According to the National Institute of Neurological Disorders and Stroke, there are more than 600 neurologic disorders that affect approximately 50 million Americans each year. The $91 billion dollars spent by Medicare on Alzheimer's disease and other dementias in 2005 is projected to increase to $189 billion by 2015 [4]. The existence of neural stem cells (NSC's) and neurogenesis makes neural regeneration a viable option. The ethical barriers of using embryonic stem cells, rejection of the transplanted cells, and possible tumor formation, are only a few of the problems that face stem cell transplantation, a widely considered option to repopulate the brain with cells. A noninvasive pharmaceutical approach that can promote neuron regeneration and recovery would be the key to curing many neurodegenerative diseases. The development of MS-818 as a non-invasive enhancer of the proliferation process of NS Cs is revolutionary for the treatment of neurodegenerative diseases. Due to the fact that its mechanism of action remains unknown, the full pharmacological potential of MS- 818 has not been fully exploited [8]. Isolating protein targets of MS-818 is key in identifying the molecular pathways responsible for its mechanism of action. UV-Vis analysis of MS-818 showed absorbance at 275-nm, and this data was used to calculate coupling yield. MS-818 coupled to the NHS-activated sepharose beads of the affinity column with 83% efficiency. Proteins were isolated from human embryonic kidney cells (HEK 293 cells) and applied to the column. Bradford assay confirmed that bound proteins eluted in a concentration dependent manner when an MS-818 gradient was applied to the column. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the eluate revealed two sets of polypeptides migrating at 37-75 kDa and 100-150 kDa. In addition, some trace polypeptides in the sub-35 kDa region could be seen. Although we have not yet identified specific proteins that MS-818 interacts with, we were able to successfully to isolate such proteins.

