The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC) transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22 interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique interactors belong to a wide variety of functional categories. There were 11 interactors involved in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4 interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2 interactors involved in stress response, and 1 interactor in each of the following categories: cell wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p play an important role in zinc homeostasis because deletion of these ABC transporters results in sensitivity to zinc shock.

Genetic mapping of complex traits has been successful over the last decade, with over 2,000 regions in the genome associated to disease. Yet, the translation of these findings into a better understanding of disease biology is not straightforward. The true promise of human genetics lies in its ability to explain disease etiology, and the need to translate genetic findings into a better understanding of biological processes is of great relevance to the community. We hypothesized that integrating genetics and protein- protein interaction (PPI) networks would shed light on the relationship among genes associated to complex traits, ultimately to help guide understanding of disease biology. First, we discuss the design, testing and implementation of a novel in silico approach (“DAPPLE”) to rigorously ask whether loci associated to complex traits code for proteins that form significantly connected networks. Using a high-confidence set of publically available physical interactions, we show that loci associated to autoimmune diseases code for proteins that assemble into significantly connected networks and that these networks are predictive of new genetic variants associated to the phenotypes in question. Next, we study variation in the electrocardiographic QT-interval, a heritable phenotype that when prolonged is a risk factor for cardiac arrhythmia and sudden cardiac death. We show that a large proportion of QT-associated loci encode proteins that are members of complexes identified by immunoprecipitations in mouse cardiac tissue of proteins known to be causal of Mendelian long-QT syndrome. For several of the identified proteins, we show they affect cardiac ion channel currents in model organisms. Using replication genotyping in 17,500 individuals, we use the complexes to identify genome-wide significant loci that would have otherwise been missed. Finally, we consider whether PPIs can be used to interpret rare and de novo variation discovered through recent technological advances in exome-sequencing. We report a highly connected network underlying de novo variants discovered in an autism trio exome-sequencing effort, and we design, test and implement a novel statistical framework (“DAPPLE/SEQ”) to analyze rare inherited variants in the context of PPIs in a way that significantly boosts power to detect association.

Presented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.

E2A-PBX1 is expressed as a consequence of a recurring chromosomal translocation seen in 5% of acute lymphoblastic leukemia cases. We recently reported that substitution of a leucine residue (L20A) within the N-terminal transcriptional activation domain (AD1) of E2A-PBX1 markedly impairs binding to the KIX domain of CBP/p300 and, importantly, leukemia induction in a mouse bone marrow transplantation model. Since both the protein-protein interaction and consequent leukemogenesis rely on a focal contact point and might therefore be susceptible to antagonism by small molecules, we devised a cell-free assay based on fluorescence anisotropy (FA) to detect binding of a fluorescently labeled peptide derived from AD1 of E2A-PBX1 (FITC-E2A) with recombinantly expressed KIX domain. The optimized FA assay reveals a dissociation constant of 2 µM for the wild-type interaction and correctly detects disruption of the complex by naphthol AS-E phosphate, a compound previously shown to antagonize KIX binding. The optimized FA assay was used to screen the Prestwick, Spectrum and Chembridge libraries containing 12400 compounds in total. Of the initial 43 positive hits from the libraries, 10 caused a reproducible decrease in FA. Since intrinsic small molecule fluorescence can produce false positive results in the FA-based screen, intrinsically fluorescent compounds were excluded from further analysis unless they could be shown to bind to KIX. Two hits, L1 and C2, were intrinsically fluorescent but demonstrated KIX interactions and one hit, P9, was not intrinsically fluorescent. These three compounds were tested for their ability to inhibit binding of a larger portion of E2A (residues 1 to 483) to full length CBP in a pull down assay with only compound P9 demonstrating efficacy. Further characterization of P9 by NMR showed no binding to KIX, however evaluation by FA showed binding to FITC-E2A with a 20 µM affinity. A cell-based cytotoxicity assay demonstrated that compound P9 was slightly more toxic on leukemic cells that express E2A-PBX1, compared to leukemic cells lacking E2A-PBX1 expression. Mammalian two-hybrid analysis did not provide details of the effects of P9 on the E2A:KIX interaction. We expect the identification of a novel compound, P9, capable of disrupting the oncogenic E2A-PBX1:CBP interaction, to guide the development of effective, less toxic leukemia drugs and provide new tools for elucidating the molecular mechanisms of leukemia induction by E2A-PBX1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-08-31 11:13:19.517

Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin. HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction. In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.

Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin. HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction. In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.

The p53 protein is a critical tumor suppressor that is mutated in over half of all human cancers. It plays essential roles in maintaining genomic integrity by modulating the cellular response to various types of genotoxic stress. Associating with over 270 proteins to date, one of the mechanisms pivotal to p53’s multifaceted activities is protein-protein interactions. As to how most of these molecules bind to and affect p53 function remains unclear. Here we present a combined structural and biophysical approach to study three p53-interacting partners: BRCA1, IFI16 and p53 affinity reagent in an attempt to elucidate the basis of how these proteins recognize, bind to and alter p53’s biochemical functions. We have biophysically characterized the central region of BRCA1 and examined how it acts as a disordered scaffold to mediate association with p53 and other proteins. Having a putative role as a tumor suppressor, we have determined the crystal structures of the HIN-A and HIN-B domains of IFI16 and find that they interact with the C-terminus and DNA-binding core domain of p53, respectively, and enhance the DNA binding and transactivation activities of p53. Most cancer hot spot mutations of p53 are localized in the core domain and are thermally destabilized. Attaining molecules that stabilize the p53 fold has therefore been regarded as an attractive approach for cancer therapy. Lastly, using a phage-displayed library, evidence is presented to demonstrate a proof-of-principle for generating synthetic affinity reagents to potentially restore the function of tumor-derived p53 core domain mutants.

The p53 protein is a critical tumor suppressor that is mutated in over half of all human cancers. It plays essential roles in maintaining genomic integrity by modulating the cellular response to various types of genotoxic stress. Associating with over 270 proteins to date, one of the mechanisms pivotal to p53’s multifaceted activities is protein-protein interactions. As to how most of these molecules bind to and affect p53 function remains unclear. Here we present a combined structural and biophysical approach to study three p53-interacting partners: BRCA1, IFI16 and p53 affinity reagent in an attempt to elucidate the basis of how these proteins recognize, bind to and alter p53’s biochemical functions. We have biophysically characterized the central region of BRCA1 and examined how it acts as a disordered scaffold to mediate association with p53 and other proteins. Having a putative role as a tumor suppressor, we have determined the crystal structures of the HIN-A and HIN-B domains of IFI16 and find that they interact with the C-terminus and DNA-binding core domain of p53, respectively, and enhance the DNA binding and transactivation activities of p53. Most cancer hot spot mutations of p53 are localized in the core domain and are thermally destabilized. Attaining molecules that stabilize the p53 fold has therefore been regarded as an attractive approach for cancer therapy. Lastly, using a phage-displayed library, evidence is presented to demonstrate a proof-of-principle for generating synthetic affinity reagents to potentially restore the function of tumor-derived p53 core domain mutants.

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008