Effects of ligand binding and conformational switching on intracellular stability of human thymidylate synthase.

Thymidylate synthase (TS) is the target in colon cancer therapeutic protocols utilizing such drugs as 5-fluorouracil and raltitrexed. The effectiveness of these treatments is hampered by emerging drug resistance, usually related to increased levels of TS. Human TS (hTS) is unique among thymidylate synthases from all species examined as its loop 181-197 can assume two main conformations related by rotation of 180 degrees. In one conformation, "active", the catalytic Cys-195 is positioned in the active site; in the other conformation, "inactive", it is at the subunit interface. Also, in the active conformation, region 107-128 has one well-defined conformation while in the inactive conformation this region assumes multiple conformations and is disordered in crystals. The native protein exists in apparent equilibrium between the two conformational states, while the enzyme liganded with TS inhibitors assumes the active conformation. The native protein has been reported to bind to several mRNAs, including its own mRNA, but upon ligation, RNA binding activity is lost. Ligation of TS by inhibitors also stabilizes it to turnover. Since currently used TS-directed drugs stabilize the active conformation and slow down the enzyme degradation, it is postulated that inhibitors of hTS stabilizing the inactive conformation of hTS should cause a down-regulation in enzyme levels as well as inactivate the enzyme.

[1]  G. Maley,et al.  Identification of an RNA binding site for human thymidylate synthase. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  G. Maley,et al.  Crystal structure of a deletion mutant of human thymidylate synthase Δ (7–29) and its ternary complex with Tomudex and dUMP , 2001, Protein science : a publication of the Protein Society.

[3]  N. Xuong,et al.  Crystal structure of Escherichia coli thymidylate synthase containing bound 5-fluoro-2'-deoxyuridylate and 10-propargyl-5,8-dideazafolate. , 1990, Journal of molecular biology.

[4]  F A Quiocho,et al.  Structural basis for sequence-nonspecific recognition of 5'-capped mRNA by a cap-modifying enzyme. , 1998, Molecular cell.

[5]  D. Santi,et al.  The catalytic mechanism and structure of thymidylate synthase. , 1995, Annual review of biochemistry.

[6]  G. Peters,et al.  Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[7]  N. Horie,et al.  Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase. , 1995, Cell structure and function.

[8]  P C Elwood,et al.  Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[9]  G. Watanabe,et al.  Different lengths of a polymorphic repeat sequence in the thymidylate synthase gene affect translational efficiency but not its gene expression. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[10]  G. Aherne,et al.  Comparison of thymidylate synthase (TS) protein up-regulation after exposure to TS inhibitors in normal and tumor cell lines and tissues. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[11]  R. Stroud,et al.  Conformational dynamics along an enzymatic reaction pathway: thymidylate synthase, "the movie". , 2003, Biochemistry.

[12]  D. Steadman,et al.  Genetic complementation and resistance to 5-fluoro-2'-deoxyuridine in thymidine auxotrophs expressing a highly defective mutant of human thymidylate synthase. , 1999, Biochemical pharmacology.

[13]  L. Lebioda,et al.  Structure of Human Thymidylate Synthase Suggests Advantages of Chemotherapy with Noncompetitive Inhibitors* , 2001, The Journal of Biological Chemistry.

[14]  R. Stroud,et al.  Crystal structures of a unique thermal-stable thymidylate synthase from Bacillus subtilis. , 1998, Biochemistry.

[15]  F. Berger,et al.  Ligand-mediated Induction of Thymidylate Synthase Occurs by Enzyme Stabilization , 1999, The Journal of Biological Chemistry.

[16]  P. Johnston,et al.  Colorectal liver metastasis thymidylate synthase staining correlates with response to hepatic arterial floxuridine. , 1999, Clinical cancer research : an official journal of the American Association for Cancer Research.

[17]  G. Maley,et al.  Crystal structures of rat thymidylate synthase inhibited by Tomudex, a potent anticancer drug. , 1999, Biochemistry.

[18]  G. Maley,et al.  Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA. , 2000, Nucleic acids research.

[19]  D. M. Greenberg,et al.  Studies on the conversion of deoxyuridylic acid to thymidylic acid by a soluble extract from rat thymus. , 1958, Archives of biochemistry and biophysics.

