Glycogen synthase kinase-3 (GSK3) is a ubiquitously expressed protein kinase with key roles in controlling proliferation, differentiation and survival of a wide variety of mammalian cells. In most cells, GSK3 is active in the absence of growth factor signaling and acts to inhibit cell proliferation and induce apoptosis. In cardiomyocytes, GSK3 plays a novel role as a negative regulator of cardiac hypertrophy, and it appears that GSK3 plays a central role as an inhibitor of cardiac hypertrophy induced by a variety of stimuli. In the present study, we sought to further elucidate the role of GSK3 in cardiomyocyte hypertrophy by studying the effects of inhibition of GSK3 in the absence of other hypertrophic stimuli. By combining global expression profiling with computational predictions and experimental analysis of transcription factor binding sites, we have identified hypertrophy-related genes that are controlled directly by GSK3 and have found that CREB is a major transcriptional target of GSK3 in cardiomyocytes. In addition, we find that inhibition of GSK3 is sufficient to induce the re-expression of fetal development genes characteristic of hypertrophy, but not sufficient to induce the full hypertrophic phenotype of cardiomyocyte growth.

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain.

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An der Ausbildung der individuellen Persönlichkeitseigenschaften eines Menschen sowie an der Entstehung von Persönlichkeitsstörungen und anderen psychischen Erkrankungen sind sowohl genetische Faktoren als auch Umwelteinflüsse beteiligt. Mittels Assoziationsstudien kann man prüfen, ob zwischen einzelnen genetischen Varianten und Persönlichkeitsmerkmalen bzw. psychischen Störungen ein Zusammenhang besteht. Im Rahmen der vorliegenden Arbeit wurden funktionelle Polymorphismen in zwei Kandidatengenen, der Dopamin-β-Hydroxylase (DBH) und der neuronalen NO-Synthase (NOS1), im Hinblick auf eine Assoziation mit Persönlichkeitsvariablen und Persönlichkeitsstörungen untersucht. Diese Enzyme spielen eine wichtige Rolle im noradrenergen bzw. nitrinergen System, die beide an der Steuerung des Verhaltens entscheidend beteiligt sind. Des Weiteren wurde geprüft, ob der Polymorphismus im Promotorbereich des DBH-Gens mit affektiven Störungen assoziiert ist. Die Genotypisierung wurde bei 642 Probanden mit Persönlichkeitsstörungen und 182 Patienten mit affektiven Störungen durchgeführt; die Kontrollgruppen umfassten 387 Personen (DBH-Polymorphismus) bzw. 494 Personen (NOS1-Polymorphismen). Eine Assoziation des -1021C→T-Polymorphismus des DBH-Gens mit affektiven Störungen ließ sich nicht nachweisen, obwohl Vorbefunde einen Zusammenhang zwischen dem mit einer niedrigen Plasmaaktivität der Dopamin-β-Hydroxylase assoziierten T/T-Genotyp und affektiven Störungen nahegelegt hatten. Diese Assoziation findet sich jedoch möglicherweise nur bei Subgruppen affektiver Störungen wie z. B. Depressionen mit psychotischer Symptomatik. Eine hochsignifikante Assoziation zeigte sich zwischen dem T/T-Genotyp und dem Auftreten von zwei oder mehr Persönlichkeitsstörungen, so dass dieser Genotyp als Risikofaktor für die Entwicklung von Persönlichkeitsstörungen angesehen werden kann. Des Weiteren ist der T/T-Genotyp mit verschiedenen Subskalen von Neuroticism, Agreeableness und Novelty Seeking assoziiert, die sich auf impulsives, feindseliges und wenig zielgerichtetes Verhalten beziehen. Dies bestätigt die Ergebnisse früherer Studien, die einen Zusammenhang zwischen dem noradrenergen System und impulsiven Verhaltensweisen gezeigt haben. Zahlreiche Studien weisen auch auf eine Verbindung zwischen Veränderungen des NOS1-Gens und impulsivem, aggressivem Verhalten hin. Im Rahmen dieser Arbeit konnte eine Assoziation beider NOS1-Polymorphismen mit Cluster-B-Persönlichkeitsstörungen beobachtet werden, die Impulsivität als ein gemeinsames Merkmal aufweisen. Es fand sich jedoch keine Assoziation mit Persönlichkeitsdimensionen, die impulsives und aggressives Verhalten widerspiegeln. In einer Weiterführung der vorliegenden Studie mit größeren Probandenzahlen wurde erneut der Zusammenhang zwischen einem dieser NOS1-Polymorphismen, dem Exon-1f-VNTR, und Persönlichkeitsvariablen sowie dem durch gesteigerte Impulsivität gekennzeichneten Aufmerksamkeitsdefizit- und Hyperaktivitätssyndrom (ADHS) bei