<p>Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is <i>N</i>-sulfated by the enzyme <i>N</i>-deacetylase <i>N</i>-sulfotransferase (NDST). <i>N</i>-Sulfation occurs in domains where further modifications (including <i>O</i>-sulfations) take place, giving the chain a complex sulfation pattern.</p><p>In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that <i>N</i>-deacetylation is the rate-limiting step in HS <i>N</i>-sulfation and that two different NDST molecules can work on the same GlcN unit.</p><p>By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for <i>N</i>-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on <i>N</i>-deacetylase activity. </p><p>We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. </p><p>Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones.</p><p>.</p>

<p>Peroxisome is a ubiquitous eukaryotic organelle with a single-layer membrane. It maintains various functions that differ depending on the species and cell types, as well as the environmental or developmental conditions.</p><p>In the first part of this thesis, the peroxisomal protein content was systematically analyzed in different organs in mouse from different ages using proteomic approaches. Thirty-one peroxisomal proteins were identified and ten putative peroxisomal proteins were suggested. The results indicate that peroxisomal proteins show a tissue-specific functional response to the aging process that is probably dependent on their differential regeneration capacity. Besides, alteration in the fatty acid metabolism could alter membrane protein functions; decrease in catalase expression in kidney may contribute to oxidative stress and isoprenoid biosynthesis could contribute to decline in bile salt synthesis. The ability to detect changes in the peroxisomal proteome associated with organ impairment during the course of aging would provide a conceptual framework to understand the role of peroxisome in aging.</p><p>In the second part, peroxisome proteomics was used as a novel approach in marine pollution assessment. The peroxisomal protein expression profiles were obtained and identified from mussel Mytilus sp. exposed to different pollutants, in both laboratory and field experiments. The identified proteins were involved in α- and β–oxidation pathways, xenobiotics and amino acid metabolism, cell signalling, oxyradical metabolism, peroxisomal assembly, respiration and cytoskeleton pathway, etc. Generally, these findings suggest that protein expression signatures could become a valuable tool to monitor the presence of pollutants in marine environment.</p>

<p>The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. </p><p>A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. </p><p>In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.</p>

Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex. In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs. The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.

Chromatin is a highly dynamic, regulatory component in the process of transcription, repair, recombination and replication. The BRG1 and SNF2H proteins are ATP-dependent chromatin remodeling proteins that modulate chromatin structure to regulate DNA accessibility for DNA-binding proteins involved in these processes. The BRG1 protein is a central ATPase of the SWI/SNF complexes involved in chromatin remodeling associated with regulation of transcription. SWI/SNF complexes are biochemically hetero-geneous but little is known about the unique functional characteristics of the various forms. We have shown that SWI/SNF activity in SW13 cells affects actin filament organization dependent on the RhoA signaling pathway. We have further shown that the biochemical composition of SWI/SNF complexes qualitatively affects the remodeling activity and that the composition of biochemically purified SWI/SNF complexes does not reflect the patterns of chromatin binding of individual subunits. Chromatin binding assays (ChIP) reveal variations among subunits believed to be constitutive, suggesting that the plasticity in SWI/SNF complex composition is greater than suspected. We have also discovered an interaction between BRG1 and the splicing factor Prp8, linking SWI/SNF activity to mRNA processing. We propose a model whereby parts of the biochemical heterogeneity is a result of function and that the local chromatin environment to which the complex is recruited affect SWI/SNF composition. We have also isolated the novel B-WICH complex that contains WSTF, SNF2H, the splicing factor SAP155, the RNA helicase II/Guα, the transcription factor Myb-binding protein 1a, the transcription factor/DNA repair protein CSB and the RNA processing factor DEK. The formation of this complex is dependent on active transcription and links chromatin remodeling by SNF2H to RNA processing. By linking chromatin remodeling complexes with RNA processing proteins our work has begun to build a bridge between chromatin and RNA, suggesting that factors in chromatin associated assemblies translocate onto the growing nascent RNA.

Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is N-sulfated by the enzyme N-deacetylase N-sulfotransferase (NDST). N-Sulfation occurs in domains where further modifications (including O-sulfations) take place, giving the chain a complex sulfation pattern. In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that N-deacetylation is the rate-limiting step in HS N-sulfation and that two different NDST molecules can work on the same GlcN unit. By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for N-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on N-deacetylase activity. We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones. .