The aim of the project was to identify positions and amino acids that contribute to improved structure and stability of bispecific ADAPT proteins. During the 20 weeks project period, different amino acid substitutions were analysed to evaluate the effect on the three-helical structure and stability of bispecific ADAPTs targeting human serum albumin (HSA) and tumor necrosis factor α (TNFα). Furthermore, the study also included identification of which amino acid substitutions that affect the simultaneous binding ability of the anti-TNFα ADAPT. The amino acids substitutions that demonstrated improved stability was further evaluated in two other bispecific ADAPT proteins targeting epithelial cell adhesion molecule (EpCAM), in terms of structure and stability. The TNFα-targeting ADAPT variants was produced in Escherichia coli (E. coli), purified through affinity chromatography using a HSA-coupled matrix and was further analysed and evaluated using SDS-PAGE, circular dichrosim, size-exclusion chromatography and surface plasmon resonance to detect expression levels, yields, thermal stability, secondary structure, and simultaneous binding to TNFα and HSA. Furthermore, the production, purification and evaluation were redone with other bispecific ADAPTs targeting EpCAM, to be able to draw more general conclusions. The outcome showed which amino acids substitutions in the scaffold that improve the structure and stability of the TNFα- and EpCAM-binding ADAPT protein variants, respectively. Some of the ADAPT variants targeting TNFα showed improved stability and increased melting temperature. One of the variants with most potential from these mutants was ADAPT_TNFα5_F21K, both able to refold after heat treatment and demonstrated a higher melting temperature in the same order as the original binder. The variant bound HSA but not TNFα, thus consequently was not able to bind TNFα and HSA simultaneously. The variants ADAPT_TNFα5_V17I and ADAPT_TNFα5_M22Q both demonstrated a clear alpha-helix structure, were able to refold after heat treatment and demonstrated simultaneous binding to TNFα and HSA. The melting temperature for ADAPT_TNFα5_V17I was the same as for the original binder (59°C) and ADAPT_TNFα5_M22Q showed a decreased melting temperature (45°C) compared to the original binder. The amino acid substitutions that improve the stability of the original binder was combined and two variants withthese mutations were designed. Unfortunately, these variants could not express in E. coli cells and were not able to be produced. For the EpCAM targeting mutants one variant, ADAPT_EpCAM_02_X11N, showed huge improvements of the stability and structure compared to the original binder ADAPT_EpCAM_02. This variant improved the melting temperature with 24°C compared to the original binder and was able to refold after heat treatment, which the original binder did not have the ability to do. However, ADAPT_EpCAM_02_X11N was not able to simultaneously bind EpCAM and HSA, demonstrating that the mutation also had an effect on the binding ability. In the variant ADAPT_EpCAM_08 the mutation Y5I improved the melting temperature with 14°C compared to the original binder and was able to refold after thermal denaturation. However, the simultaneous binding to EpCAM and HSA was negatively affected. The project results have contributed to better understanding of the bispecific ADAPT proteins, which enables further development of the scaffold. The amino acid positions in the scaffold that showed to be important for ADAPT structure and stability will be used in the design of a new ADAPT-library, from which new binders with improved structure and stability hopefully can be selected, which might have the potentially to be used as future therapeutics. / Syftet med projektet var att identifiera positioner och aminosyror som bidrar till ökad struktur och stabilitet hos bispecifika ADAPT proteiner. Under projektperioden på 20 veckor har olika aminosyrasubstitutioner i ett bispecifikt TNFα/HSA-bindande ADAPT protein analyserats med syftet att undersöka om dessa substitutioner förbättrar stabiliteten och strukturen hos proteinet. Vidare analyserades hur dessa aminosyrasubstitutioner påverkar ADAPTs förmåga att binda HSA och TNFα samtidigt. De aminosyrasubstitutioner som visade på förbättrad stabilitet, utvärderades vidare med avseende på struktur, stabilitet och bindning i två andra bispecifika ADAPTs riktade mot EpCAM. De TNFα-bindande ADAPT-varianterna producerades i E. coli, renades fram med affinitetskromatografi med en HSA-kopplad matris, analyserades och utvärderades med metoder som SDS-PAGE, cirkulär dichroism, gelfiltrering och surface plasmon resonance för att detektera uttrycks-nivåer, utbyten, termisk stabilitet, sekundärstruktur och om proteinerna upprätthöll bindning till både HSA och TNFα. Vidare utvärderas de aminosyrasubstitutionerna som visade på förbättrad stabilitet även i andra bispecifika ADAPTs riktade mot EpCAM för att kunna dra mer generella slutsatser. Resultaten visade vilka aminosyrasubstitutioner i proteinet som förbättrade strukturen och stabiliteten hos de TNFa- respektive EpCAM-bindande ADAPT-protein varianterna. Några av ADAPT-TNFa varianterna visade förbättrad stabilitet och ökad smälttemperatur. ADAPT_TNFα5_F21K hade en tydlig alpha-helix struktur och kunde återveckas efter värmebehandling. Varianten hade en smälttemperatur i samma storleksordning som den ursprungliga TNFα-bindande ADAPT varianten. ADAPT_TNFα_F21K hade dock inte förmågan att binda simultant till TNFα och HSA. Varianterna ADAPT_TNFα_V17I och ADAPT_TNFα_M22Q visade en tydlig alpha-helix struktur, kunde återveckas efter värmebehandling och visade simultan bindning till TNFα och HSA. ADAPT_TNFα_V17I hade samma smälttemperatur som den ursprungliga bindaren (59°C) medan ADAPT_TNFα_M22Q visade en minskad smälttemperaturen (45°C) jämfört med original-bindaren. Två varianter bestående av kombinationer av de aminosyrasubstitutioner som visade på förbättrad stabilitet skapades. Dessa kunde tyvärr inte uttryckas och produceras i E.coli-celler. Gällande de EpCAM-bindande ADAPT-proteinerna var det en variant, ADAPT_EpCAM_02_X11N, som visade stor förbättring i stabilitet och struktur jämfört med originalbindaren ADAPT_EpCAM_02. Denna variant hade 24°C högre smälttemperatur jämfört med originalbindaren och kunde återveckas efter värmebehandling, vilket inte original-bindaren kunde. Dock kunde inte ADAPT_EpCAM_02_X11N binda simultant till EpCAM och HSA. Detta tyder på att denna mutation har en negativ effekt på ADAPTs bindingskapacitet. Varianten ADAPT_EpCAM_08_Y5I visade en förbättrad smälttemperatur på 14°C jämfört med original-bindaren ADAPT_EpCAM_08 och kunde återveckas efter värmebehandling. Dock resulterade även denna mutation i en negativ effekt på den simultana bindningen till EpCAM ochHSA. Projektets resultat har bidragit till en bättre förståelse av de bispecifika ADAPT-proteinerna och möjliggör en vidareutveckling av scaffoldet. Aminosyranspositionerna som visade sig vara viktiga för ADAPTs struktur och stabilitet kommer användas för design av ett ny ADAPT-bibliotek, från vilket nya bindare med förbättrad struktur och stabilitet förhoppningsvis kan bli selekterade. Dessa nya, förbättrade bindare, ökar de bispecifika ADAPT proteiners användningsmöjligheter inom terapi.

