Glyoxalase 11, the second enzyme of the glyoxalase system, hydrolyzes S-D-lactoylglutathione (SLG) to regenerate glutathione (GSH) and liberate free D-lactate. It was found that GTP binds with Gil from rat liver and inhibits Gil activity. Preincubation experiments showed that the binding is relatively tight, since more than 15 minutes are required to release GTP from the complex following dilution. Inhibition kinetics studies indicate that GTP is a "partially competitive inhibitor"; Thus, it would appear that the binding sites for substrate (SLG) and inhibitor (GTP) are different, but spatially close. Glyoxalase 11 binds to a GTP affinity medium, and with polyacrylamide gel electrophoresis, Gil has a higher relative mobility when GTP is present (ATP has no effect). The functional consequences of GTP binding with a specific site on Gil are still unclear. It is speculated that Gil may interact with tubulin by serving as a dissociable GTP carrier, delivering GTP to the tubulinGTP binding site, and thus facilitating tubulin polymerization.

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Communication is a ubiquitous component of life. While complexity and sophistication vary, both unicellular and multicellular organisms constantly interact with their environment. Unicellular organisms, once thought to be asocial, have since been demonstrated to display a multitude of social interactions and hierarchies. For example, quorum sensing enables a bacterial population to modulate gene expression in response to cell-population density, initiating social behavior and the exchange of resources. In eukaryotes, unicellular ascomycete fungi use mating GPCRs to detect secreted peptide pheromones, initiating changes in gene expression required for mating. An overview of communication in unicellular organisms is presented in Chapter 1. In general, these communication systems are characterized by a high degree of fidelity, and as such have been harvested by synthetic biologists to organize communication in synthetic systems. Quorum sensing modules have been employed for pattern formation and to coordinate biosynthesis processes across a community. However, fungal mating remains underutilized as a source of synthetic biology tools. In this dissertation, we leverage fungal mating G-protein-coupled receptors (GPCRs) and their peptide ligands to build responsive yeast systems. We use genome-mining to identify additional fungal peptide-GPCR pairs, which are then characterized in the yeast Saccharomyces cerevisiae. In Chapter 2, we exploit the high specificity and sensitivity of fungal mating GPCRs to design a yeast whole-cell biosensor that produces a visible output in response to detection of peptide biomarkers. In Chapter 3, we genome-mine additional peptide-GPCR pairs and use them as orthogonal signaling channels to build synthetic yeast communities. Finally, in Chapter 4, we use these synthetic yeast communities to provide sense-and-respond capabilities to an Engineered Living Material (ELM).

Lau Pui Ngan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 171-181). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Abbreviations --- p.viii / Publications Based on work in this thesis --- p.xii / Chapter Chapter 1 --- Introduction and project overview --- p.1 / Chapter 1.1 --- Ghrelin structure and its synthesis --- p.3 / Chapter 1.2 --- Types of growth hormone secretagogues (GHSs) --- p.6 / Chapter 1.3 --- Characterization of GHS-R --- p.7 / Chapter 1.3.1 --- Cloning of GHS-Rla and GHS-Rlb --- p.7 / Chapter 1.3.1.1 --- GHS-R subtypes --- p.7 / Chapter 1.3.1.2 --- Properties of GHS-R subtypes --- p.7 / Chapter 1.3.1.3 --- Evidence of non-GHS-Rla stimulated by ghrelin and GHSs --- p.9 / Chapter 1.3.1.4 --- Distribution of GHS-R --- p.10 / Chapter 1.3.2 --- Signal transduction pathways of GHS-R --- p.11 / Chapter 1.3.3 --- Comparison between human and seabream GHS-R --- p.12 / Chapter 1.4 --- Is adenosine a partial agonist