Browsing by Author "Chou, Kai-Ming"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    BASE EXCISION REPAIR APURINIC/APYRIMIDINIC ENDONUCLEASES IN APICOMPLEXAN PARASITE TOXOPLASMA GONDII
    (2012-03-19) Onyango, David O.; Sullivan, William J., Jr.; Chou, Kai-Ming; Georgiadis, Millie M.; Queener, Sherry F.; Vasko, Michael R.
    Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. Toxoplasma infection is a serious threat to immunocompromised individuals such as AIDS patients and organ transplant recipients. Side effects associated with current drug treatment calls for identification of new drug targets. DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen species produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host immune response. Most of the oxidative DNA damage is repaired through the base excision repair (BER) pathway, of which, the apurinic /apyrimidinic (AP) endonucleases are the rate limiting enzymes. Toxoplasma possesses two different AP endonucleases. The first, TgAPE, is a magnesium-dependent homologue of the human APE1 (hAPE1), but considerably divergent from hAPE1. The second, TgAPN, is a magnesium-independent homologue of yeast (Saccharomyces cerevisiae) APN1 and is not present in mammals. We have expressed and purified recombinant versions of TgAPE and TgAPN in E. coli and shown AP endonuclease activity. Our data shows that TgAPN is the more abundant AP endonuclease and confers protection against a DNA damaging agent when over-expressed in Toxoplasma tachyzoites. We also generated TgAPN knockdown Toxoplasma tachyzoites to establish that TgAPN is important for parasite protection against DNA damage. We have also identified pharmacological inhibitors of TgAPN in a high-throughput screen. The lead compound inhibits Toxoplasma replication at concentrations that do not have overt toxicity to the host cells. The importance of TgAPN in parasite physiology and the fact that humans lack APN1 makes TgAPN a promising candidate for drug development to treat toxoplasmosis.
  • Thumbnail Image
    Item
    CONTRIBUTIONS OF TM5, ECL3 AND TM6 OF HUMAN BCRP TO ITS OLIGOMERIZATION ACTIVITIES AND TRANSPORT FUNCTIONS
    (2012-03-16) Mo, Wei; Safa, Ahmad R.; Zhang, Jian-Ting; Chou, Kai-Ming; Hocevar, Barbara A.; Smith, Martin L.
    Human BCRP is one of the major ATP-binding cassette transporters involved in the development of multidrug resistance in cancer chemotherapy. Overexpression of BCRP in the tumor cell plasma membrane and apical membrane of the gastrointestinal tract leads to decreased intracellular accumulation of various anticancer drugs as well as reduced drug bioavailability. BCRP has been shown to exist on the plasma membrane as higher forms of homo-oligomers. In addition, the oligomerization domain of BCRP has been mapped to the carboxyl-terminal TM5-ECL3-TM6 and this truncated domain, when co-expressed with the full-length BCRP, displays a dominant inhibitory activity on BCRP function. Thus, the oligomerization of BCRP could be a promising target in reversing multidrug resistance mediated by BCRP. To further dissect the oligomerization domains of human BCRP and test the hypothesis that TM5, ECL3, and TM6 each plays a role in BCRP oligomerization and function, we engineered a series of BCRP domain-swapping constructs with alterations at TM5-ECL3-TM6 and further generated HEK293 cells stably expressing wild-type or each domain-swapping construct of BCRP. Using co-immunoprecipitation and chemical cross-linking, we found that TM5, ECL3, and TM6 all appear to partially contribute to BCRP oligomerization, which are responsible for the formation of oligomeric BCRP. However, only TM5 appears to be a major contributor to the transport activity and drug resistance mediated by BCRP, while ECL3 or TM6 is insufficient for BCRP functions. Taken together, these findings suggest that homo-oligomeric human BCRP may be formed by the interactions among TM5, ECL3 and TM6, and TM5 is a crucial domain for BCRP functions and BCRP-mediated drug resistance. These findings may further be used to explore targets for therapeutic development to reverse BCRP-mediated drug resistance and increase the bioavailability of anti-cancer drugs for better treatment of multidrug resistant cancers.