Research Interests:

Our laboratory is focused on the MDR caused by over-expression of MRP1 protein. The research interests are: 1. How does MRP1 protein couple ATP binding/hydrolysis to anticancer drug transport? 2. What is the relationship between the structure and function of MRP1 protein? 3. Can we find a way to combat MDR caused by over-expression of MRP1 protein in cancer patients?

  1. How does MRP1 protein couple ATP binding/hydrolysis to anticancer drug transport? For ATP-dependent anticancer drug transport to occur, the anticancer drug binding and releasing must occur at different sites with different affinities, accompanied with variant conformational changes of the MRP1 protein. Anticancer drug binding must occur at high affinity sites whereas releasing must occur at low affinity sites. We are interested in where are the drug binding sites. We are also interested in whether anticancer drug binding will induce conformational changes of MRP1 protein and enhances ATP binding. Interestingly, we have found that the two nucleotide binding domains (NBDs) of MRP1 play different roles during ATP-dependent anticancer drug transport. ATP binding, conformational changes induced by the ATP binding at these two unequal NBDs, ATP hydrolysis and the coupling of ATP binding/hydrolysis to anticancer drug transport across biological membrane are the areas of our research interests. 
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  3. What is the relationship between the structure and function of MRP1 protein? Cells over-expressing either P-gp or MRP1 become MDR. Both of these proteins couple ATP binding/hydrolysis to anticancer drug transport across the biological membrane. However, the ways they pump anticancer drug out of the cell are different. P-gp pumps the hydrophobic anticancer drugs out of cells directly, whereas MRP1 transports the glutathione-S-conjugated hydrophobic anticancer drugs out of the cells. Thus, the variant properties of these proteins reflect the structure differences. We have purified MRP1 protein, determined the ATPase activity of the purified protein and made “spherulite” crystal. However, due to the nature of big membrane-bound protein, we have never made real crystal of MRP1 protein. We have made cysteine-less MRP1 construct. We will introduce cysteine codon to the desired sites to do cross-linking and modeling of the transmembrane domains and nucleotide binding domains. In the meantime, the residues involved in ATP binding and hydrolysis, based on the crystal structures of prokaryotic ABC transporters, such as HisP, MalK and HlyB etc., have been mutated to either a similar or dissimilar amino acid to study the relationship between the structure and function of MRP1 protein. 
  4. Can we find a way to combat MDR caused by over-expression of MRP1 protein in cancer patients? MRP1 co-transports its substrates, including anticancer drugs and endogenous phospholipids, with glutathione (GSH, in a covalent or non-covalent manner) across the biological membrane. If the rate of de novo biosynthesis of GSH cannot catch up the rate of MRP1-mediated GSH export, the GSH content should be decreased in MRP1-over-expressing cells. Interestingly, cells over-expressing MRP1 protein have much lower GSH contents than their corresponding control cells, presumably due to MRP1 mediated co-transport of endogenous MRP1 substrate with GSH. The intracellular GSH contents in MRP1 expressing cancer cells were significantly decreased upon treatment with some special MRP1 substrates. In addition, upon treatment with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO), the GSH content in MRP1-expressing cells decreased much faster than their corresponding control cells, presumably due to the synergetic effects of BSO and MRP1 protein. GSH is the major reducing agent that scavenges reactive oxygen species (ROS). Excess amount of ROS is toxic to the cell and may induce apoptosis. We plan to develop a special approach to selectively kill the MRP1-over-expressing MDR cancer cells by employing BSO, special MRP1 substrates and anticancer drugs.