at GHS-Rla? --- p.15 / Chapter 1.5 --- Physiological effects of ghrelin --- p.17 / Chapter 1.6 --- Apoptosis --- p.19 / Chapter 1.6.1 --- Introduction --- p.19 / Chapter 1.6.2 --- Apoptosis versus necrosis --- p.19 / Chapter 1.6.3 --- Mechanisms of apoptosis --- p.20 / Chapter 1.6.4 --- Methods to study apoptosis --- p.23 / Chapter 1.6.5 --- Different types of apoptotic inducers --- p.24 / Chapter 1.7 --- Apoptotic and anti-apoptotic pathways regulated by GPCRs --- p.27 / Chapter 1.7.1 --- Bcl-2 family pathway --- p.27 / Chapter 1.7.2 --- Caspase pathway --- p.27 / Chapter 1.7.3 --- ERK pathway --- p.28 / Chapter 1.7.4 --- PI3K/Akt pathway --- p.29 / Chapter Chapter 2 --- Materials and solutions --- p.31 / Chapter 2.1 --- Materials --- p.31 / Chapter 2.2 --- "Culture medium, buffer and solutions" --- p.37 / Chapter 2.2.1 --- Culture medium --- p.37 / Chapter 2.2.2 --- Buffers --- p.37 / Chapter 2.2.3 --- Solutions --- p.38 / Chapter Chapter 3 --- Methods --- p.41 / Chapter 3.1 --- Maintenance of cell lines --- p.41 / Chapter 3.1.1 --- Human Embryonic kidney (HEK293) cells --- p.41 / Chapter 3.1.2 --- HEK293 cells stably expressing black seabream growth hormone secretagogues receptors (HEK-sbGHS-Rla and HEK-sbGHS-Rlb) --- p.41 / Chapter 3.2 --- Preparation of plasmid DNA --- p.42 / Chapter 3.2.1 --- Preparation of competent E. coli --- p.42 / Chapter 3.2.2 --- Transformation of DNA into competent cells --- p.42 / Chapter 3.2.3 --- Small-scale and large-scale plasmid DNA preparation --- p.43 / Chapter 3.2.4 --- Confirmation of the purity and the identity of the plasmid DNA --- p.43 / Chapter 3.3 --- Transient transfection of mammalian cells --- p.45 / Chapter 3.4 --- Development of stable cell lines --- p.46 / Chapter 3.4.1 --- Determination of the optimum concentration of each antibiotic used in selection of clones --- p.46 / Chapter 3.4.2 --- Development of monoclonal stable cell line --- p.46 / Chapter 3.4.3 --- Confirmation the expression of 2myc-hGHS-Rla and myc-hGHS-Rlb --- p.48 / Chapter 3.5 --- Measurement of phospbolipase C activity --- p.49 / Chapter 3.5.1 --- Introduction --- p.49 / Chapter 3.5.2 --- Preparation of columns --- p.49 / Chapter 3.5.3 --- [3 H]-inositol phosphate assay --- p.49 / Chapter 3.5.4 --- Measurement of [3H]-inositol phosphates production --- p.50 / Chapter 3.5.5 --- Data analysis --- p.50 / Chapter 3.6 --- Determination of transient transfection efficiency --- p.51 / Chapter 3.7 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.52 / Chapter 3.7.1 --- RNA extraction and first strand cDNA production --- p.52 / Chapter 3.7.2 --- PCR and visualization of amplicons --- p.52 / Chapter 3.7.3 --- Real-time PCR --- p.59 / Chapter 3.7.3.1 --- Construction of standard curve --- p.60 / Chapter 3.7.3.2 --- Data analysis --- p.60 / Chapter 3.8 --- Measurement of caspase-3 activity --- p.65 / Chapter 3.8.1 --- Determination of caspase-3 activity using colorimetric assay --- p.65 / Chapter 3.8.1.1 --- Introduction --- p.65 / Chapter 3.8.1.2 --- Induction of apoptosis --- p.65 / Chapter 3.8.1.3 --- Preparation of cell lysates --- p.65 / Chapter 3.8.1.4 --- Quantification of caspase-3 activity by measuring pNA absorbance --- p.66 / Chapter 3.8.1.5 --- Data analysis --- p.67 / Chapter 3.8.2 --- Determination of caspase-3 activity using bioluminescence resonance energy transfer (BRET2) assay --- p.67 / Chapter 3.8.2.1 --- Introduction --- p.67 / Chapter 3.8.2.2 --- Quantification of caspase-3 activity using BRET2 assay --- p.68 / Chapter 3.8.2.3 --- Data analysis --- p.69 / Chapter 3.8.3 --- Determination of caspase-3 activity using fluorescence resonance energy transfer (FERT) assay --- p.70 / Chapter 3.8.3.1 --- Introduction --- p.70 / Chapter 3.8.3.2 --- Quantification of caspase-3 activity using FRET assay --- p.70 / Chapter 3.8.3.3 --- Data analysis --- p.71 / Chapter Chapter 4 --- Results --- p.72 / Chapter 4.1 --- Characterization of GHS-R --- p.72 / Chapter 4.1.1 --- Properties of