Multidrug resistance protein 1 (MRP1) is an integral membrane protein belonging to the ATP-binding cassette (ABC) superfamily that utilizes ATP binding and hydrolysis to transport various endogenous substrates and/or xenobiotics across membranes against a concentration gradient. The overall goal of my research was to examine the nucleotide and protein interactions of MRP1 using various biochemical methods. In the first study, Cu2+(Ph)3 which promotes cross-linking of two nearby Cys residues and limited proteolysis were used to study conformational changes of MRP1 at different stages of ATP binding and hydrolysis at the nucleotide binding domains (NBDs). The limited trypsin digestion patterns indicated that some Cys residues of MRP1 could be cross-linked in the nucleotide-free state and that the Cys cross-linked MRP1 was more susceptible to trypsinolysis. Furthermore, binding of ATP, AMP-PNP, and trapping of ADP by MRP1 prevented the cross-linking events from occurring, but binding of ATPγS did not. However, the ATPγS-bound MRP1, like nucleotide-free MRP1, showed enhanced sensitivity towards trypsinolysis. These studies show that the two ATP analogs, AMP-PNP and ATPγS, interact with MRP1 in different ways. In the second study, the interaction of MRP1 with other cellular proteins was examined. An in vivo chemical cross-linking approach combined with affinity purification and MS analysis was initially used to identify protein partners directly interacting with MRP1. When this approach proved unsuccessful, a second approach involving immunoaffinity purification of MRP1-containing complexes followed by MS analysis was adopted. Six potential candidate interacting protein partners of MRP1 were identified via this approach and two of them, FUS and drebrin, were further characterized by co-immunoprecipitation and colocalization experiments. FUS seems unlikely to be an important binding partner of MRP1 since confocal and subcellular fractionation studies showed it to be exclusively localized in the nucleus. On the other hand, drebrin depletion by siRNA knock-down resulted in a moderate decrease in MRP1 overall expression levels although the membrane localization of MRP1 remained unchanged. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-12-08 17:44:52.767

Influenza A virus is well known for its severe clinical consequences. Structurally, this virus is made up of a lipid bilayer embedded with HA, NA and M2 proteins and a core containing eight viral ribonucleoprotein (RNP) complexes. In a typical RNP complex, the nucleoprotein binds with RNA in a non specific manner. The nucleoprotein plays a vital role in transcription, replication, and packaging of RNA during infection. This study aims that NP of A/PR/8/34(H1N1) virus and A/NY/55/2004(H3N2) virus interact with different host proteins depending on cell lines and virus strains. Monoclonal antibodies targeting the nucleoprotein of these viruses have been used for immunoprecipitation and the interacting proteins were identified by mass spectrometry. Tow proteins from the cytoplasm (elongation factor 1 sigma, and Mov10 protein) and 3 proteins from the nucleus (heat shock protein70, hnRNP K protein, and anti alpha actinin 4) were found in all the viral infected cells, and were chosen for validation study. This study will help to understand the virus-host interactions in a better way and may open the gateway for the synthesis of new antiviral drugs which can block these interactions, hence controlling the infection.

The BTB domain is a well-conserved protein-protein interaction motif. There are 43 BTB-ZF transcription factors in the human proteome. Many of these transcription factors play crucial roles in cancer and developmental processes. The purpose of this project is to identify lists of interactors of the BTB domains of six BTB-ZF proteins with high confidence using a mass spectrometry based approach. The BTB domains BCL6, PLZF, Kaiso, LRF, FAZF, and Miz1 were studied. This study was able to identify 142 putative interactors. The list of putative interactor proteins participates in a wide array of biological functions. Selected putative interactors of the BCL6BTB were also validated using biochemical techniques. In conclusion, this project was able to provide an analysis of the protein-protein interactions mediated by the BTB domains of six BTB-ZF transcription factors. The information generated is valuable to guide future functional and structural studies of the BTB domains.