erwachsenen Patienten untersucht. In dieser Studie wurde in der Kontrollgruppe eine nur bei Frauen statistisch signifikante Assoziation des kurzen Allels mit niedrigen Conscientiousness-Werten, die als Anzeichen für ein hohes Maß an Impulsivität verstanden werden können, beobachtet. Auch eine Assoziation des kurzen Allels mit ADHS konnte nachgewiesen werden, was die Bedeutung dieses Polymorphismus bei der Entstehung impulsiver Verhaltensweisen weiter untermauert. Zur Aufdeckung der genetischen Grundlage von Persönlichkeitseigenschaften und psychischen Erkrankungen bedarf es der Identifizierung weiterer genetischer Risikovarianten und deren Untersuchung in großen Assoziationsstudien mit einer hohen Probandenzahl. Um den Zusammenhang zwischen genetischen Varianten und Persönlichkeit bzw. Verhalten zu erhellen, müssen zudem komplexe Interaktionen verschiedener Gene und der Einfluss von Umweltfaktoren einbezogen werden. / Genetic factors and environmental influences contribute to human personality dimensions as well as to the development of personality disorders and other psychiatric diseases. Genetic association studies are used to test for a correlation between genetic variants and personality traits or psychiatric disorders. In this thesis, the association between functional polymorphisms in two candidate genes, the dopamine beta-hydroxylase gene (DBH) und the neuronal nitric oxide synthase gene (NOS1), and personality variables und personality disorders was investigated. These enzymes play an important role in the noradrenergic and nitrinergic system, respectively, which are both critically involved in the regulation of behaviour. Furthermore, we tested for an association between the promotor polymorphism in the DBH gene and affective disorders. Genotyping was performed on 642 subjects with personality disorders and 182 subjects with affective disorders; the control groups consisted of 387 individuals (DBH polymorphism) and 494 individuals (NOS1 polymorphisms), respectively. No association was observed between DBH -1021C→T genotype and affective disorders although previous findings had suggested a link between the T/T genotype (which is associated with lower dopamine beta-hydroxylase plasma activity) and affective disorders. However, this association may be limited to subgroups of affective disorders, e.g. unipolar psychotic depression. A highly significant association was detected between the T/T genotype and the co-occurrence of two or more personality disorders. Thus, this genotype can be regarded as a risk factor for the development of personality disorders. Furthermore, the T/T genotype is associated with various neuroticism, agreeableness and novelty seeking subscales related to impulsive, hostile and little goal-directed behaviour. This is in line with previous studies that showed a correlation between the noradrenergic system and impulsive behaviours. Numerous studies suggest a link between genetic variations in the NOS1 gene and impulsive, aggressive behaviour. In the present study, an association was detected between both NOS1 polymorphisms and Cluster B personality disorders which share the feature of impulsivity. However, no association was observed with personality dimensions reflecting impulsive and aggressive behaviour. A subsequent study with larger sample sizes investigated the correlation between one of the NOS1 polymorphisms, the Exon1f VNTR, and personality variables as well as adult attention-deficit/hyperactivity disorder (ADHD), a disorder characterized by increased impulsivity. In healthy controls, an association was detected between the short allele and low levels of conscientiousness which indicate a high degree of impulsivity; this effect was statistically significant only for women. The short allele was also associated with ADHD which underscores the significance of this polymorphism in the development of impulsive behaviours. Identification of further genetic risk variants and their investigation in association studies with large sample sizes is needed to unravel the genetic basis of personality traits und psychiatric diseases. Furthermore, complex interactions of different genes as well as the influence of environmental factors must be taken into account in order to elucidate the relationship between genetic variants and personality and behaviour.