GHS-Rla --- p.72 / Chapter 4.1.1.1 --- Constitutively active receptor --- p.72 / Chapter 4.1.1.2 --- Characterization of epitope-tagged hGHS-Rla --- p.73 / Chapter 4.1.2 --- Properties of GHS-Rlb --- p.75 / Chapter 4.1.3 --- Conclusions --- p.75 / Chapter 4.2 --- Effect of co-transfection of HEK293 cells --- p.85 / Chapter 4.2.1 --- Effect of balancing DNA concentrations transfected into HEK293 cells --- p.85 / Chapter 4.2.2 --- Effect of balancing DNA concentration using another Gq-coupled receptor --- p.87 / Chapter 4.2.3 --- Effect of Gi- and Gs-coupled receptor on GHS-Rla signaling --- p.88 / Chapter 4.2.4 --- Potentiating effect of co-transfection appeared using different transfection reagents --- p.88 / Chapter 4.2.5 --- Co-transfection improves transfection efficiency --- p.89 / Chapter 4.2.6 --- Discussions --- p.91 / Chapter 4.3 --- Development of cell lines stably expressing hGHS-Rla or hGHS-Rlb --- p.102 / Chapter 4.3.1 --- Advantages of using a monoclonal cell line --- p.102 / Chapter 4.3.2 --- Sensitivity of HEK293 cells to antibiotics --- p.102 / Chapter 4.3.3 --- Production of polyclonal stable cell line --- p.103 / Chapter 4.3.4 --- Monoclonal stable cell line selection --- p.104 / Chapter 4.3.5 --- Discussions --- p.105 / Chapter 4.4 --- Effect of adenosine on GHS-Rla signaling --- p.111 / Chapter 4.4.1 --- Adenosine acts as partial agonist --- p.111 / Chapter 4.4.2 --- Effect of substance P analog on adenosine-mediated GHS-Rla signaling --- p.112 / Chapter 4.4.3 --- Effect of adenosine deaminase (ADA) on adenosine- and ghrelin-stimulated GHS-Rla signaling --- p.113 / Chapter 4.4.4 --- Specificity of ADA --- p.115 / Chapter 4.4.5 --- Conclusions --- p.116 / Chapter 4.5 --- Role of GHS-R in apoptosis --- p.124 / Chapter 4.5.1 --- Different methods to measure caspase-3 activity --- p.124 / Chapter 4.5.1.1 --- Colorimetric assay --- p.124 / Chapter 4.5.1.1.1 --- Time course for staurosporine and etoposide in HEK293 cells --- p.125 / Chapter 4.5.1.1.2 --- Effect of 2myc-hGHS-Rla on staurosporine- and etoposide-induced caspase-3 activity --- p.127 / Chapter 4.5.1.1.3 --- Time course for staurosporine and etoposide in sbGHS-R monoclonal stable cell line --- p.128 / Chapter 4.5.1.1.4 --- Effect of sbGHS-Rs on staurosporine- and etoposide- induced caspase-3 activityin HEK 293 cells --- p.129 / Chapter 4.5.1.1.5 --- Effect of sbGHS-Rs on staurosporine- induced caspase-3 activity in sbGHS-R monoclonal stable cell line --- p.130 / Chapter 4.5.1.1.6 --- Differences between epitope-tagged and non-tagged sbGHS-Rs in staurosporine- induced caspase-3 activity --- p.131 / Chapter 4.5.1.1.7 --- The role of epitope-tagged sbGHS-Rlbin staurosporine-induced caspase-3 activity --- p.132 / Chapter 4.5.1.1.8 --- Effect of staurosporine and etoposide on GHS-Rla signaling --- p.133 / Chapter 4.5.1.2 --- BRET2 assay --- p.135 / Chapter 4.5.1.3 --- FRET assay --- p.136 / Chapter 4.5.1.4 --- Conclusions --- p.136 / Chapter 4.6 --- Determination of GHS-R amount in terms of mRNA --- p.155 / Chapter 4.6.1 --- Determination of GHS-R amount in stable cell lines --- p.155 / Chapter 4.6.2 --- Transfected DNA amount match with stable cell lines --- p.155 / Chapter Chapter 5 --- "Discussion, Conclusions and Future Plan" --- p.159 / Chapter 5.1 --- General Discussion and Conclusions --- p.159 / Chapter 5.2 --- Future Plan and Experimental Design --- p.168 / References --- p.171

