个人简介
Our research involves a comprehensive range of studies of the structure, mechanism of action, regulation of expression and subcellular trafficking of several members of the ATP Binding Cassette (ABC) superfamily of transmembrane transporters. We are particularly interested in the Multidrug Resistance Protein (MRP) subfamily of ABC transporters, specifically, ABCC/MRP1, 2 and 3, and another member of the superfamily, ABCA1.Ê
研究领域
Our research involves a comprehensive range of studies of the structure, mechanism of action, regulation of expression and subcellular trafficking of several members of the ATP Binding Cassette (ABC) superfamily of transmembrane transporters. We are particularly interested in the Multidrug Resistance Protein (MRP) subfamily of ABC transporters, specifically, ABCC/MRP1, 2 and 3, and another member of the superfamily, ABCA1.
The ABC transporters are a large and ancient superfamily of integral membrane proteins which characteristically span the membrane multiple times. Typically, they use the energy of ATP hydrolysis to actively transport substrates across various cell membranes. These substrates range in size and structure from ions and small molecule drugs to large polypeptides. Several members of the superfamily, including some MRPs, have been implicated in the clinical development of multidrug resistance, a major obstacle to successful systemic treatment of many solid and haematological tumors. In addition, mutations in a number of ABC proteins are responsible for inherited disorders, such as cystic fibrosis, congenital hyperinsulinemia of infancy, the connective tissue disease Pseudoxanthoma elasticum and macular degeneration (Stargardt’s Disease), as well as certain types of hyperbilirubinemia and other disorders involving cholesterol disposition, such as Tangier disease, which is caused by defects in ABCA1.
Multidrug Resistance Proteins (MRPs)
The MRPs comprise one of the largest subfamilies of the ABC superfamily. We discovered the first of these proteins, human MRP1, in 1992 (see Cole et al,. Science (1992), below). Since then, the human MRP family has grown to include 12 additional members. MRP1 has been detected in many multidrug resistant cell lines and tissue samples from various types of cancer. The protein transports an exceptionally broad range of compounds. These include structurally diverse natural product drugs and toxins and their anionic conjugates, as well as many other anionic conjugates of physiological importance. One of the reasons that MRP1 can transport such a range of substrates is that in addition to ATP dependent direct transport of substrates, it is also capable of an ATP dependent co-transport mechanism involving reduced glutathione. As a consequence, high levels of MRP1 expression may have implications for the redox state of the cell. The two closest relatives of MRP1, MRP2 and MRP3, share some but not all of MRP1’s transport characteristics. By making various hybrid and mutant MRPs, we have been able to gain considerable insight into the way in which the proteins recognize their substrates and to begin to understand how they couple the hydrolysis of ATP to the transport process. Some of the MRPs, including MRP1, 2 and 3, are unusual among ABC proteins in that they contain a third membrane spanning domain in addition to the two typically found in other members of the superfamily. We are pursuing a variety of approaches to better understand the higher order structure of the 17 transmembane helices in these three domains and to elucidate how they communicate with the protein’s two cytosolic ATP binding domains. We are also using a number of molecular imaging techniques to identify regions of MRP1 that are important for targeting the protein to the basolateral regions of the plasma membrane and for controlling its recycling through various membrane compartments (see figure for confocal laser microscopic images of fluorescently tagged MRP1 localized with various subcellular markers). Finally, we have cloned the human, mouse and rat MRP1/mrp1 genes and are investigating mechanisms involved in their basal transcription, as well as how they may respond to oxidative and other forms of stress.
ABCA1
Mouse ABCA1 was identified in 1994 during a search for ABC transporters expressed in macrophages. Initial interest focussed on the possible role of the transporter in the macrophage mediated removal of remnants of cells undergoing programmed cell death, or apoptosis. Recognition of the physiological importance of the protein broadened significantly with the discovery in 1999 that defects in the ABCA1 gene were the underlying cause of Tangier disease, a rare inherited disorder that severely impairs the process by which cholesterol is transported from peripheral tissues back to the liver, so called reverse cholesterol transport. The role of ABCA1 in this process is believed to be to facilitate the efflux of free cholesterol to acceptor apolipoproteins such as ApoA1, which is the major protein component of high density lipoprotein. Precisely how ABCA1 stimulates the transfer of cholesterol to ApoA1 is not fully understood. Like the MRPs, ABCA1 has a number of unusual structural characteristics that distinguish it from most ABC transporters. We are now applying the techniques we have developed to study the MRPs to investigate the transport mechanism of ABCA1 and to determine the functional roles of some of its distinguishing structural features.