[20]  M. Karin,et al.  Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation , 1996, Molecular and cellular biology.

[21]  H. McLeod,et al.  Ethnic variation in the thymidylate synthase enhancer region polymorphism among Caucasian and Asian populations. , 1999, Genomics.

[22]  R. Stroud,et al.  Lessons and conclusions from dissecting the mechanism of a bisubstrate enzyme: thymidylate synthase mutagenesis, function, and structure. , 2003, Biochemistry.

[23]  R. Stroud,et al.  Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate. , 1990 .

[24]  K. Keyomarsi,et al.  The thymidylate synthase inhibitor, ICI D1694, overcomes translational detainment of the enzyme. , 1993, The Journal of biological chemistry.

[25]  U. McDermott,et al.  The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates. , 2002, Cancer research.

[26]  P. Johnston,et al.  Thymidylate synthase gene and protein expression correlate and are associated with response to 5-fluorouracil in human colorectal and gastric tumors. , 1995, Cancer research.

[27]  R. Stroud,et al.  Pairwise specificity and sequential binding in enzyme catalysis: thymidylate synthase. , 1990, Biochemistry.

[28]  R. Stroud,et al.  The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits. , 1999, Biochemistry.

[29]  G. Maley,et al.  Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells , 1994, Molecular and cellular biology.

[30]  Jörg Stappert,et al.  β‐catenin is a target for the ubiquitin–proteasome pathway , 1997 .

[31]  G. Maley,et al.  Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex , 1999, Molecular and Cellular Biology.

[32]  T. Maniatis,et al.  Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. , 1995, Genes & development.

[33]  L. Lebioda,et al.  Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254. , 1999, Molecular pharmacology.

[34]  S. B. Gates,et al.  Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases. , 2001, Journal of molecular biology.

[35]  C. Allegra,et al.  Identification of in vivo target RNA sequences bound by thymidylate synthase. , 1996, Nucleic acids research.

[36]  G. Maley,et al.  In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase. , 2000, Nucleic acids research.

[37]  W. Washtien.  Increased levels of thymidylate synthetase in cells exposed to 5-fluorouracil. , 1984, Molecular pharmacology.

[38]  K. Keyomarsi,et al.  Mechanism of the cytotoxic synergism of fluoropyrimidines and folinic acid in mouse leukemic cells. , 1988, The Journal of biological chemistry.

[39]  R. Stroud,et al.  Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site. , 1995, Biochemistry.

[40]  S. Groshen,et al.  Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy , 2001, The Pharmacogenomics Journal.

[41]  A. Goldberg,et al.  Proteasome inhibitors: valuable new tools for cell biologists. , 1998, Trends in cell biology.

[42]  R D Klausner,et al.  The iron-responsive element-binding protein: localization of the RNA-binding site to the aconitase active-site cleft. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Stephen W. Byers,et al.  Serine Phosphorylation-regulated Ubiquitination and Degradation of β-Catenin* , 1997, The Journal of Biological Chemistry.

[44]  Robert R. Rando,et al.  Aminoglycoside antibiotics are able to specifically bind the 5'-untranslated region of thymidylate synthase messenger RNA. , 1999, Biochemistry.

[45]  F. Berger,et al.  Mechanisms of acquired resistance to thymidylate synthase inhibitors: the role of enzyme stability. , 1999, Molecular pharmacology.

[46]  R M Stroud,et al.  Atomic structure of thymidylate synthase: target for rational drug design. , 1987, Science.

[47]  K. Danenberg,et al.  Evidence for a sequential interaction of the subunits of thymidylate synthetase. , 1979, The Journal of biological chemistry.

[48]  T. Seno,et al.  Conditional thymidine auxotrophic mutants of mouse FM3A cells due to thermosensitive thymidylate synthase and their prototrophic revertants. , 1981, The Journal of biological chemistry.

[49]  M. Yin,et al.  Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors. , 1997, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[50]  P. Danenberg,et al.  Thymidylate synthase inhibitors. , 1999, Seminars in oncology.

[51]  Kazuki Kurimoto,et al.  Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein , 1999, Nature.

[52]  P. Johnston,et al.  Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. , 1993, Advances in experimental medicine and biology.

[53]  L. Lebioda,et al.  Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug. , 2001, Biochemistry.