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Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2007

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Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2008

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Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2001

Multidrug resistance (MDR) is a major problem in the treatment of cancer. MDR is often induced by the overexpression of multidrug transport proteins of which the ATP-binding cassette (ABC) transmembrane proteins P-glycoprotein (Pgp) and the multidrug resistance associated protein 1 (MRP1) are the best characterized. Both proteins are located in the plasma membrane and are responsible for the active efflux of a broad spectrum of substrates (allocrites), including clinically important anti-cancer drugs. Studies to use the occurrence of MDR-related transporters such as Pgp and MRP1 as a tool in survival prognosis of cancer patients show variable outcome, and depend on the type of cancer. Since not all types of MDR can be correlated to the presence of known transport proteins, the identification and characterization of new transport proteins is a prerequisite in the development of cancer treatments. An interesting new member of the ABC transport proteins recently identified is the half-transporter MXR, also named BCRP or ABCP. MXR confers resistance to various important anti-cancer drugs, such as mitoxantrone, anthracyclines (doxorubicin), and the DNA topoisomerase I inhibitors CPT-11 and topotecan. Only two modulators have been reported to specifically inhibit MXR-mediated transport. Since modulators often show profound pharmacokinetic interaction with anti-cancer drugs, other MXR-specific modulators need to be identified. |Functional characterization of (transporter) proteins in mammalian cells is often hampered by the complexity of the regulation of gene expression, mRNA stability and protein lifetime. In this aspect, bacterial cells offer a much simpler system for characterization of activity and specificity. Equally important, bacterial expression allows the purification of larger quantities of protein that facilitates their characterization using purified proteoliposomes and eliminates possible cross-activities of other transporters. Furthermore, genetic manipulations can be exploited readily within the bacterial system.|We plan to overproduce MXR in Lactococcus lactis and purify the transport protein in large quantities. A first characterization of the transporter will be performed by transport assays using whole cells, purified membrane vesicles and proteoliposomes made of purified MXR protein and lipids. Transport and binding will be followed by the use of fluorescently or radioactively labeled allocrites. Modulators of transport activity will be identified by inhibition of these activities. As a second line of research, we will isolate the nucleotide-binding domain of MXR to test allocrites for stimulatory capability of the intrinsic ATPase activity. Results from our group with the nucleotide-binding domain of MRP1 show that its ATPase activity can be stimulated by MRP-specific substrates. Thirdly, we will characterize MXR on its ability to dimerize. Even though the homodimer seems to be functional, there are indications that heterodimer formation induces a higher resistance level. The putative partner for MXR in this heterodimer is unknown as yet. We plan to apply a bacterial two-hybrid system to investigate homodimer formation and to screen a mammalian cDNA bank for possible partners of MXR in heterodimer formation using MXR as bait.|In order to circumvent MXR-mediated MDR in cancer cells, the half-transporter needs to be characterized in more detail with respect to the allocrite and modulator specificity. Moreover, since the functional unit of the transporter appears to be a dimer, more information is needed concerning the dimerization and the identity of putative partner half-transporters. The characterization of MXR is a prerequisite to attack MDR in cancer cells in an efficient manner.


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2007

ATP synthase is a membrane protein that uses energy from the transmembrane proton electrochemicai gradient to form ATP from ADP and phosphate [l]Th e enzyme is composed of two major domains, the transmembrane F0 domain and the membrane excluded F1 domain [Z] The current project focuses on the subunit c of the protein, which is an essentiai part of the F0 domain and participates in transmembrane proton conduction. Structural studies indicate that the subunit c arranges into ring structures comprising of 10 -12 subunits in different organisms However, littie is known about the type of interactions that affect the formation of c-rings in the ATPase complex In this project, we further investigate the stability and self aggregation propensity of the c subunit, Molecular dynamics simulations wil1 be performed using a coarse-grain force field [3] t0 define the protein and its membrane environment Simulations wil1 be carried out from a single monomer as weli as the pre-assembied modelled ring structure of the c subunit The stability and dynamics of these systems wil1 be probed and the results wil1 be compared to atomistic simulations [4] Further, simulations wil1 be performed on multiple copies of the monomer to probe the association of these peptides The free energy profile of association of two peptides wil1 be caicuiated. Preliminary results indicate that the subunit has a tendency to assocate in the membrane into dimers and trimers. However, to investigate the more complex decarner interactions, longer time scales and larger length scales need to be investigated


Grant
Agency: Narcis | Branch: Project | Program: Completed | Phase: Physics, Chemistry and Medicine | Award Amount: | Year: 2005

To study the relationships between societal visions on technology and food and industrial and academic genomics research.

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