Atherosclerosis is a multifactorial inflammatory disease of the arterial wall and its clinical manifestations, including myocardial infarction and stroke, are the leading causes of death in western societies. Recent data has suggested that disruption of protein homeostasis in a cell's endoplasmic reticulum (ER), a condition known as ER stress, is associated with the progression of atherosclerosis. Furthermore, signaling by the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β mediates pro-atherogenic processes. This thesis examines the role of ER stress and GSK3α/β signaling in atherosclerosis. Initially, three apolipoprotein-E deficient (ApoE-/-) mouse models of accelerated atherosclerosis were established. Relative to ApoE-/- mice fed a chow diet, pro-atherogenic conditions promoted hepatic steatosis, atherosclerosis, ER stress and GSK3β activity. A subset of mice from each group were given the GSK3α/β inhibitor valproate. Valproate supplementation suppressed hepatic steatosis, atherosclerosis and GSK3β activity in each mouse model without altering ER stress levels. This study revealed a role for ER stress and GSK3α/β in multiple murine models of atherosclerosis. Next, we investigated ER stress and GSK3α/β signaling in macrophage foam cell formation. In macrophages, ER stress induced GSK3α/β activity in a protein kinase R-like endoplasmic reticulum kinase (PERK) dependent manor. GSK3α/β inhibition attenuated ER stress induced lipid accumulation and the expression of distal components of the PERK pathway. Overexpression of constitutively active GSK3β induced foam cell formation. In mice, valproate supplementation attenuated PERK signaling in peritoneal macrophages and macrophages within atherosclerotic lesions. Together, these results point to GSK3α/β being a downstream component of the PERK pathway and that PERK-GSK3α/β signaling mediates ER stress induced foam cell formation. Lastly, we investigated the tissue and homolog specific functions of GSK3α and GSK3β in atherosclerosis. In high fat diet (HFD) fed low-density lipoprotein receptor deficient (LDLR-/-) mice, deletion of GSK3α or GSK3β in hepatocytes did not alter liver lipid content or atherosclerosis. Myeloid cell deletion of GSK3α, but not GSK3β, attenuated HFD induced atherosclerosis. Mechanistically, deletion of GSK3α in macrophages promotes the anti-atherogenic M2 macrophage phenotype by modulating signal transducer and activator of transcription (STAT)-3 and STAT6 phosphorylation and activation. Together, the data presented in this thesis suggest; 1) GSK3α/β inhibition attenuates atherosclerosis in multiple mouse models, 2) PERK-GSK3α/β signaling regulates macrophage foam cell formation and 3) myeloid cell GSK3α mediates atherosclerosis and macrophage phenotype. / Thesis / Doctor of Philosophy (PhD)

CDP-ribitol synthase is a bifunctional reductase and cytidylyltransferase that catalyzes the transformation of D-ribulose 5-phosphate, NADPH and CTP to CDP-ribitol, a repeating unit presem in the virulence-associated polysaccharide capsules of Haemophilus influenzae type a and b (Follens et al., 1999, J Bacterial. 181: 2001). In the work described here, we investigated the order of the reactions catalyzed by CDPribitol synthase and conducted experiments to resolve the question of substrate channeling in this bifunctional enzyme. It was determined that the synthase first catalyzed the reduction ofo-ribulose 5-phosphate followed by cytidylyltransfer to oribitol 5-phosphate. Steady state kinetic measurements revealed a 650-fold kinetic preference for cytidylyltransfer too-ribitol 5-phosphate over o-ribulose 5-phosphate. Rapid mixing studies indicated quick reduction of o-ribulose 5-phosphate with a lag in the cytidylyltransfer reaction consistent with a requirement for the accumulation of Km quantities of o-ribitol 5-phosphate. Signature motifs in the C-terminal and N-terminal sequences of the enzyme (short chain dehydrogenase/reductase and nucleotidyltransferase motifs, respectively) were targeted with site directed mutagenesis to generate variants that were impaired for only one of the two activities (K386A and R18A impaired for reduction and cytidylyltransfer, respectively). Release and free diffusion of the metabolic intermediate o-ribitol 5-phosphate was indicated by the finding that equimolar mixtures of K386A and R18A variants were efficient for bifunctional catalysis. Taken together these findings suggest that bifunctional turnover occurs in distinct active sites of CDP-ribitol synthase with reduction of n-ribulose 5-phosphate, release and free diffusion of the metabolic intermeditate n-ribitol 5-phosphate followed by cytidylyltransfer. / Thesis / Master of Science (MS)

Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h post-infection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that limit parasite development. However, the Plasmodium-derived factors that can induce AsNOS expression and the signaling pathways responsible for transduction in A. stephensi have not been identified until completion of the work described herein. In my studies, I have determined that P. falciparum glycosylphosphatidylinositol (PfGPIs) can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Based on related work in mammals, I hypothesized that parasite-derived AsNOS-inducing factors signal through the insulin signaling pathway and the NF-kappaB-dependent Toll and Immune deficiency (Imd) signaling pathways. In support of this hypothesis, I have determined that signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi protein kinase B (Akt/PKB) and DSOR1 (mitogen activated protein kinase kinase, MEK)/Extracellular signal-regulated protein kinase (ERK), kinases which are associated with the insulin signaling pathway. However, signaling by P. falciparum and PfGPIs is distinctively different from signaling by insulin and these parasite signals are not insulin-mimetic to A. stephensi cells. In other studies, treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced AsNOS expression by P. falciparum merozoites in A. stephensi cells. This result suggested the involvement of Toll and Imd pathways in parasite signaling of mosquito cells. Knockout of Pelle, a proximal signaling protein in the Toll pathway, increased AsNOS expression following parasite stimulation, suggesting that the Toll pathway may negatively regulate signaling by Plasmodium-derived AsNOS-inducing factors. In contrast, knockout of TGF-beta-activated kinase 1 (Tak1), a proximal signaling protein in the Imd pathway, reduced AsNOS expression by 20% relative to the control, suggesting that the Imd pathway is required for signaling by Plasmodium-derived AsNOS-inducing factors. Despite the NO-rich environment of the midgut, Plasmodium development is not completely inhibited. This observation suggests that Plasmodium may have efficient detoxification systems during sexual development in A. stephensi. To identify Plasmodium defense genes that may defend parasites against nitrosative stress caused by AsNOS induction, expression of several antioxidant defense genes known to function in nitrosative stress defense in a variety of organisms were examined during sporogonic development. Notably, increased expression levels of P. falciparum peroxiredoxins containing 1 or 2 cysteines (1-cys or 2-cys PfPrx) were associated with periods of parasite development just prior to and during parasite penetration of midgut epithelium, an event associated with significant AsNOS induction in the midgut. The provision of N omega-L-arginine (L-NAME), a known inhibitor of NOS enzyme activity, to A. stephensi with Plasmodium culture by artificial bloodmeal significantly reduced expression of 1-cys and 2-cys PfPrx indicating that these gene products may function to protect parasites against nitrosative stress induced by AsNOS. / Ph. D.

