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Role of N-terminal residues in the
ubiquitin-independent degradation of human thymidylate synthase.
Pena MM, Xing YY, Koli S, Berger FG.
Thymidylate
synthase (TS) catalyzes 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. 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 26S 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 current study, we have verified this conclusion
by showing that proteasomal degradation of TS is maintained when all
lysine residues, which are typically the sites of ubiquitin ligation,
are replaced by arginine. 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 to the control of thymidylate synthase
expression, and to the regulation of protein degradation in general,
are discussed.
PMID: 16259621 [PubMed - as supplied by publisher]
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