation by the Proteasome

“…In addition, as is also shown in Fig. 3, A and C, the molecule was stabilized by a Pro-2 Ala replacement within the hTS moiety at the N terminus; this substitution is known to promote N-␣-acetylation and to stabilize wild-type hTS (25,26). Thus, the IDR of fibrinogen-␣ is capable of promoting degradation of full-length hTS and operates by a similar mechanism as the native IDR within hTS.…”

“…Previous studies in our laboratory have identified the human enzyme (denoted hTS) as a ubiquitinindependent proteasomal substrate (24,25). Degradation of hTS is governed by a 45-residue region at its N-terminal end, which is composed of a flexible, intrinsically disordered region (IDR) spanning the first 27 amino acids, followed by an amphipathic ␣-helix (helix A (hA)) at residues 31-42 (26).…”