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Conseil, G., Deeley, R.G. and Cole, S.P.C. Functional importance of three basic residues clustered at the cytosolic interface of transmembrane helix 15 in the multidrug and organic anion transporter MRP 1 (ABCC1). J. Biol. Chem. 281(1): 43-50 (2006).
Zhang, D-W., Nunoya, K., Vasa, M., Gu, H-M., Cole, S.P.C. and Deeley, R.G. Mutational analysis of polar amino acid residues within predicted transmembrane helices 10 and 16 of multidrug resistance protein 1 (ABCC1): effect on substrate specificity. Drug Metab. Disp 34: 539-546 (2006). (ASPET James R. Gillette Drug Metabolism Best Paper of 2006)
Deeley, R.G. and Cole, S.P.C. Substrate recognition and transport by multidrug resistance protein (MRP) 1 (ABCC1). FEBS Lett. 580: 1103-1111 (2006) (invited, peer-reviewed).
Tam, S-P., Mok, L., Chimini, G., Vasa, M. and Deeley, R.G. ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of the oxysterol by intact cells. Am J Physiol Cell Physiol: 291(3): C490-502 (2006).
Rothnie, A., Callaghan, R., Deeley R.G. and Cole, S.P.C. Role of GSH in estrone sulphate binding and translocation by the multidrug resistance protein 1 (MRP1, ABCC1). J. Biol. Chem. 281(20): 13906-13914 (2006).
Cole, S.P.C. and Deeley, R.G. Transport of glutathione and glutathione conjugates by MRP1. Trends Pharm. Sci. 27: 438-446 (2006) (invited, peer-reviewed).
Deeley, R.G., Westlake, C. and Cole, S.P.C. Transmembrane transport of endo- and xenobiotics by membrane ATP-binding cassette multidrug resistance proteins. Physiol. Rev. 86: 849-899 (2006) (invited, peer-reviewed).
Létourneau, I.J., Slot, A.J., Deeley, R.G. and Cole, S.P.C. Mutational analysis of a highly conserved proline residue in MRP1, MRP2 and MRP3 reveals a partially conserved function. Drug Metab. Disp. 35: 1372-1379 (2007).
DeGorter, M.K., Conseil, G., Deeley, R.G., Campbell, R.L. and Cole S.P.C. Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1). Biochem. Biophys. Res. Commun. 365: 29-34 (2008).
Slot, A.J., Wise, D.D., Deeley, R.G., Monks, T.J. and Cole, S.P.C. Modulation of human MRP1 (ABCC1) and MRP2 (ABCC2) transport by endogenous and exogenous glutathione-conjugated catechol metabolites. Drug Metab. Disp. 36: 552-560 (2008).
Létourneau, I.J., Nakajima, A., Deeley, R.G. and Cole, S.P.C. Role of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter. Biochem. Pharmacol. 75: 1659-1669 (2008).
Bandler, P.E., Westlake, C.J., Grant, C.E., Cole, S.P.C. and Deeley, R.G. Identification of regions in human multidrug resistance protein (MRP) 2 required for apical membrane localization. Mol. Pharmacol. 74: 9-19 (2008).
Rothnie, A., Conseil, G., Lau, A.Y.T., Deeley, R.G. and Cole, S.P.C.. Mechanistic differences between GSH transport by MRP1 (ABCC1) and GSH modulation of MRP1-mediated transport. Mol. Pharmacol. 74: 1630-1640 (2008).
Grant, C.E., Gao, M., DeGorter, M.K., Cole, S.P.C. and Deeley, R.G. Structural determinants of substrate specificity differences between human multidrug resistance protein (MRP) 1 (ABCC1) and MRP3 (ABCC3). Drug Metab. Dispos. 36: 2571-2581 (2008).
Qin, L., Zheng, J., Grant, C.E., Jia, Z., Cole, S.P.C. and Deeley, R.G. Residues responsible for the asymmetric function of the nucleotide binding domains of multidrug resistance protein (MRP) 1. Biochemistry 47: 13952-13965 (2008).
Conseil, G., Rothnie, A., Deeley, R.G. and Cole, S.P.C. Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1). Mol. Pharmacol. 75: 397-406 (2009).
Maeno, K., Nakajima, A., Conseil, G., Rothnie, A., Deeley, R.G., and Cole, S.P.C. Molecular basis for reduced estrone sulphate transport and altered modulator sensitivity of TM6 and TM17 mutants of MRP1 (ABCC1). Drug Metab. Dispos. 37: 1411-1420 (2009).
Rosenberg, M.F., Oleschuk, C.J., Wu, P., Mao, Q., Deeley, R.G., Cole, S.P.C. Cole and Ford, R.C.. Structure of a human multidrug transporter in an inward-facing conformation. J. Struct. Biol. 170: 540-547 (2010).
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