Flock House virus (FHV) is a (+) ssRNA virus that belongs to the Nodaviridae family. The viral genome is composed of two viral RNA’s: RNA 1 and RNA 2. Deletion and mutation studies in the N- and C-terminus of protein alpha have identified protein regions required for the packaging of FHV viral RNAs. Residues 2-31 on the N-terminus have been attributed with RNA2 recognition, while residues at positions 32-50 were required for the packaging of RNA1. The C-terminus is needed for packaging of both viral RNAs. The identified protein regions involved in packaging viral RNAs bind random cellular RNA with high affinity and standard methods of identifying RNA-protein interactions such as gel shift mobility assays will be unable to discriminate between specific and unspecific binding. Due to the difficulty in differentiating between specific and unspecific binding a new method for studying RNA-protein interactions was developed using a surface based detection approach. The surface based system monitors real-time binding, whereby specific and unspecific RNA-protein interactions will be distinguished through comparison of relative association rates for each binding interaction. A well studied RNA-protein interaction, the HIV-1 Rev-RRE, was used to develop the methodology for the surface based system. Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein Rev that binds to HIV-1 mRNA of the Rev responsive element (RRE). Rev-RRE interaction regulates viral gene expression by controlling the export of spliced and unspliced mRNAs into the cytoplasm. The high-affinity and specificity of the Rev-RRE binding has been well characterized and was used as a model system to gauge the sensitivity of the surface based detection system, which can be further used to characterize various RNA-protein interactions. The surface based system uses diffractive optics to detect real time binding of molecules to receptors that are immobilized on a flat sensor surface. An avidin coated sensor surface was applied to couple the small biotinylated Rev peptide to the surface followed by binding its complementary RRE RNA. The binding interactions of the 30 nucleotide RRE to the immobilized 23 residue Rev peptide were successfully monitored using the avidin sensor. The Rev-RRE interaction was heavily influenced by the immobilization technique and steric hindrance at the sensor surface.

Flock House virus (FHV) is a (+) ssRNA virus that belongs to the Nodaviridae family. The viral genome is composed of two viral RNA’s: RNA 1 and RNA 2. Deletion and mutation studies in the N- and C-terminus of protein alpha have identified protein regions required for the packaging of FHV viral RNAs. Residues 2-31 on the N-terminus have been attributed with RNA2 recognition, while residues at positions 32-50 were required for the packaging of RNA1. The C-terminus is needed for packaging of both viral RNAs. The identified protein regions involved in packaging viral RNAs bind random cellular RNA with high affinity and standard methods of identifying RNA-protein interactions such as gel shift mobility assays will be unable to discriminate between specific and unspecific binding. Due to the difficulty in differentiating between specific and unspecific binding a new method for studying RNA-protein interactions was developed using a surface based detection approach. The surface based system monitors real-time binding, whereby specific and unspecific RNA-protein interactions will be distinguished through comparison of relative association rates for each binding interaction. A well studied RNA-protein interaction, the HIV-1 Rev-RRE, was used to develop the methodology for the surface based system. Human immunodeficiency virus type 1 (HIV-1) encodes a regulatory protein Rev that binds to HIV-1 mRNA of the Rev responsive element (RRE). Rev-RRE interaction regulates viral gene expression by controlling the export of spliced and unspliced mRNAs into the cytoplasm. The high-affinity and specificity of the Rev-RRE binding has been well characterized and was used as a model system to gauge the sensitivity of the surface based detection system, which can be further used to characterize various RNA-protein interactions. The surface based system uses diffractive optics to detect real time binding of molecules to receptors that are immobilized on a flat sensor surface. An avidin coated sensor surface was applied to couple the small biotinylated Rev peptide to the surface followed by binding its complementary RRE RNA. The binding interactions of the 30 nucleotide RRE to the immobilized 23 residue Rev peptide were successfully monitored using the avidin sensor. The Rev-RRE interaction was heavily influenced by the immobilization technique and steric hindrance at the sensor surface.

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.

