Proteins are among the most abundant biological macromolecules. The cellular machinery is coupled to exact structural shape and properties of the more than 100 000 different proteins. Still, proteins can sometimes completely change their character and as a result trigger neuro degenerative disease. Exactly what happens is yet poorly understood but misfolding and aggregation leading to toxic gain of function is probable causes, i.e. the protein adopts new noxious properties. In 1993 the protein superoxide dismutase (SOD) was found to be associated with the neuro degenerative disease ALS. Up to date more than 100 mutations in SOD have been associated with ALS. However, the mutations are scattered all over the structure and no common denominator for the disease mechanism has been found. This work has been focused on the molecular mechanism of the toxic gain-of - function of mutant SOD from the perspective of protein folding and structural stability. To facilitate the studies of SOD and its ALS associated mutations, an expression system resulting in increased copper content was developed. Coexpression with the copper chaperone for superoxide dismutase (yCCS) leads to increased expression levels, especially for the destabilised ALS mutants. Through thermodynamic studies, I show that with the exception of the most disruptive mutations the holo protein is only marginally destabilised, whereas all mutations show a pronounced destabilisation on the apo protein. Kinetic studies suggest further that the dimeric apoSOD folds via a three-state process where the dimerisation proceeds via a marginally stable monomer. The apoSOD monomer folds by a two-state process. The disulphide bond is not critical for the folding of the apoSOD monomer although it contributes significantly to its stability. Interestingly, in the absence of metals, reduction of the disulphide bond prevents the formation of the dimer. A mutation can affect the protein stability in various ways: either from destabilisation of the monomer (case 1), weakening of the dimer interface (case 2) or, in the worst case, from a combination of both (case 1+2). Thus, therapeutic strategies to prevent the noxious effects of mutant SOD must include both mechanisms. An important finding in this study is that we can see a correlation between the stability for each mutation and the mean survival time. This could be an opening in the development of therapeutic substances that counteract the defect in SOD upon mutation.

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

Cytochrome cd1 nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the d1 haem iron of the enzyme. Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions. Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction. Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty. Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.

Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing. Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy in man with stromal fibrosis as one of the main features. Carcinoma cells synthesized no or little collagen I protein. Pro-α1(I) collagen mRNA was expressed by stromal cells throughout the tumor, but expression of procollagen type I protein was restricted to stromal cells situated close to nests of carcinoma cells. These data suggest that the carcinoma cells stimulated collagen type I deposition by increasing pro-α1(1) collagen mRNA translation. Cocultures, of the human ATC cell line KAT-4, with fibroblasts under conditions that allow the study of stimulatory factors on collagen mRNA translation, showed that the KAT-4 cells stimulated collagen type I protein synthesis in fibroblasts. Specific inhibitors of PDGF and TGF-β1 and -β3 were able to inhibit this carcinoma cell-induced stimulation of collagen type I synthesis. These findings suggest that tumor cells were able to stimulate collagen mRNA translation in stromal fibroblasts by, at least in part, transferring PDGF and/or TGF-β1 and -β3. Xenograft transplantation of different ATC cell lines into athymic mice demonstrated that the low collagen producing carcinoma cell lines were less tumorigenic compared to non-collagen producing carcinoma cell lines. The morphology of tumors derived from non-collagen producing ATC cell lines showed a well demarked stroma surrounding carcinoma cell nests. TGF-β1 and -β3 were found to play a role in generating a high tumor interstitial fluid pressure (TIPF) in experimental KAT-4 tumors. A specific inhibitor of TGF-β1 and -β3 was able to lower TIPF and reduce tumor growth after a prolonged period of treatment, suggesting that TGF-β1 and -β3 have a role in maintaining a stroma that support tumor growth.

The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed. The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. N,N’-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, i.e., a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power. A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.

