University, Diss., 2004--Konstanz. / Erscheinungsjahr an der Haupttitelstelle: 2003.

D-4F, an ApoA-I mimetic peptide, alleviates mechanical hyperalgesia after administration to rodents suffering from inflammatory and neuropathic pain. D-4F scavenges proalgesic oxidized lipids – as such an anti-inflammatory drug – but also activates ATP-binding cassette transporters (Abca1 and Abcg1) – as such an anti-atherosclerotic drug. Aim of the project was to investigate the impact of neuropathy and its treatment with D-4F on the expression of Abca1/Abcg1 as well as cytokines. / D-4F, ein ApoA-I-mimetisches Peptid, lindert mechanische Hyperalgesie nach Verabreichung an Nagetiere, die an entzündlichen und neuropathischen Schmerzen leiden. D-4F fängt proalgetische oxidierte Lipide ab – als ein solches entzündungshemmendes Medikament – ​​aktiviert aber auch ATP-bindende Kassettentransporter (Abca1 und Abcg1) – als ein solches anti-atherosklerotisches Medikament. Ziel dieser Arbeit war es, den Einfluss von Neuropathie und deren Behandlung mit D-4F auf die Expression von Abca1/Abcg1 sowie Zytokinen zu untersuchen.

ATP Binding Cassette (ABC) transporters are trans-membrane proteins that exist in all phyla. Mutations in this family of proteins can cause inherited diseases like Cystic Fibrosis. ABC transporters consist of dimers of nucleotide binding domains (NBDs) and transmembrane domains (TMDs). NBDs regulate ABC transporters by binding to and hydrolyzing ATP. Although NBD-ATP interactions, NBD-TMD interactions and NBD-water interactions are known to be crucial to the function of these proteins, it is still not clear what structural and energetic factors are involved in the NBD-NTP interactions, how NBD and TMD interact with each other, how water is involved in the functions of ABC transporters and what are the structures and energetics of protein bound water. Molecular modeling and molecular dynamics (MD) simulations were conducted to interpret and predict the structural and energetic factors in control and action of two ABC transporters, CvaB and SUR2B. Water is essential for ABC transporters to carry out their functions, to increase the accuracy of simulations. Therefore, water potentials in molecular modeling and dynamics simulations were improved based on the calculation of water structures from protein surface. Previous study showed the NBDs of ABC transporter CvaB bind tighter to GTP than to ATP at lower temperature but not at high temperature. The MD simulations in this study suggested the velocity of water molecules initiates the temperature dependent functional change of proteins. Previous study found that Ser1387 in the NBD of SUR2B, an ABC transporter in vascular smooth muscles, is critical to Kir6.1/SUR2B channel. The molecular modeling and dynamics simulation conducted on SUR2B showed that Ser1387 is located at a region that contacts a TMD. Upon the phosphorylation, the interaction between the NBD and TMD was enhanced which led to an inter domain movement. Water is essential for ABC transporters to carry out their functions, to increase the accuracy of simulations, and, therefore, the structures and energetics of protein bound water were studied. The water radial distribution function for protein bound water was calculated from 105 atomic resolution protein crystal structures and was found to be sharper than that observed for bulk water.

Multidrug resistance protein (MRP)1, a member of the ABCC branch of the ATP-binding cassette (ABC) superfamily of transporters, can confer resistance to a broad spectrum of chemotherapeutic agents. In addition to the core functional unit of ABC transporters that consists of two membrane spanning domains (MSD) and two nucleotide binding domains (NBD), MRP1 contains a third MSD (MSD0) resulting in the following arrangement: NH2-MSD-(MSD-NBD)2. In lieu of high-resolution structural information for MRP1, cysteine scanning mutagenesis (CSM) was applied to MRP1 and involves the development of a functional template devoid of cysteines into which paired cysteines can be introduced. Previous attempts to create a functional, cys-less template of MRP1 demonstrated that cysteines in MSD0 were structurally and functional important (1;2). However, given that MRP1 lacking MSD0 remains functional, a partially functional, cys-less MRP1 lacking this domain has been expressed in yeast (3-5). Given these results, with the ultimate goal of applying CSM to MRP1 in its entirety, we investigated the endogenous cysteines within MSD0 and co-expressed MRP1 half-molecules and validated these potential CSM templates by transport and ATP binding/hydrolysis assays. Mutation of cysteines within the core of MRP1 had detrimental effects on MRP1 transport activity and further mutation of cysteines by domain revealed that wild-type activity was retained in an MSD0-less MRP1 dual lacking cysteines in both NBDs. This construct was used for introduction of cysteines on juxtaposed faces of the NBD1:NBD2 heterodimer at positions 775 and 1329; comparable residues in the related Cystic Fibrosis Transmembrane Regulator (CFTR/ABCC7) have been suggested to be evolutionarily coupled and joined by a hydrogen bond, maintained in structures of related proteins (6). Unfortunately, functional assays revealed that introduction of cysteines at these positions greatly reduced transport activity of MRP1 and diminished trapping of nucleotide at both NBDs. Finally, alanine substitution of the seven cysteines in MSD0 was not without effect and cellular trafficking assays, co-expression studies and SDS-PAGE analysis suggested an altered conformation of this domain. In addition, a disulfide pair of Cys7 and Cys32 was suggested by these experiments in MSD0 and further supported by examination of these mutants in full-length MRP1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-02-17 12:51:38.13

Thesis (doctoral)--Universität, Düsseldorf, 2000.

Konstanz, Univ., Diss., 2004.

Zugl.: Aachen, Techn. Hochsch., Diss., 2009

Universiẗat, Diss., 2007--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Universiẗat, Diss., 2005--Würzburg.

Universiẗat, Diss., 2004--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.