Based on what we have found, we proposed a model for Ni presenting by HypB involved in hydrogenase maturation. HypB binds two Ni ions in the apo- and GDP-bound form. Ni binding also induces dimerization of HypB. Upon GTP binding, HypB can bind an extra Ni ion at the dimeric interface. GTP hydrolysis will release the extra Ni ion, which may be subsequently inserted into hydrogenases during the maturation process. / Furthermore, two Ni binding sites were determined in a monomeric HypB. One is the cluster including C92, H93 and C122, the other is composed of H97 and H101. Upon GTP-dependent dimerization, HypB can bind an extra Ni ion. Our results have shown that the C92/H93/C122 is involved in binding the extra Ni ion, and such binding requires both cysteine residues in the reduced form. Since the GTP-induced dimerization of HypB is coupled to bind an extra Ni, so HypB could act as a GTP-mediated switch that regulate one Ni release from the GTP-bound form to the GDP-bound form. / In the future, we will attempt to crystallize AfHypB in complex with GDP, GTP analogue and AfHypA. Availability of good quality crystals will pave way for the structure determination of AfHypA and AfHypA/HypB complex. And the results obtained will provide a better understanding of the mechanism of functional interaction between HypA and HypB and how HypA and HypB play a role in Ni ion delivery for hydrogenase maturation. / The assembly of the [NiFe]-hydrogenases requires incorporation of Ni ions into the enzyme's metallocenter, which process requires the GTPase activity of HypB and HypA. Due to the essential role in assembly of the active site of hydrogenases, the two proteins were defined as hydrogenase maturation factors. To better understand the mechanism of GTP hydrolysis-dependent Ni delivery accomplished by HypB and HypA, our work focuses on the structure-function study of AfHypB from Archaeoglobus fulgidus and the functional interaction between AfHypA and AfHypB. / The intrinsic GTPase activity of AfHypB is very low, suggesting that AfHypB requires a G-protein activating protein (GAP) to activate its GTPase activity. Although AfHypB can interact with AfHypA to form 1:1 heterodimer, our data suggests that AfHypA is not a GAP for AfHypB. In addition, the FRET results showed that AfHypA could serve as a GEF (G-protein exchange factor) to activate the AfHypB from GDP-bound form to GTP-bound form and facilitate the dissociation of AfHypB dimer in the presence of GMPPNP. / Up to now, we have solved the structure of apo-AfHypB by X-ray crystallography. Crystals of AfHypB were grown using the hanging-drop-vapor-diffusion method and diffracted to ∼2.3 A. It belonged to space group P2(1)2(1)2, with unit cell dimensions a=72.49, b=82.33, c=68.66 A, alpha=beta=gamma=90°. Two molecules of AfHypB were found in an asymmetric unit. Structural comparison between the apo-AfHypB and GTP-bound HypB from M jannachii showed that the GTP binding broke the salt-bridge between K43 and D66, and induced conformational changes in the switch I loop and helix-3, which caused the HypB to form dimer and bind an extra Ni ion. The GTP-bound form of HypB was ready for Ni presenting. And GTP hydrolysis could induce the conformational changes of HypB in the switch I loop and helix-3, which dissociate the HypB dimer into the monomeric GDP-bound form. / Li, Ting. / Adviser: K. B. Wong. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 98-104). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In a phage binding assay, phage clone (3p5A190) expressing a surrogate mas ligand displayed punctate binding and were internalized in cell expressing native mas and GFP-tagged variants. However, the number of bound and internalized phages in cells expressing mas-GFP was substantially less than the cells expressing mas-(Gly10Ser5)GFP and native mas. In parallel, biotinylation experiment quantitatively showed that the extent of mas-(Gly10Ser 5)-GFP translocation was higher than that of mas-GFP. Consistently, cells expressing mas-(Gly10Ser5)-GFP and native mas showed a rapid and sustained increase of intracellular calcium levels upon MBP7 stimulation. By contrast, cells expressing mas-GFP only response to higher concentration of MBP7 challenge and showed a delayed increase of intracellular calcium level. Moreover, cells expressing native mas had a higher proportion (80%) of cells responsive to MBP7 stimulation; in contrast to only 10∼20% of cells expressing mas fusion proteins. / MBP7-like motif was identified in human facilitative