Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.

The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts.

Neural stem and progenitor cells (NSPC) can differentiate to neurons and glial cells. NSPC are easily propagated in vitro and are therefore an attractive tool for tissue regeneration. Traumatic brain injury (TBI) is a common cause for death and disabilities. A fundamental problem following TBI is tissue loss. Animal studies aiming at cell replacement have encountered difficulties in achieving sufficient graft survival and differentiation. To improve outcome of grafted cells after experimental TBI (controlled cortical impact, CCI) in mice, we compared two transplantation settings. NSPC were transplanted either directly upon CCI to the injured parenchyma, or one week after injury to the contralateral ventricle. Enhanced survival of transplanted cells and differentiation were seen when cells were deposited in the ventricle. To further enhance cell survival, efforts were made to reduce the inflammatory response to TBI by administration of ibuprofen to mice that had been subjected to CCI. Inflammation was reduced, as monitored by a decrease in inflammatory markers. Cell survival as well as differentiation to early neuroblasts seemed to be improved. To device a 3D system for future transplantation studies, NSPC from different ages were cultured in a hydrogel consisting of hyaluronan and collagen. Cells survived and proliferated in this culturing condition and the greatest neuronal differentiating ability was seen in cells from the newborn mouse brain. NSPC were also used in a model of peripheral nervous system injury, and xeno-transplanted to rats where the dorsal root ganglion had been removed. Cells survived and differentiated to neurons and glia, furthermore demonstrating their usefulness as a tool for tissue regeneration.

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

The kinetic mechanism of an α/β hydrolase fold epoxide hydrolase from potato, StEH1, has been studied with the aims of explaining the underlying causes for enantio- and regioselectivity, both being important for product purity. Further effort has been laid upon understanding the causes of a hysteretic behavior discovered in the measurements leading to Paper I. The enantioselectivity was investigated with substrates differing only in substituent size at one carbon of the oxirane ring structure. In catalysis with trans-stilbene oxide and styrene oxide, enantioselectivity is the result of differences in alkylation rates. In pre-steady state measurement with trans-2-methylstyrene oxide (2-MeSO), a rate-limiting step involving slow transitions, referred to as hysteresis, was discovered. With this substrate enantioselectivity is proposed to be a consequence of the catalytic rate of (1R,2R)-enantiomer being more influenced by the hysteretic behavior than was the rate of the other enantiomer. In steady-state measurements with (1R,2R)-2-MeSO, at different temperatures and pH, hysteretic cooperativity was displayed. It can be concluded that this behavior is dependent on the relationship between kcat and the rate of transition between two Michaelis complexes. From the differences in pH dependence of kcat/KM in formation of the two diols resulting from low regioselectivity in catalysis of (1R,2R)-2-MeSO, it is suggested that hysteresis is a result of the substrates placed in different conformational modes within the active site cavity. Regioselectivity is proposed to be the result of specific interactions between the catalytically important Tyr and the substrate, with a link between KM-values and degree of regioselectivity. Furthermore, the hysteretic kinetic model proposed can explain hysteresis, cooperativity and regioselectivity resulting from StEH1 catalyzed hydrolysis of (1R,2R)-2-MeSO.