Work on a wide variety of metabolic pathways indicates that these systems are often, if not always, organized as multienzyme complexes. Enzyme complexes have the potential to increase catalytic efficiency and provide unique mechanisms for the regulation of cellular metabolism. The flavonoid biosynthetic pathway of Arabidopsis is an excellent model for studying the organization, localization, and regulation of enzyme complexes at the cellular level. Flavonoids are specialized metabolites that perform many important physiological roles in plants. Protein interactions among several key flavonoid enzymes have been described. Moreover, at least two of the flavonoid enzymes have a dual cytoplasmic/nuclear localization. These results indicate that flavonoid enzymes assemble into one or more distinct complexes at different intracellular locations. The current study integrates a new technology, mass spectrometry, with well-established affinity chromatography methods to further characterize the organization and composition of the Arabidopsis flavonoid enzyme complex. One of the key flavonoid enzymes, chalcone isomerase (CHI), was used in these experiments to detect interacting enzymes. Recombinant thioredoxin (TRX), or TRX-CHI, was produced in E. coli, then purified by metal affinity chromatography, and covalently coupled to an activated resin, Affi-Gel 10. Extracts prepared from 4-day-old wild type or CHI-deficient lines of Arabidopsis were then passed over the column and the bound proteins eluted with sodium dedocyl sulfate (SDS). This eluate was then subjected to a liquid chromatography (LC) - mass spectrometry (MS) protocol developed for the analysis of complex peptide mixtures. An Agilent LC system coupled with an LTQ-MS instrument (Thermo Electron, San Jose, CA) was used for this purpose. Data analysis was performed with the Bioworks software package. Different optimization strategies were performed to improve the affinity chromatography, sample preparation, and the LC separation method. A novel approach has been developed for the MS analysis of biological samples containing contaminants such as salts and detergents. Protein extracts prepared from wild type Landsburg and mutant tt5(86) were analyzed by LC-MS/MS. A total of 491 proteins were identified for Landsburg and 633 for tt5(86) extracts using a combination of data filters and p-value sorting. All detected proteins had p<0.001 and most were identified by at least 2 unique peptides. / Master of Science