GLUT1 and GLUT7 indicating that mas might interact with glucose transporter (GLUT) and regulate cellular glucose uptake. GLUT4 was found to be expressed endogenously in the CHO cell by RT-PCR, but expression of insulin receptor was not detectable. Although no statistical difference was detected in basal glucose uptake among control cells Vc0M80 and cells with different levels of mas expression, cells expressing mas-(Gly10Ser5)-GFP showed a high glucose uptake in response to insulin. Furthermore, basal 2-DOG uptake in Mc0M80 cells was not affected by pretreatment with various kinase inhibitors or transient expression of Rho variants. By contrast, MBP7 was found to induce a significant elevation of glucose uptake specifically in Mc0M80 cells transiently transfected with GLUT1. / Orphan G protein-coupled receptor (GPCR) mas was initially isolated from a human epidermal carcinoma. Previous study from our lab identified a surrogate ligand---MBP7 (mas binding peptide 7) for mas, and suggested that GFP tagging might affect the receptor activity of mas. In this project, three stable CHO cell lines expressing native mas, mas-GFP and mas-(Gly10Ser 5)-GFP were used to characterize receptor activity of mas. / To summarize, direct GFP tagging at the C-terminus of mas decreased its interactions with ligand and downstream signaling molecules. Partial recovery of mas receptor activity by adding a peptide linker was confirmed by phage binding, membrane fusion protein translocation and calcium response. In addition, mas was possibily coupled with GLUT1 to affect cellular glucose uptake via signaling pathways yet to be fully characterized. / Sun, Jingxin. / Adviser: Cheung Wing Tai. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0104. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 150-170). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Chan, Kwok Ho. / Thesis submitted in: November 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgements --- p.II / Abstract --- p.III / 摘要 --- p.IV / Table of Contents --- p.V / Abbreviations --- p.XIII / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- GTPase in general --- p.1 / Chapter 1.2 --- G proteins and GTP switch --- p.2 / Chapter 1.3 --- Structural similarities in GTPase --- p.3 / Chapter 1.4 --- G proteins in bacteria --- p.3 / Chapter 1.5 --- Background information of the protein family EngB --- p.4 / Chapter 1.6 --- Basic information of EngB in Thermotoga maritima --- p.5 / Chapter 1.7 --- Objectives of this work --- p.6 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemical reagents --- p.8 / Chapter 2.1.2 --- Buffers / Chapter 2.1.2.1 --- Preparation of buffers --- p.10 / Chapter 2.1.2.2 --- Buffers for common use --- p.11 / Chapter 2.1.3 --- Expression strains and plasmids --- p.14 / Chapter 2.1.4 --- Primer list --- p.14 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Preparation of competent cells --- p.15 / Chapter 2.2.2 --- Cloning / Chapter 2.2.2.1 --- Cloning of target genes by PCR --- p.15 / Chapter 2.2.2.2 --- Agrose gel electrophoresis --- p.17 / Chapter 2.2.2.3 --- Extraction and purification of DNA from agarose gel --- p.17 / Chapter 2.2.2.4 --- Restriction digestion of DNA --- p.18 / Chapter 2.2.2.5 --- Ligation of digested insert and expression vector --- p.18 / Chapter 2.2.2.6 --- Transformation and plating out transformants for miniprep --- p.19 / Chapter 2.2.2.7 --- Verification of insert by PCR --- p.20 / Chapter 2.2.2.8 --- Mini-preparation of plasmid DNA --- p.21 / Chapter 2.2.2.9 --- Confirmation of miniprep product by restriction enzyme digestion..