Weak affinity interaction of monoclonal antibodies and carbohydrate antigens can be detected and quantified by alterations in the antibodies' intrinsic tryptophan fluorescence. These weak/transient binding events have been monitored by total internal reflection fluorescence (TlRF) by facilitating the change in intrinsic tryptophan fluorescence. This immunosensor followed instant changes in the antigen concentration with rapid association- and dissociation rate constants reaching equilibrium in a short time, without the need for regeneration. Furthermore, in a competition assay with extrinsic fluorescence labeling, it was established that Förster/fluorescence resonance energy transfer (FRET) can be applied for weak and transient interactions. By entrapping components in small semipermeable capsules, aconvenient flow system was fabricated allowing on-line measurements of maltose. Quantification of maltose concentration was achievable in the mM-range without need for regeneration.High specificty for maltose was exhibited in crude food-samples with quantification in accordance with batch analysis. Furthermore, a monoclonal antibody was developed for potential use as a glucose immunosensor for diabetes. Its ability to interact with glucose was determined by competitive weak affinity chromatography (WAC) to approximately 19 mM in dissociation constant. This antibody was developed to bind monosaccharides, especially glucose, by utilizing crossreation with a carbohydrate dextran polymer. Selectivity for glucose was greater than for the similar monosaccharides, mannose and galactose. This antibody, or a fragment, in a fluorescence platform is an alternative to monitor glucose in vivo where other glucose-binders might fail. / Att känna igen en motståndare är viktigt i många sammanhang, inte minst i kroppens immunförsvar som är utvecklat för att angripa främmande ämnen i kroppen. Antikroppen spelar en central roll i immunförsvaret där den lär sig att känna igen sin motståndare (antigen) och därmed binda sitt antigen. De antikroppsproducerande cellerna kan användas i laboratoriet för att producera antikroppar som härstammar från försöksdjur. I denna avhandling har antikroppar använts som binder betydligt svagare till antigenet än vad man i de flesta analyser använder sig av för att t.ex. detektera sjukdomar. Antikroppar som binder till olika typer av socker, däribland maltsocker (maltos) och blodsocker (glukos) har studerats. Dessa antikroppar har använts för att undersöka hur hårt de binder till sitt antigen beroende på temperatur och om antikropparna kan känna igen liknande motståndare (korsreaktivitet). Fördelen med dessa svaga bindningar är att antikroppen snabbt kan binda in och släppa sitt antigen istället för att nästan permanent sitta på sitt antigen, som vid starka bindningar. Bindningens styrka (affinitet) har i avhandlingen studerats med hjälp av fluorescensteknik och affinitets-separation. Den maltosbindande antikroppen har använts tillsammans med fluorescensteknik för att designa två olika biosensorer (immunosensorer). Immunosensorerna kan mäta förändringen av maltoskoncentration över tid, vilket är attraktivt i t.ex. livsmedelsindustrin när man vill mäta maltoshalten kontinuerligt under tillverkningen. Den glukosbindande antikroppen har använts i affinitets-separation för att bestämma dess affinitet mot glukos och olika polymerer av glukos. En glukosbindande antikropp är åtråvärt för att t.ex. kontinuerligt mäta koncentrationen av blodsocker genom huden hos diabetiker och därmed minska antalet blodprover man idag behöver ta.

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5. TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5. We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript. Paper 4: Manuscript.

Heparan sulfate (HS) is a sulfated glycosaminoglycan expressed by all cells in the body. It is found at the cell surface and in the extracellular matrix where it binds a large amount of various ligands including growth factors and morphogens. HS is important for building up morphogen gradients during embryonic development and to act as coreceptors for signaling molecules. Many different Golgi enzymes are involved in the biosynthesis of HS. It is known that some of these enzymes interact with each other but not how the whole biosynthesis machinery works or how the cell regulates the structure of the HS that it produces. In this thesis, cells and mice deficient in two of these biosynthetic enzymes, glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST1) and the isoform NDST2 have been studied. NDSTs perform the first modifications during biosynthesis where they replace N-acetyl groups on N-acetyl-glucosamine units with sulfate groups. It is known that deficiency of NDST1 is lethal, while lack of NDST2 only results in abnormal connective tissue type mast cells. Here it is shown that deficiency of both NDST1 and NDST2 is embryonically lethal. The embryonic stem (ES) cells extracted from the inner cell mass of double knockout blastocysts show in addition an impaired differentiation capacity compared to wild-type ES cells and fail completely to differentiate into cardiac muscle cells which NDST1-/-, NDST2-/- and wild-type ES cells all do. Cultured mast cells that lack NDST2 produce heparin that is low-sulfated compared to wild-type HS. To our surprise, we could show that mast cells deficient in NDST1 instead produce a more highly sulfated heparin than wild-type cells. We use a model that predicts that the biosynthesis enzymes work together in a multienzyme complex, the GAGosome, to explain our results. We hypothesize that NDST1 has a higher affinity for the GAGosome than NDST2 which only in the absence of NDST1 gets incorporated into the enzyme complex. When all GAGosomes contain NDST2, a more highly sulfated glycosaminoglycan chain will be synthesized. A splice variant of NDST1, NDST1S, has also been studied. We could show that NDST1S lacks enzyme activity but that it probably has the capacity to incorporate into GAGosomes. Overexpression of NDST1S results in altered structure of the HS produced by the cells. We speculate that expression of the splice variant during development may be one way to regulate HS structure.

The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved. In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase. In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches. In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance. In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage. / QC20100629