Molecular analysis of membrane transporters implicated in drug resistance
Description
Thesis (Ph.D.)--Georgetown University, 2008.; Includes bibliographical references. Resistance to cancer chemotherapeutics and antimalarial drugs is a major obstacle to the successful treatment of these diseases. Membrane transporters have been identified as contributing to this drug resistance. The HuMDR1 protein is thought to reduce accumulation of chemotherapeutics by ATP-dependent transport of the toxic compounds out of the cell. Though previously believed to be a major component of clinical cancer drug resistance, HuMDR1 is now accepted as playing only a minor role, secondary to other mechanisms. The Plasmodium falciparum protein homologue, PfMDR1, may be acting in a similar manner within the malarial parasite, affecting the partitioning of antimalarial drugs amongst cellular compartments. However, the evolving picture of quinoline antimalarial drug resistance may point to a mere modulatory role for PfMDR1 in comparison to another membrane protein, PfCRT, which has been proven to be causative of some drug resistance phenotypes but through an unknown mechanism. Since the protein is native to a subcellular organelle within an intracellular parasite, molecular level analysis of PfMDR1 would benefit from heterologous expression in a simpler system. This thesis reports the successful inducible overexpression of PfMDR1 in Pichia pastoris yeast. The tagged protein can be purified by affinity chromatography and functionally reconstituted in proteoliposomes. ATPase assays of many PfMDR1 variants show the protein to have high basal activity, with very little drug-induced responsiveness. These results support a model in which PfMDR1 acts to modulate the drug resistance profiles determined by PfCRT or to compensate for fitness losses incurred by mutation of PfCRT. All current hypotheses for the molecular mechanism by which PfCRT confers quinoline antimalarial drug resistance entail the direct interaction of the drug molecule with the protein, but evidence for these theories is inferential. This thesis reports the labeling of PfCRT with a photoactivatable chloroquine analogue. The probe is shown to be specific and labeling is efficiently competed with other antimalarial drugs, suggesting a single drug binding site is present in the protein. The photolabeling site is mapped to within 11 amino acids, and a model is proposed in which PfCRT transmembrane helices 1, 9 and 10 form a drug binding pocket.
Permanent Link
http://hdl.handle.net/10822/552859Date Published
2008Subject
Type
Collections
Metadata
Show full item recordRelated items
Showing items related by title, author, creator and subject.
-
Analysis of the cellular and molecular mechanisms of Chloroquine Resistance in Plasmodium Falciparum
(Georgetown University, 2009) -
TWO APPROACHES TO THE STUDY OF PROTEIN INTERACTIONS WITH SMALL MOLECULES: (A) STRUCTURAL ANALYSIS OF PYRIDOXAL L-PHOSPHATE BINDING ENZYMES (B) PURIFICATION, RECONSTITUTION, AND DRUG-BINDING CAPABILITIES OF THE PLASMODIUM FALCIPARUM MULTIDRUG RESISTANCE PROTEIN (PfMDR1)
(Georgetown University, 2012)Structural genomics initiatives are producing new protein structures at a rate that will soon -
Molecular Mechanisms of Taxane Resistance
(Georgetown University, 2010)Treatment with the taxanes (Paclitaxel or Docetaxel) is often the therapy of choice for women with breast cancer. In most cases, the taxanes can arrest cell proliferation at the G2/M phase of mitosis and cause cell death. ...