There are at least two models describing how the enzymes of metabolic pathways are arranged in living cells. The first is a stochastic model, where enzymes are freely-diffusing in the aqueous environment of the cell, and the second, the metabolon model, has pathway enzymes organized as enzyme complexes. Both are valid scientific hypotheses in that they make predictions that can be tested regarding pathway regulation, localization, and function. The goal of the work presented here was to test the metabolon model using the flavonoid biosynthetic pathway in Arabidopsis, which has been hypothesized to exist as a metabolic enzyme complex. Five novel mutants of the gene encoding the first enzyme of flavonoid biosynthesis, chalcone synthase (CHS), were characterized in an effort to develop tools for investigating the organization of flavonoid metabolism in Arabidopsis. A variety of mutant CHS genotypes were identified in this allelic series, including ones that displayed both null and temperature-sensitive phenotypes, based on endproduct analysis. Characterization of protein and RNA levels indicated that the stability of the CHS enzyme was reduced in some of the mutants as compared to wild type. In several of the alleles, homodimerization of CHS was also impaired. Effects of the mutations at the amino acid level were predicted from the three-dimensional crystal structure of the highly-homologous alfalfa CHS, which indicated substitutions at diverse sites on the enzyme, including ones that may disrupt folding and/or active site function. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization. In an effort to determine the in planta location of the first two enzymes in flavonoid biosynthesis, CHS and chalcone isomerase (CHI), immunolocalization experiments were performed. Results indicate that CHS and CHI are abundant in epidermal and cortex cells of the root elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. At the subcellular level, both of these enzymes were found to localize to the endoplasmic reticulum (ER), consistent with the hypothesis that the enzymes of flavonoid biosynthesis are organized as a membrane-associated enzyme complex. Analysis of the tt7(88) mutant, which lacks the cytosolic domain of the putative 'anchor' P450 enzyme, flavonoid 3'-hydroxylase, showed an altered distribution of CHS and CHI as compared to wild type, however CHS and CHI were still found to be associated with ER. These results suggest that complex interactions occur within the flavonoid enzyme complex to mediate the subcellular distribution of its constituents. Also evident from these studies was the asymmetric distribution of CHS and CHI in cortex cells of the elongation zone, a finding that may provide clues about the physiological function of flavonoids in roots. Together, these immunolocalization data support the metabolon model for the organization of flavonoid biosynthesis in Arabidopsis. In an effort to develop tools to investigate the in vivo dynamics of flavonoid biosynthesis, fusion proteins between CHS or CHI and the reporter, green fluorescent protein (GFP), were produced. Transient transfection assays in epidermal cells from onion root bulbs and Arabidopsis seedlings indicated that the GFP component of the fusion constructs was functional, as determined via GFP fluorescence. To investigate the spatial and temporal dynamics of these fusion proteins in all cell types, Arabidopsis plants stably transformed with the CHI-GFP fusion constructs were generated. The analysis of these transgenic plants should provide information regarding the localization and dynamics of flavonoid biosynthesis in vivo, and thereby serve to offer new insights into the function and regulation of this important plant metabolic pathway. Overall, the research presented here represents a significant contribution toward understanding how subcellular organization may be important in regulating metabolism. / Ph. D.

Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies. / Ph. D.

Flavonoid biosynthesis is an important secondary metabolic pathway in higher plants with a range of vital functions in plants and animals. This pathway has been developed as a model system for the study of multi-enzyme complexes. The goal of the work presented here was to structurally characterize a series of loss-of-function chalcone synthase (CHS) alleles and to define the molecular basis of the interaction between CHS and the second enzyme of flavonoid biosynthesis, chalcone isomerase (CHI). CHS proteins encoded by five previously characterized alleles were characterized by homology modeling in an effort to explain the alterations in function, stability, and dimerization exhibited by these variants. Four of the encoded proteins have a single amino acid substitution and the fifth is a truncated protein resulting from a frameshift. Models for each of these proteins were generated in silico and analyzed after molecular dynamics simulations. This analysis suggested reasons for changes in catalytic ability and stability for three of the five CHS variants. To characterize the molecular basis of the CHS-CHI interaction, a model was developed using X-ray crystallography, small-angle neutron scattering (SANS), in silico docking, molecular dynamics simulations, and yeast 2-hybrid analyses. These enzymes appear to be interacting in a manner that could facilitate the flow of intermediates from one active site to another. These experiments also identified a series of amino acids that appear to be involved in the interaction, which are currently undergoing alteration and analysis using a yeast 2-hybrid assay to verify the authenticity of the model. The data presented herein could be used in future engineering experiments to alter pathway flux to control the levels or types of flavonoid endproducts, resulting in more nutritious plants or flowers with novel pigments. These experiments advance the study of the structure of multi-enzyme complexes, an area that currently contains little information. As well, this is the first known use of SANS for the investigation of the architecture of metabolons. The techniques described herein could easily be applied to other systems in an effort to better understand the organization of multi-enzyme complexes and the implications of these assemblies on metabolic regulation. / Ph. D.