… --- p.22 / Chapter 2.2.2.10 --- Sequencing of the plasmid DNA --- p.23 / Chapter 2.2.3 --- Expression of the recombinant MBP-TM EngB protein and SBP-CBP EC EngB / Chapter 2.2.3.1 --- Transformation for protein expression --- p.23 / Chapter 2.2.3.2 --- Preparation of starter culture --- p.24 / Chapter 2.2.3.3 --- Expression of recombinant protein --- p.24 / Chapter 2.2.3.4 --- Cell harvesting --- p.24 / Chapter 2.2.3.5 --- Releasing the cell content --- p.25 / Chapter 2.2.3.6 --- Check for protein expression by SDS-PAGE --- p.25 / Chapter 2.2.4 --- Purification of TM EngB / Chapter 2.2.4.1 --- SP ion-exchange chromatography --- p.27 / Chapter 2.2.4.2 --- Thrombin digestion to remove MBP tag --- p.28 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.29 / Chapter 2.2.4.4 --- Gel filtration chromatography --- p.29 / Chapter 2.2.5 --- Purification of SBP-CBP EC EngB / Chapter 2.2.5.1 --- SP ion-exchange chromatography --- p.30 / Chapter 2.2.5.2 --- Gel filtration chromatography --- p.31 / Chapter 2.2.6 --- Protein concentration quantitation --- p.32 / Chapter 2.2.7 --- Crystallography of TM EngB / Chapter 2.2.7.1 --- Crystallization preparation --- p.32 / Chapter 2.2.7.2 --- Crystallization screening by sitting drop method --- p.32 / Chapter 2.2.7.3 --- Optimization of crystallization conditions --- p.33 / Chapter 2.2.7.4 --- X-ray diffraction --- p.33 / Chapter 2.2.8 --- Thermodynamics studies of proteins / Chapter 2.2.8.1 --- Preparation of protein sample --- p.34 / Chapter 2.2.8.2 --- Guanidine-induced denaturation experiment --- p.34 / Chapter 2.2.8.3 --- Thermal-induced denaturation experiment --- p.35 / Chapter 2.2.9 --- Binding assay to study affinity for ligands --- p.36 / Chapter 2.2.9.1 --- Using GDP analogue mant-GDP to detect formation of enzyme-ligand complex (TM EngB-mant-GDP) --- p.36 / Chapter 2.2.9.2 --- Basic information of Fluorescence spectroscopy --- p.36 / Chapter 2.2.9.3 --- Determination of λem and λex --- p.37 / Chapter 2.2.9.4 --- Studying ligand affinity by titration with ligand analogue --- p.37 / Chapter 2.2.10 --- Pull down experiment to study interacting partner of E. coli EngB --- p.38 / Chapter 2.2.10.1 --- Preparing protein extracts from E. coli --- p.38 / Chapter 2.2.10.2 --- Preparing streptavidin resin --- p.39 / Chapter 2.2.10.3 --- Binding of dual-tagged E. coli EngB to streptavidin resin --- p.39 / Chapter 2.2.10.4 --- Purifying protein using the prepared streptavidin resin --- p.40 / Chapter 2.2.10.5 --- Preparing calmodulin resin --- p.41 / Chapter 2.2.10.6 --- Binding of dual-tagged E.coli EngB to calmodulin resin --- p.41 / Chapter 2.2.10.7 --- Analysis of dual-tag affinity purified protein --- p.42 / Chapter 2.2.11 --- Silver staining of acrylamide gel / Chapter 2.2.11.1 --- Staining reagents --- p.42 / Chapter 2.2.11.2 --- Staining procedures --- p.43 / Chapter Chapter 3 --- Structure determination of T. maritima EngB by X-ray crystallography / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Generation of TM EngB expression construct --- p.45 / Chapter 3.3 --- Expression and purification of TM EngB --- p.46 / Chapter 3.4 --- TM EngB was crystallized with freshly purified TM EngB --- p.47 / Chapter 3.5 --- Data processing of diffraction data and structure refinement of TM EngB …… --- p.48 / Chapter 3.6 --- Apo-form TM EngB was obtained by unfolding and refolding --- p.49 / Chapter 3.7 --- Crystallization of apo-form TM EngB --- p.50 / Chapter 3.8 --- Data processing of diffraction data and structure refinement of apo-form TM EngB --- p.51 / Chapter 3.9 --- Producing EngB-GDP complex crystal from apo-from EngB --- p.52 / Chapter 3.10 --- TM EngB is a monomer in solution --- p.54 / Chapter 3.11 --- Summary of chapter three --- p.55 / Tables and figures of chapter three --- p.57 / Chapter Chapter 4 --- Structural details of TM EngB / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Overall fold of TM EngB --- p.67 / Chapter 4.3 --- Mode of nucleotide binding of TM EngB --- p.68 / Chapter 4.4 --- Structural differences in switch I region between chain A and chain B in crystal structure of TM EngB/GDP complex --- p.70 / Chapter 4.5 --- Structural difference between TM EngB/GDP complex and apo TM EngB --- p.73 / Chapter 4.6 --- Summary of chapter four --- p.73 / Tables and figures of chapter four --- p.76 / Chapter Chapter 5 --- Purified