Faculty & Staff Directory
Franklin G. Berger
Director for The Center for Colon Cancer Research
Department of Biology
University of South Carolina
|Office:||PSC 614||Phone Number:||(803) 777-1171 (office) (803) 777-7216 (lab) (803) 777-1231 (CCCR office)||Email:||firstname.lastname@example.org|
Background: Resistance to anti-neoplastic agents is a major impediment to the chemotherapy of cancer. The high genetic plasticity of neoplastic cells generally leads to rapid emergence of cells that are resistant to the cytotoxic effects of chemotherapeutic drugs. We have been examining the mechanism of action of agents that inhibit the pyrimidine biosynthetic enzyme thymidylate synthase (TS). This enzyme catalyzes the formation of dTMP from dUMP, and is indispensible for DNA synthesis during cell proliferation and DNA repair. Treatment of cells with fluoropyrimidine analogs (e.g., 5-fluorouracil and 5-fluoro-2'- deoxyuridine), as well as anti-folates (e.g., tomudex, AG337, and BW1843), result in generation of metabolites that inhibit TS, leading to depletion of thymidylate pools, cessation of cell growth, and eventually apoptotic and non-apoptotic cell death. Thus, these compounds have been useful in the treatment of a variety of neoplasms.
Reactive oxygen species mediate cell death in response to TS inhibition: We have shown that apoptosis in response to TS inhibitors is due to increased levels of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide. This, in turn, derives from activation of the enzyme NADPH oxidase, which generates superoxide by transfer of an electron from NADPH to molecular oxygen. Current efforts are aimed at an understanding of how thymidylate deficiencies promote NADPH oxidase activation and ROS-mediated cell death.
Ligand-mediated induction of TS concentrations: Numerous investigations have shown that the cellular concentrations of TS undergo about a 2-4-fold induction following treatment with TS inhibitors. This induction does not involve a change in TS mRNA levels, indicating that it occurs via a post-transcriptional mechanism. In vitro studies have led to the proposal that the drug-mediated increase in TS protein is due to relief of the translational repression brought on by binding of TS to its own mRNA. We have tested several predictions of this so-called autoregulatory translation model, and find that contrary to expectations, TS inhibitors do not cause a change in the extent of ribosome binding to TS mRNA. Furthermore, mutations that abolish the ability of TS mRNA to bind the enzyme have no effect on the induction. Finally, enzyme degradation measurements show that TS protein induction is associated with an increase in the stability of the TS polypeptide.
Mechanisms of intracellular TS degradation: Based on the findings described in the preceding paragraph, we have embarked on a study of the mechanisms underlying TS degradation and its alteration by inhibitory ligands. Most protein degradation within the cell is carried out by a large, multi-submunit "machine" termed the 26S proteasome. Generally, target substrates of the proteasome are recognized via covalent attachment of ubiquitin moieties to lysine residue within the target polypeptides. We have found that TS is indeed degraded by the 26S proteasome, but in a ubiquitin-independent manner. Such degradation is mediated by a 42-residue region that is located at the N-terminal end of the polypeptide and that forms a degron capable of destabilizing a heterologous protein to which it is fused. The TS degron is composed of an intrinsically disordered region (residues 1-27) followed by an amphipathic alpha-helix (residues 31-42) that cooperate to promote degradation of the polypeptide.
The disordered region has an amino acid composition that is characteristic of disordered domains in general. Comparative analysis of the primary sequence of TS from a number of mammalian species has revealed that the segment is hypervariable among species. This is driven by weakened purifying (i.e., negative) selection, as opposed to diversifying (i.e., positive) selection. Thus, while the region is capable of tolerating a higher degree of amino acid substitution than the rest of the polypeptide, it is still subject to evolutionary constraint.
Functional elements within the TS degron: Using targeted in vitro mutagenesis, we have defined specific elements within the TS degron that regulate its activity. A free, unblocked N-terminal amino group is required for degron activity. In addition, an Arg-Arg motif at residues 10-11 within the disordered domain is required. The alpha-helical segment of the degron does not function simply as an extension or scaffold for the disordered domain; rather, it provides a specific structural component that is necessary for degradation and that requires its helical conformation. Small domains from heterologous proteins can substitute for both the disordered and helical regions, indicating that the degradation-promoting function of these regions is not sequence-specific. Thus, there appears to be little sequence constraint on the ability of these regions to function as degron constituents; rather, it is the overall conformation (or lack thereof) that is critical.
Future studies are focused on identifying the specific functions of the disordered and helical domains within the TS degron, and how they mediate ubiquitin-independent degradation of the TS polypeptide by the 26S proteasome.
Destabilizing the TS polypeptide as a means of improving cytotoxicity of TS inhibitors: The fact that TS is a relatively stable protein, along with the observation that active-site inhibitors of the enzyme markedly increase its half-life, means that elevated concentrations of enzyme that are resistant to change will be maintained during drug treatment, thereby decreasing effective inhibition of dTMP synthesis during chemotherapy. In order to combat this constraint to effective TS-directed therapy, we are using chemical genetic strategies to specifically destabilize the TS molecule. Novel drugs are being designed that should promote enzyme degradation and resistance to ligand-mediated stabilization. This should enhance the effectiveness of TS as a chemotherapeutic target.
Melo, SP, KW Barbour, and FG Berger. 2011. Cooperation between an intrinsically disordered region and a helical segment is required for ubiquitin-independent degradation by the proteasome. J. Biol. Chem. Oct 21;286(42). 36559–36567. PubMed
Davis C, Price R, Acharya G, Baudino T, Borg T, Berger FG, Peña MM. 2011. Hematopoietic derived cell infiltration of the intestinal tumor microenvironment in Apc Min/+ mice. Microsc Microanal. Aug;17(4). 528-539. PubMed
Melo SP, Yoshida A, Berger FG. 2010. Functional dissection of the N-terminal of human thymidylate synthase. Biochem Journal. Oct 25;432(1). 217-226. Pubmed
Huang X, Gibson LM, Bell BJ, Lovelace LL, Pena MM, Berger FG, Berger SH, Lebioda L. 2010. Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intercellular stability. Biochemistry. Mar 23;49(11). 2475-2482. PubMed
Peña MM, Melo SP, Xing YY, White K, Barbour KW, Berger FG. 2009. The intrinsically disordered N-terminal domain of thymidylate synthase targets the enzyme to the ubiquitin-independent proteasomal degradation pathway. J Biol Chem. Nov 13;284(46). 31597-31607. PubMed
Barbour KW, Berger FG. 2008. Cell death in response to antimetabolites directed at thymidylate synthase. Cancer Chemother Pharmacol. Feb;61(2). 189-201. PubMed
Berger FG, Berger SH. 2006. Thymidylate synthase as a chemotherapeutic drug target: where are we after fifty years?. Cancer Biol Ther. Sep;5(9). 1238-1241. PubMed
Peña MM, Xing YY, Koli S, Berger FG. 2006. Role of N-terminal residues in the ubiquitin-independent degradation of human thymidylate synthase. Biochem J. Feb 15;394(Pt 1). 355-363. PubMed
Forsthoefel AM, Peña MM, Xing YY, Rafique Z, Berger FG. 2004. Structural determinants for the intracellular degradation of human thymidylate synthase. Biochemistry. Feb 24;43(7). 1972-1979. PubMed