Lectin affinity chromatography has proven to be a powerful method to separate oligosaccharides based on their stereochemical structures. This technique has not been used for the separation of glycolipids since mixtures of these compounds form micelles in aqueous solution. Since N-acetylgalactosamine (GalNAc) is commonly found in glycolipids, three GalNAc-specific lectins were selected to develop a lectin affinity chromatographic method for glycolipids. To circumvent the difficulty of working with micelles, the autoradiographic detection of ¹²⁵l-labeled lectins binding to glycolipids on thin-layer chromatograms was used to study the glycolipid-binding specificity of the lectins from Helix pomatia, Wisteria floribunda and Dolichos biflorus. All three lectins detected the Forssman glycolipid which has a terminal GalNAcα1-3 residue. The Helix pomatia and Wisteria floribunda lectins are also bound to glycolipids with GalNAcβ-linked residues. The interactions of these lectins with glycolipid derived, ³H-labeled oligosaccharides were also analyzed by affinity chromatography on agarose-immobilized lectins. Only the immobilized Helix pomatia lectin was able to specifically bind oligosaccharides with α-linked GalNAc residues. The Helix pomatia lectin was selected to develop an affinity chromatography system for the purification of intact glycolipids having terminal GalNAcα1-3 residues. This technique relies on the ability of the immobilized lectin to bind its oligosaccharide ligands in aqueous solutions of tetrahydrofuran (THF) which inhibits micelle formation and permits the separation of non-specifically bound glycolipids. Forssman glycolipid and a human blood group A-active hexaosylceramide were bound to the Helix pomatia column equilibrated in water/THF (5:95). After applying a step gradient of increasing water content (to 50% water), the specifically bound glycolipids were eluted when GalANc was included in the mobile phase. Using these chromatographic conditions, the Forssman glycolipid from the neutral lipid fraction of sheep erythrocyte stroma and the A-active glycolipids from a total extract of type A human erythrocytes were purified in the Helix pomatia column. The ability to purify human A-active glycolipids from total lipid extracts in a single chromatographic step with the Helix pomatia column was used to isolate A-active glycolipids present in erythrocytes from donors from a rare blood group B(A). The erythrocytes from B(A) subgroup of blood group B individuals, are weakly hemagglutinated by a murine monoclonal anti-A antibody although these erythrocytes should not express blood group A antigens. The Helix pomatia lectin was used to determine the presence and isolate A-active glycolipids from the neutral lipid fraction of erythrocytes from two blood group B(A) donors. However, A-active glycolipids were absent in the glycolipid extracts from erythrocytes from a third B(A) donor and plasma of all three B(A) donors as well as erythrocytes of blood group B and O donors. Based on the fact that only glycolipids and oligosaccharides with GalNAcα1-3 residues specifically bind to the Helix pomatia column, this lectin column was used to isolate the 'terminal products' of the biosynthetic pathway of the human blood group A glycolipids and glycopeptides from the human epidermoid carcinoma cell line A-431. The metabolically active A-431 cells were grown in the presence of ³H-labeled monosaccharide precursors and the Helix pomatia column was used to determine and compare the rate of incorporation of labeled precursors in the A-active glycoconjugates from these cells. / Ph. D.

<p>The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.</p><p>The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.</p><p>In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.</p>

The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein. The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization. In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.

Membrane bound AChE from calf brain caudate nucleus was solubilized by use of ion-free media in presence of 10~ M EDTA and 10“5M tetracaine. The irreversible release of AChE was more effectively inhibited by divalent ions compared to monovalent ions added to the medium. EDTA appears to chelate divalent ions released from the tissue while tetracaine competes with the same ions at the membrane. The tetracaine effect is restricted to the procaine series of local anesthetics. Small amounts of soluble AChE are present in the cytosol fraction. In fresh preparations most of the enzyme appeared in a form having a molecular weight of 80.000 daltons as determined by gel filtration. The enzyme seems to be released in this form. It is proposed that this form represents the monomer form of the enzyme. In solution the AChE aggregates seemingly together with a factor that is released from the membrane in amounts stafchio- metric to the enzyme. By treatment with DEAE-Sephadex the enzyme preparation can be made non-aggregating. A highly purified nonaggregating monomeric AChE Specific activity 17150 micromoles acetylthiocholine hydrolyzed per minute at 27° C per mg protein) was obtained by affinity chromatography. Some anomalous binding phenomena was observed during the affinity chromatographic purification. The main observation was that edrophonium eluted crude enzyme preparation adsorbed to the affinity gel in a biphasic manner. It was found that in the crude preparation there is present besides unspecific material competing with the enzyme for the affinity gels a factor that increases the affinity of AChE to certain cholinergic ligands. Since the enzyme could be titrated by the factor it seems to have a very high affinity to the enzyme and the biphasic elution curve is explained by the presence of free as well as factor- bound enzyme in the preparation. In search for compounds having a similar effect it was found that fluoride ion too increased the affinity of AChE to the same ligands as the factor. The affinity of edrophonium to the site defined by the binding of AChE to MTA-CH (65x10“5m) is lower than that defined by the enzyme inhibitory constant (1.8xlO“7M). As an explanation of this finding it is proposed that the substrate induces a conformation having high affinity to edrophonium, a conformation that has a comparatively low relaxation rate. Thus acetylcholinesterase may be added to the list of enzymes that have hystere- tic properties. / <p>S. 1-54: sammanfattning, s. 55-100: 4 uppsatser</p> / digitalisering@umu

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC₅₀ 16-640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a 'gene-like sequence' of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC₅₀ = 6.25 μM in a susceptible cell line, compared with > 100 μM for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes.