The BTB domain is a well-conserved protein-protein interaction motif. There are 43 BTB-ZF transcription factors in the human proteome. Many of these transcription factors play crucial roles in cancer and developmental processes. The purpose of this project is to identify lists of interactors of the BTB domains of six BTB-ZF proteins with high confidence using a mass spectrometry based approach. The BTB domains BCL6, PLZF, Kaiso, LRF, FAZF, and Miz1 were studied. This study was able to identify 142 putative interactors. The list of putative interactor proteins participates in a wide array of biological functions. Selected putative interactors of the BCL6BTB were also validated using biochemical techniques. In conclusion, this project was able to provide an analysis of the protein-protein interactions mediated by the BTB domains of six BTB-ZF transcription factors. The information generated is valuable to guide future functional and structural studies of the BTB domains.

Influenza A virus is well known for its severe clinical consequences. Structurally, this virus is made up of a lipid bilayer embedded with HA, NA and M2 proteins and a core containing eight viral ribonucleoprotein (RNP) complexes. In a typical RNP complex, the nucleoprotein binds with RNA in a non specific manner. The nucleoprotein plays a vital role in transcription, replication, and packaging of RNA during infection. This study aims that NP of A/PR/8/34(H1N1) virus and A/NY/55/2004(H3N2) virus interact with different host proteins depending on cell lines and virus strains. Monoclonal antibodies targeting the nucleoprotein of these viruses have been used for immunoprecipitation and the interacting proteins were identified by mass spectrometry. Tow proteins from the cytoplasm (elongation factor 1 sigma, and Mov10 protein) and 3 proteins from the nucleus (heat shock protein70, hnRNP K protein, and anti alpha actinin 4) were found in all the viral infected cells, and were chosen for validation study. This study will help to understand the virus-host interactions in a better way and may open the gateway for the synthesis of new antiviral drugs which can block these interactions, hence controlling the infection.

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Understanding protein-protein interaction (PPI) is important in order to understand cellular processes. X-ray crystallography and mutagenesis, expensive methods both in time and resources, are the most reliable methods for detecting PPI. Computational approaches could, therefore, reduce resources and time spent on detecting PPIs. During this master thesis a method, cProQPred, was created for scoring how realistic coarse PPI models are. cProQPred use the machine learning method Random Forest trained on previously calculated features from the programs ProQDock and InterPred. By combining some of ProQDock’s features and the InterPred score from InterPred the cProQPred method generated a higher performance than both ProQDock and InterPred. This work also tried to predict the quality of the PPI model after refinement and the chance for a coarse PPI model to succeed at refinement. The result illustrated that the predicted quality of a coarse PPI model also was a relatively good prediction of the quality the coarse PPI model would get after refinement. Prediction of the chance for a coarse PPI model to succeed at refinement was, however, without success.

Cellular pH homeostasis plays crucial roles in cellular functions, and it is now widely recognized that Na⁺/H⁺ exchangers are among the most prominent players in this process. Although recently described mammalian Na⁺/H⁺ exchanger NHE7 has attracted much attention, its biological functions remain largely unknown. Most proteins exist as protein complexes in the cell and elicit their unique functions in collaboration with their binding partners. Therefore, identification and characterization of binding proteins will often unveil unexpected functions of the protein of interest. To begin to elucidate biological roles of the novel class of Na⁺/H⁺ exchanger NHE7, yeast two-hybrid screening was conducted and several binding candidates were identified. Among these candidates, I show that Secretory Carrier Membrane Proteins (SCAMPs) are novel NHE7 binding proteins and that SCAMPs regulate endocytic trafficking of NHE7 from the recycling endosomes to the trans-Golgi network (TGN). In agreement with this finding, I found that NHE7 can also be targeted to the plasma membrane and then internalized. Caveolins, structural proteins for caveolae, were identified as NHE7-binding proteins and it was initially hypothesized that caveolins might regulate NHE7-internalization. Interestingly, caveolins bound to NHE7 through a novel binding domain and facilitated its association to caveolae/lipid rafts, but did not affect NHE7-internalization. I also show that SCAMP2 associates with the heterotrimeric G protein β subunit (Gβ) and regulates the ERK1/2 signaling. Moreover, NHE7 was found to associate with both SCAMP2 and Gβ in the cell, suggesting that ERK1/2 signaling mediated by the SCAMP2-Gβ complex might regulate NHE7.