This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The focus of this project is on the structure elucidation of membrane transporter proteins that underlie the molecular processes of apoptosis evasion and drug resistance in cancer cells. Mitochondria mediate programmed cell death through a complex balancing of pro- and anti-apoptotic factors that respond to a variety cellular growth and homeostatic signals. The primary regulators of this process are known to be members of the Bcl-2 family of proteins (including Bid, Bax and Bak);however the precise mechanism remains molecularly uncharacterized. Exciting novel participants in mitochondrial apoptosis are the mitochondrial carrier homologues (MtCH1 &MtCH2), members of the solute carrier super-family of transport proteins. MtCH2 was found to mediate tBid association with the mitochondrial membrane and is properly positioned to be either a mediator of Bax/Bak conformational change and membrane integration, or perhaps even an additional constituent of MAC channels which release cytochrome C from mitochondria as the committing step towards apoptosis. Many new techniques have recently emerged for the structure determination of integral membrane proteins, proteins that traditionally have been recalcitrant to high resolution structural analysis. We use Mistic-fusion technology, which we continue to develop, to produce MtCH1 &MtCH2 recombinantly in bacteria culture at high yields. Our lab will utilize the data obtained from these studies for the structure-based design of novel therapeutic agents to target these proteins. Our primary approach is the use of X-ray crystallography, in combination with biochemical and biophysical assays, to determine the structural and functional mechanisms by which these critical molecules contribute to carcinogenesis, thus uncovering new approaches to cancer treatment.
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