Nickel insertion into the [NiFe]-hydrogenase requires the accessory protein HypB, which is a GTPase. The GTPase domain of Escherichia coli (E. coli) HypB undergoes dimerization in the presence of GTP. To determine the role of HypB dimerization in hydrogenase biosynthesis, a double mutation L242A/L246A was introduced into full-length E. coli HypB, and the protein was expressed and characterized both in vitro and in vivo. Gel filtration experiments demonstrated that L242A/L246A HypB was monomeric as expected. The inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric L242A/L246A HypB has a similar Ni(II)-binding behavior as that of wild type HypB. Upon the expression of L242A/L246A HypB in vivo the hydrogenase activity is approximately half of the activity of the wild-type control. These experimental results suggest that dimerization of HypB does have a, but not critical, role in hydrogenase biosynthesis.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

GPCRs are known to form dimeric structures, and this affects their pharmacological properties. The ?2AR and AT1aR are GPCRs that are involved the regulation of the adrenergic and renin-angiotensin systems. The ?2AR is polymorphic at position 164, affecting its responsiveness to adrenergic ligands. Both receptors have been shown to form dimers, but little is known on how dimerization affects their trafficking and signalling following ligand treatments. Plasma membrane localization, arrestin-2 recruitment, and G-protein interactions were determined between receptor dimers using molecular biological techniques. This study demonstrates that the formation of heterodimers can change the expected response to ligand treatments, along with associated trafficking events. It was determined that ligands bind to dimers, resulting in conformational changes to the dimeric complexes. Both the ?2AR and AT1aR are targeted in cardiovascular disease and this research demonstrates the importance of dimerization when prescribing drug therapies to avoid potential unwanted drug side effects.

Abstract FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture. The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored. FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery. Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement. FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit. Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard. Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.

No description available.

Hepatitis C, a life threatening disease, caused by the hepatitis C virus (HCV) currently affects over 170-200 million people worldwide (~3% of global human population), more than five times the percentage of total HIV infections. HCV infection has been shown to be a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma and is the leading cause of liver transplantation in the U.S. HCV has escaped every therapeutic target to date by means of its error-prone RNA polymerase, which allows it to mutate prolifically. The current standard anti-HCV therapy, which is pegylated interferon a combined with ribavirin, is difficult to tolerate, and more than 50% of HCV patients are refractory to it. No protective vaccine or therapeutic antibody is available, making the need for the development of an efficacious immunoprophylactic and therapeutic agent imperative. HCV is an enveloped virus with a positive sense RNA genome of ~9.6 kilobases (kb), which carries a large open reading frame (ORF), flanked by 5'- and 3'- untranslated regions (UTRs). Interestingly, within the highly mutational HCV RNA, there are a limited number of 100% conserved and functionally vital motifs, located in the 5' UTR, coding region and in the 3' UTR. Within the HCV genome, these motifs have been proposed to be involved in multiple exclusive interactions with each other and furthermore, these interactions have been demonstrated to be essential for HCV replication and/or translation of the viral proteins. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. It is a non-covalent, homodimeric and N-glycosylated protein, which is regulated in a tissue-specific manner and is dependent on an activator protein, apolipoprotein CII. Dissociation of active LPL dimers to monomers leads to loss of activity. This was previously found to be an important event in the rapid regulation of LPL in tissues. The mechanisms involved in the processing of LPL to active dimers, as well as in LPL inactivation through monomerization, were unknown. We have investigated the folding properties of the LPL protein, in particular the requirements for LPL to attain its active quaternary structure and to remain in the native conformation. On expression of LPL in insect cells we found that most of the LPL protein was synthesized in an inactive monomeric form. By co-expression of LPL with human molecular haperones, especially with calreticulin (CRT), the activity of LPL increased greatly, both in the cells and in the media. The effect of CRT on LPL activity was not due to increased levels of the LPL protein, but was due to an increased proportion of active dimeric LPL. Co-immunoprecipitation experiments showed direct interaction between LPL and CRT supporting the idea that this ER-based molecular chaperone supports the formation of active LPL dimers. We showed that, bis-ANS, the aromatic hydrophobic probe 1,1.-bis(aniline)-4,4.- bis(naphthalene)-8,8.disulfonate, can be used to obtain specific information about the interaction of LPL with lipid substrates and with apoCII. Bis-Ans was found to be a potent inhibitor of LPL activity, but apoCII prevented the inhibition. Our results suggest that bis-Ans binds to three exposed hydrophobic sites, of which one is at or close to the binding site(s) for apoCII. In studies of the mechanisms responsible for the spontaneous inactivation of LPL, we showed that active LPL is a dynamic dimer in which the subunits rapidly exchange partners. The rapid equilibrium between dimers and monomers exists even under conditions where LPL is relatively stable. This supports the idea that the dimer is in equilibrium with dimerization-competent, possibly active monomers. This dimerization-competent intermediate was also implicated in studies of the inactivation kinetics. The inactive LPL monomer was found to have a stable, defined conformation irrespective of how it was formed. The main differences in conformation between the inactive monomer and the active dimer were located in the middle part of the LPL subunit. Experiments with bis-Ans demonstrated that more hydrophobic regions were exposed in the inactive monomer, indicating a molten globule conformation. We concluded that the middle part of the LPL subunit is most likely engaged in the formation of the active LPL dimer. The dimerization-competent LPL monomer is a hypothetical conformational state, because it has not been possible to isolate it. To study complete refolding of LPL we used fully denatured LPL and were able to demonstrate that the recovery of LPL activity was about 40% when the denaturant was diluted by a buffer containing 20% human serum and 2M NaCl. Further studies identified calcium as the component in serum that was crucial for the reactivation of LPL. The refolding of LPL was shown to involve at least two steps, of which the first one was rapid and resulted in folded, but inactive monomers. The second step, from inactive monomers to active dimers, was slow and calcium-dependent. Also inactive monomers isolated from human tissue were able to recover activity under the influence of calcium. We proposed that calcium-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity. In conclusion, LPL is a relatively unstable enzyme under physiological conditions due to its noncovalent dimeric structure. The energy barrier for folding to the active dimer is high and requires the presence of calcium ions and molecular chaperones to be overcome. The dimeric arrangement is probably essential to accomplish rapid down-regulation of LPL activity according to metabolic demand, e.g. in adipose tissue on fasting.

Protein arginine methyltransferase, PRMT, is a family of epigenetic enzymes that methylate arginine residues on histone and nonhistone substrates which result in a monomethylation, symmetric dimethylation or asymmetric dimethylation via the transfer of a methyl group from S-adenosyl-L-methionine (SAM). We discovered a novel interaction between two PRMT isoforms: PRMT1 interacts and methylates PRMT6. In this study site-directed mutagenesis was performed on selected arginines identified from tandem mass spectrometric analysis to investigate major methylation sites of PRMT6 by PRMT1. In combination with radiometric methyltransferase assays, we determined two major methylation sites. Methylations at these sites have significant effects on the nascent enzymatic activity of PRMT6 in H4 methylation. PRMTs have the ability to homodimerize which have been linked to methyltransferase activity. We designed dimerization inhibitors (DMIs) to further investigate the need for dimerization for enzyme activity. Preliminary results suggest that the monomeric form of PRMT1 retains methyltransferase activity comparable to that of the uninhibited PRMT1.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-109).