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Toxicology

Paumi Laboratory

Research Interests:

Determining the biological role of multidrug resistance proteins (MRPs) in cellular metabolism and detoxification and mechanisms of posttranslational Regulation

The multidurug resistance-associated protein (MRP, also called the ABCC) subfamily of the ATP-binding cassette (ABC) transporters promote multidrug resistance and the detoxification of exogenous toxins such as heavy metals (Cd+2, As+3, Hg+2, and Pb+2). In mammalian cells the MRPs promote the efflux of leukotrienes and glutathione conjugated prostaglandins, and in yeast the MRPs are required for the formation of the red pigment of ade2 mutants, suggesting that they may also play a key role in protecting cells from endogenous toxins or the over accumulation of metabolic pathway intermediates.

The goal of my research is to answer important, unsolved questions about how MRP proteins perform their physiological roles in drug disposition and metabolism and to determine how and to what extent is MRP function regulated. To answer these questions I have used the yeast, Saccharomyces cerevisiae, as a model system to study the physiological function of the MRPs. Recently, I have used the genetic and biochemical versatility of yeast to identify and characterize genes that regulate MRP function. Ultimately, I intend to extend my studies into a mammalian cell system where I will determine if human homologues of the newly identified regulators of MRP function have similar roles for the human MRPs. By combining the genetic power available in yeast with in vitro biochemical assays and in vivo cellular assays I have been able to ask questions about MRP physiological function that previously could not be tested. The skills I have developed during my postdoc in yeast in combination with the skills I developed as a graduate student in mammalian cell culture will provide me with the tools necessary to answer questions that would be difficult to answer in either a yeast or mammalian model system alone.

A major research goal of our lab is to determine the role of MRP1 in refractory acute lymphoblastic leukemia. Treatment of high risk acute lymphoblastic leukemia patients whom are positive for the Philadelphia chromosome mutation (ALL Ph+) which results in the fusion of BCR and Abl kinase (BCR/Abl), patients over the years have been extremely difficult. Recently, the standard hyperCVAD (Cyclophosphamide, Vincristine, Dexamethasone, and Doxorubicin) treatment for these patients was modified to include Gleevec (imatinib). The addition of Imatinib has been extremely successful and has increased the complete remission (CR) rate from 60 to 96% and the 2 year survival rate from about 40% of 85%. The mechanism by which Imatinib increases the efficacy of treatment is unclear. Cyclophosphamide metabolites, doxorubicin, and vincristine are all substrates of multidrug resistance protein 1 (MRP1/ABCC1). Recent studies have shown that increased expression and function of MRP1 is associated with a much poorer prognosis, a reduced CR, and a decreased 2 year survival (over 65% reduction) in ALL patients. Therefore, it is reasonable to believe that MRP1 plays a role in refractory ALL Ph+ due to increase drug resistance. Our work focus's on determining how MRP1 function in ALL Ph+ cells is regulated and identifying regulators of MRP1 in ALL Ph+. Inhibition of the MRP1 regulators will very likely increase chemotherapeutic efficacy.

A second project in the lab is understanding the role of MRP1 in cellular response to oxidative stress. In this project we are using three model carcinogens that induce oxidative stress, arsenite, hydrogen peroxide, and salt stress, to better understand the role of yeast Ycf1p and human MRP1 mediated resistance to ROS formation and oxidative stress. Recently we have shown that Ycf1p function is upregulated in response to salt stress. Interestingly deletion of Ycf1p does not increase cellular sensitivity to either high salt or hydrogen persoxide. It is unclear as the exact role of Ycf1p in response to salt stress and oxidative stress. We are currently in the process of determining the role of Ycf1p in salt stress and determining the mechanism by which yeast compensate for a loss of function in Ycf1p. We have expanded our studies to include the human MRP1 protein and have asked what the role of MRP1 is in the cellular response to oxidative stress. We specifically are interested in determining the role of MRP1 in chemotherapeutic induced oxidative stress and in response to environmental carcinogens including heavy metals and ROS producing toxins.

The current approaches that I am using to examine the role of the MRPs in drug detoxification and metabolism, and dissect cellular mechanisms of MRP regulation are to: 1) Carry out genetic and protein-interactor (yeast two-hybrid) screens, in Saccharomyces cerevisiae, to identify proteins that regulate MRP function in vivo and 2) determine the mechanism by which these genetic and protein-interactors regulate MRP function by a combination of in vivo cellular based assays and in vitro biochemical assays. Yeast are amenable to genetic and biochemical manipulation and therefore are an excellent tool in which to study MRP function and regulation. Further, with the recent addition of iMYTH, a membrane based yeast two-hybrid assay designed to identify protein interactors for membrane proteins, yeast are an extremely attractive model organism in which to study the membrane tethered MRPs. As a postdoctoral fellow in the lab of Dr. Susan Michaelis I compiled an extensive set of yeast tools which I have used to study the yeast MRP, Ycf1p. In collaboration with Dr. Igor Stagljar, I used iMYTH to identify protein-interactors of Ycf1p. Through a series of genetic and biochemical studies I have showed that one Ycf1p-protein interactor, Tus1p (A GEF for the small GTPase Rho1p), promotes Ycf1p transport activity via the small GTPase, Rho1p. Our studies suggest that Tus1p tethers Ycf1p and Rho1p together and via an as of yet undefined mechanism, activated Rho1p increases Ycf1p function. These studies have established that iMYTH is a robust technology which I can use to dissect MRP function and that we have identified a novel role for GEFs in regulating MRP function in vivo. For human MRP1 we have developed a number of tissue culture models to follow up on our yeast studies and ask exciting and new questions as to the role of human MRP1 in cellular resistance to environmental toxins and chemotherapeutics.

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