Magnesium, the second most abundant cation in the cell, is involved in a diverse range of biochemical activities. This project focuses on the mechanism of magnesium import into the cell through the action of Alr1p. Alr1p resides in the plasma membrane of yeast and belongs to the CorA-Alr1p-Mrs2p family of magnesium channels. Potential regulators of CorA were found through genetic screening and yeast two-hybrid screens have pulled out interactors of Alr1p. Interactors that influence Alr1p and its conformation will, with very high probability, also change the channel’s ability for magnesium import. Membrane proteins are not easily amenable to traditional yeast two-hybrid screens due to their hydrophobic nature. The goal of this thesis is to identify interactors of Alr1p using iMYTH, a modified yeast two-hybrid method. Of the eighteen Alr1p interactors identified, Vma3p and Vma11p, which are both subunits of the V-ATPase, showed the most promise for further Alr1p interaction characterizations.

Magnesium, the second most abundant cation in the cell, is involved in a diverse range of biochemical activities. This project focuses on the mechanism of magnesium import into the cell through the action of Alr1p. Alr1p resides in the plasma membrane of yeast and belongs to the CorA-Alr1p-Mrs2p family of magnesium channels. Potential regulators of CorA were found through genetic screening and yeast two-hybrid screens have pulled out interactors of Alr1p. Interactors that influence Alr1p and its conformation will, with very high probability, also change the channel’s ability for magnesium import. Membrane proteins are not easily amenable to traditional yeast two-hybrid screens due to their hydrophobic nature. The goal of this thesis is to identify interactors of Alr1p using iMYTH, a modified yeast two-hybrid method. Of the eighteen Alr1p interactors identified, Vma3p and Vma11p, which are both subunits of the V-ATPase, showed the most promise for further Alr1p interaction characterizations.

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. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

There are now numerous examples of post-translational modification with geranylgeranyl or farnesyl substituents. Once thought of as solely a mechanism for association of proteins with membranes, other functional aspects of protein prenylation have come to be appreciated. Although, in almost all instances, such proteins are membrane associated, they are often found to also engage in protein-protein interactions. In some instances, such interactions are critical aspects of prenylated protein trafficking. In this review, the role of prenylation in mediating protein-protein interactions will be considered. The hypothesis will be developed that such interactions occur through recognition of the prenyl group and a second domain, on the prenylated protein, by a heterodimeric protein partner.

No description available.

No description available.

This project applies structure-activity relationship (SAR), structure-based and database mining approaches to study ligand-protein interactions. To support these studies, we have developed a relational database system called EDinburgh University Ligand Selection System (EDULISS 2.0) which stores the structure-data files of +5.5 million commercially available small molecules (+4.0 million are recognised as unique) and over 1,500 various calculated molecular properties (descriptors) for each compound. A user-friendly web-based interface for EDULISS 2.0 has been established and is available at http://eduliss.bch.ed.ac.uk/. We have utilised PubChem bioassay data from an NMR based screen assay for a human FKBP12 protein (PubChem AID: 608). A prediction model using a Logistic Regression approach was constructed to relate the assay result with a series of molecular descriptors. The model reveals 38 descriptors which are found to be good predictors. These are mainly 3D-based descriptors, however, the presence of some predictive functional groups is also found to give a positive contribution to the binding interaction. The application of a neural network technique called Self Organising Maps (SOMs) succeeded in visualising the similarity of the PubChem compounds based on the 38 descriptors and clustering the 36 % of active compounds (16 out of 44) in a cluster and discriminating them from 95 % of inactive compounds. We have developed a molecular descriptor called the Atomic Characteristic Distance (ACD) to profile the distribution of specified atom types in a compound. ACD has been implemented as a pharmacophore searching tool within EDULISS 2.0. A structure-based screen succeeded in finding inhibitors for pyruvate kinase and the ligand-protein complexes have been successfully crystallised. This study also discusses the interaction of metal-binding sites in metalloproteins. We developed a database system and web-based interface to store and apply geometrical information of these metal sites. The programme is called MEtal Sites in Proteins at Edinburgh UniverSity (MESPEUS; http://eduliss.bch.ed.ac.uk/MESPEUS/). MESPEUS is an exceptionally versatile tool for the collation and abstraction of data on a wide range of structural questions. As an example we carried out a survey using this database indicating that the most common protein types which contain Mg-OATP-phosphate site are transferases and the most common pattern is linkage through the β- and γ-phosphate groups.