O uso de DNA plasmidial (pDNA) visando a aplicações terapêuticas tem aumentado nos últimos anos. A cromatografia aparece como a técnica de purificação mais comum para obtenção de amostras de pDNA com o elevado grau de pureza exigido. Porém, as resinas cromatográficas disponíveis apresentam ainda uma série de desafios, nomeadamente no desenvolvimento de ligantes específicos e matrizes capazes de acomodar este tipo de molécula. Relativamente à apuração de novos ligantes, alguns estudos têm mostrado o potencial do aminoácido arginina para estabelecer interações específicas e preferenciais com o pDNA. Por outro lado, resinas monolíticas surgem como suportes interessantes devido às suas excelentes propriedades de transferência de massa e altas capacidades de adsorção. Neste estudo, diferentes ligantes baseados em arginina (arginina, di-arginina e tri-arginina) foram imobilizados em resinas de agarose previamente ativadas. Um primeiro estudo de adsorção em batelada foi realizado a fim de avaliar e compreender os mecanismos envolvidos no processo de adsorção dos ácidos nucleicos pDNA e RNA em resina com o aminoácido arginina. Na sequência, apresentamos uma proposta inovadora para o uso de ligantes de arginina em resinas de agarose, em um único passo de purificação em modo negativo a seguir ao passo de concentração por isopropanol. A capacidade da resina para o pDNA foi substancialmente maior do que a obtida para o mesmo tipo de resina no modo positivo, com notória vantagem de capacidade no uso de di-arginina face a arginina com rendimentos próximos de 100% do plasmídeo carregado. Os ligantes di-arginina e tri-arginina foram também imobilizados em resinas monolíticas. Em comparação com o aminoácido arginina, a imobilização dos homopeptídeos nas resinas monolíticas levou ao aumento da capacidade de adsorção (cerca de 2,5 vezes superior) e especicificidade de interações, mostrando-se como uma estratégia promissora para processos de purificação de pDNA. / The use of plasmid DNA (pDNA) for therapeutic applications has increased in recent years. Chromatography appears as the most common purification technique to obtain samples of pDNA with the high degree of purity required. However, the available chromatographic resins still present a series of challenges, namely in the development of specific ligands and matrices capable of accommodating this type of molecule. Regarding the determination of new ligands, several studies have shown the potential of the arginine amino acid to establish specific and preferential interactions with the pDNA. On the other hand, monolithic resins appear as interesting approaches due to their excellent mass transfer properties and high adsorption capacities. In this study, different arginine based ligands (arginine and di-arginine) were firstly immobilized on activated agarose resins. The first part of the work describes the adsorption equilibrium of plasmid DNA adsorption process, as well as the interaction with its main impurity (RNA) on arginine supports in a batch format, in order to compare and gather crucial information about adsorption mechanisms involved in this type of affinity system. Then, a new use for chromatographic bead matrixes based on arginine ligands was proposed, working as an adsorption matrix pDNA purification in negative mode after isopropanol concentration of the sample. The arginine based supports capacity for pDNA under negative mode for pDNA was substantially higher than that obtained with the same type of resin in the conventional positive mode, with a notable advantage of using di-arginine with recovery yields near 100%. The homopeptides (di-arginine and tri-arginine) were also immobilized on functionalized monolithic resins (BIA Separations, Slovenia). Effectively, the immobilization of the arginine homopeptides made the monolithic resins more functional compared to the (mono)arginine based resin, exhibiting greater binding capacities (around 2,5 times higher) and interaction intensities, proving to be a promising strategy for purification processes of pDNA.

No description available.