TM EngB is Active for binding guanine nucleotide but inactive for GTPase hydrolysis activity / Chapter 5.1 --- Introduction --- p.88 / Chapter 5.2 --- Studying ligand affinity by competitive binding experiment --- p.88 / Chapter 5.3 --- GDP binds to TMEngB with higher affinity than GTPyS --- p.91 / Chapter 5.4 --- TM EngB showed very low intrinsic GTPase activity --- p.92 / Chapter 5.5 --- Discussion --- p.93 / Tables and figures of chapter five --- p.95 / Chapter Chapter 6 --- Thermostability of EngB of T. maritima / Chapter 6.1 --- Introduction --- p.98 / Chapter 6.2 --- Guanidine hydrochloride - induced unfolding --- p.98 / Chapter 6.3 --- Thermal-induced unfolding --- p.99 / Chapter 6.4 --- Structural comparison of thermophilic and mesophilic EngB --- p.100 / Chapter 6.5 --- Discussion --- p.102 / Tables and figures of chapter six --- p.105 / Chapter Chapter 7 --- Construction of a dual-tag affinity pull-down system for finding interacting partner of EngB / Chapter 7.1 --- Introduction --- p.112 / Chapter 7.2 --- Preparation of dual-tagged E.coli EngB / Chapter 7.2.1 --- Cloning of SBP-CBP-EC EngB expression construct --- p.113 / Chapter 7.2.2 --- Expression and purification of SBP-CBP-EC EngB --- p.114 / Chapter 7.3 --- Pull down using dual tagged E.coli EngB as bait to isolate potential interacting partners of EngB --- p.114 / Chapter 7.4 --- Discussion --- p.115 / Tables and figures of chapter seven --- p.117 / Chapter Chapter 8 --- Conclusion --- p.122 / References --- p.124

The Rho family of GTPases plays a crucial role in the regulation of diverse cellular processes, including proliferation and actin cytoskeletal rearrangement to promote cell migration. However, dysregulation of RhoGTPases has been associated with disease, particularly cancers such as leukemia. Despite this, RhoGTPases are rarely mutated in cancer. Rather, dysregulation of their regulatory proteins through mutation or overexpression contributes to disease pathogenesis. RhoGTPases are activated through Rho guanine nucleotide exchange factors (GEFs). Although over eighty RhoGEFs have been identified that activate the 25 RhoGTPases, the pathological role of the majority of these proteins remains unclear. Further, whereas the majority of RhoGEFs are activated through tyrosine phosphorylation, a small subset can be activated through heterotrimeric G proteins, including through GΒ;Γ; subunits. However, the mechanism by which GΒ;Γ; induces RhoGEF activation remains unclear. PLEKHG2 is a Dbl family RhoGEF that was originally identified as a gene upregulated in a leukemia mouse model, and later shown to be activated by heterotrimeric G protein Β;Γ; subunits. However, its function and activation mechanisms remain elusive. Here we show that, as compared to primary human T cells, the expression of PLEKHG2 is upregulated in leukemia cell lines. Downregulation of PLEKHG2 by siRNAs specifically inhibited GΒ;Γ;-stimulated Rac and Cdc42, but not RhoA activation. Consequently, inhibition of PLEKHG2 blocked actin polymerization, protrusion formation, and leukemia cell migration in response to SDF1alpha;. Additional studies indicate that GΒ;Γ; likely activates PLEKHG2 by binding the N-terminus of PLEKHG2. This interaction results in the release of autoinhibition imposed by the C-terminus within a region encompassing the catalytic DH domain. As a result, overexpressing either the N-terminus of PLEKHG2 that binds GΒ;Γ; or the C-terminus that autoinhibits PLEKHG2 blocked GΒ;Γ;-stimulated Rac and Cdc42 activation and the ability of leukemia cell to form membrane protrusions and to migrate. Together, our results have demonstrated that PLEKHG2 functions as a novel GΒ;Γ; -stimulated RhoGEF that could contribute to chemokine-induced leukemia cell dissemination and leukemia pathogenesis.

Thesis (M.S.)--University of Montana, 2006. / Title from title screen. Description based on contents viewed Mar. 9, 2007. Includes bibliographical references (p. 21-22).