Virus mRNAs can be divided into functional regions. The focus of this thesis will be to investigate the function of one of these regions, the 3’ untranslated region (UTR). The 3’UTR of HCV contains a U-rich element and the late 3’UTR of HPV-1 contains an AU-rich element. The roles of these regions in translation and stability of HCV and HPV have been studied. A method was established for studying translation of HCV mRNA in living cells. Noninfectious minivirus clones were synthesised in vitro and were transfected into cells by electroporation. This made it possible to bypass the nucleus and to transfer RNA directly into the cell cytoplasm. We found that HCV mRNAs that are translated from the HCV internal ribosome entry site (IRES) are inefficiently translated in comparison to capped and polyadenylated cellular mRNAs. Interestingly, the addition of a cap and a poly(A) tail resulted in a tremendous increase in the initiation of translation at the HCV IRES. This was the result of a discontinuous scanning or shunting mechanism. We also found that the 3’UTR had a small but not significant effect on the virus mRNA translation. Next, we set up an in vitro stability assay to investigate if HCV 3’UTR affects the stability of the virus mRNA. We found that the HCV 3’UTR is very unstable but interaction with the cellular La protein protects the mRNA from premature degradation. In parallel experiments, we studied translation and stability of the HPV-1 late mRNAs. By studying an AU-rich sequence in the 3’UTR, we mapped two minimal inhibitory sequence elements, UAUUUAU and UAUUUUUAU that reduced mRNA half-life. We found that the same motifs in the AU-rich element inhibit mRNA translation, demonstrating that the AU-rich element acts via a bimodal mechanism to reduce mRNA stability and inhibit translation.

The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression. The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.

Bikunin, a chondroitin sulfate-containing protein of 25 kDa, has protease inhibitory activity and occurs in the plasma in free and complexed form. In inter-α-inhibitor (IαI) and pre-a-inhibitor (PαI) it is covalently linked through its chondroitin sulfate (CS) chain to two or one other polypeptide of about 80 kDa – heavy chains 1 and 2 (H1, H2) and heavy chain 3 (H3) – respectively. Bikunin and the heavy chains are synthesized as precursors, which are proteolytically cleaved and assembled into IαI and PαI in the secretory pathway. The C-terminal extension (CTX) of the heavy chains seems to mediate its own cleavage and theassembly of the complexes. The heavy chains of the IαI family become transferred to hyaluronan during ovulation and inflammation. In this thesis, the biosynthesis of PαI, the plasma clearance of bikunin and the binding of IαI to collagen were studied. We found that in H3, a short segment on the N-terminal side of the CTX cleavage site is required for cleavage. Furthermore, the H3 could become linked to free CS chains primed by a xyloside, showing that the bikunin protein core is not needed for coupling. We also identified His649 as a residue essential for coupling, but not for cleavage. Bikunin labelled with a residualizing agent, 125I-tyramine cellobiose, was injected into mice to identify tissues involved in its uptake. Half of the radioactivity was recovered in the kidneys, 10% in the liver, and the rest distributed in other tissues. We determined the half-life of bikunin in rat plasma using two independent methods: injection of 125I-bikunin, or hepatectomy followed by assessing the rate of disappearance of endogenous bikunin. Both methods yielded half-time values of 5-7 minutes. Removal of the CS chain did not affect the clearance rate of bikunin. IαI and its heavy chains were found to bind to collagen with dissociation constants greater than 2 μM and 0.4-0.6 μM, respectively and this binding was independent of divalent metal ions. We suggest that the interaction of IαI with collagen may play a modulatory role in cell migration or in remodelling of the extracellular matrix.

Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N2), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium Anabaena PCC 7120. Apart from performing oxygenic photosynthesis, Anabaena PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. NtcA from Anabaena PCC 7120 was heterologously expressed in E. coli and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA in vitro using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N8 – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif. Furthermore, an NtcA binding site was found in the promoter region of the hupSL gene, encoding an uptake hydrogenase in Nostoc punctiforme (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.