Peroxisome is a ubiquitous eukaryotic organelle with a single-layer membrane. It maintains various functions that differ depending on the species and cell types, as well as the environmental or developmental conditions. In the first part of this thesis, the peroxisomal protein content was systematically analyzed in different organs in mouse from different ages using proteomic approaches. Thirty-one peroxisomal proteins were identified and ten putative peroxisomal proteins were suggested. The results indicate that peroxisomal proteins show a tissue-specific functional response to the aging process that is probably dependent on their differential regeneration capacity. Besides, alteration in the fatty acid metabolism could alter membrane protein functions; decrease in catalase expression in kidney may contribute to oxidative stress and isoprenoid biosynthesis could contribute to decline in bile salt synthesis. The ability to detect changes in the peroxisomal proteome associated with organ impairment during the course of aging would provide a conceptual framework to understand the role of peroxisome in aging. In the second part, peroxisome proteomics was used as a novel approach in marine pollution assessment. The peroxisomal protein expression profiles were obtained and identified from mussel Mytilus sp. exposed to different pollutants, in both laboratory and field experiments. The identified proteins were involved in α- and β–oxidation pathways, xenobiotics and amino acid metabolism, cell signalling, oxyradical metabolism, peroxisomal assembly, respiration and cytoskeleton pathway, etc. Generally, these findings suggest that protein expression signatures could become a valuable tool to monitor the presence of pollutants in marine environment.

The Ras superfamily of GTPases embraces six major branches of proteins: the Ras, Rab, Ran, Arf, Rho and Miro subfamilies. The majority of GTPases function as binary switches that cycle between active GTP-bound and inactive GDP-bound states. This thesis will focus primarily on the biological functions of the Rho and Miro proteins. The Rho GTPases control the organization of the actin cytoskeleton and other associated activities, whereas the Miro GTPases are regulators of mitochondrial movement and morphology. A diverse array of cellular phenomena, including cell movement and intracellular membrane trafficking events, are dependent on cytoskeletal rearrangements mediated by Rho GTPases. Although human Rho GTPases are encoded by 20 distinct genes, most studies involving Rho GTPases have focused on the three representatives RhoA, Rac1 and Cdc42, which each regulate specific actin-dependent cellular processes. In an effort to compare the effects of all Rho GTPase members in the same cell system, we transfected constitutively active Rho GTPases in porcine aortic endothelial (PAE) cells and examined their effects on the organization of the actin cytoskeleton. We identified a number of previously undetected roles of the different members of the Rho GTPases. Moreover, we demonstrated that the downstream effectors of Rho GTPases have a broader specificity than previously thought. In a screen for novel Ras-like GTPases, we identified the Miro GTPases (Mitochondrial Rho). In our characterization of Miro, we established that these proteins influence mitochondrial morphology and serve functions in the transport of mitochondria along the microtubule system. Additionally, we provided evidence that Miro can be under control of calcium signaling pathways. Mitochondria are highly dynamic organelles that undergo continuous change in shape and distribution. Defects in mitochondrial dynamics are associated with several neurodegenerative diseases. In conclusion, our findings have contributed to a deeper understanding of the biological roles of Rho and Miro GTPases.

Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3’ end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5’ end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using site-directed mutagenesis and fluorescence spectroscopy. We identified tryptophan 475, located within the RNA recognition motif (RRM) of PARN, as crucial for cap binding. A crystal structure of PARN bound to cap revealed that cap binding is mediated by the nuclease domain and the RRM of PARN. Tryptophan 475 binds the inverted 7-Me-guanosine residue through a stacking interaction. Involvement of the nuclease domain in cap binding suggests that the cap site and the active site overlap. Mutational analysis showed that indeed amino acids involved in cap binding are crucial for hydrolytic activity of PARN. Taken together, we show that PARN is an allosteric enzyme that is activated by the cap structure and that the allosteric cap binding site in one PARN subunit corresponds to the active site in the other PARN subunit.

Heparan sulfate proteoglycans are proteins with long, unbranched heparan sulfate (HS) polysaccharide chains attached to them. They are found on cell surfaces and in basement membranes where they exert their action by interacting with a wide range of enzymes and signaling molecules and are thereby involved in a range of various processes both during embryonic development and in adult physiology. A great part of the biological functionality of proteoglycans can be directly related to the polysaccharide part. HS chains display very variable sulfation patterns where highly sulfated regions are responsible for a large part of the biological activity. The biosynthesis of HS is a complex process in which a number of enzymes are involved. Better comprehension of how this process is regulated could reveal clues to how formation of HS sulfation patterns occurs, and thereby how HS functionality is controlled. This thesis is focusing on regulation of one of the enzymes responsible for HS sulfation, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), in an attempt to understand these mechanisms better. Different aspects of NDST regulation were studied in three projects: I) “Heparin/heparan sulfate biosynthesis: Processive formation of N-sulfated domains”, where the sulfate donor PAPS is shown to influence the manner in which NDST modifies the substrate, affecting the domain structure of the polysaccharide. II) “Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant”, where a splice variant of NDST1 which appears to influence NDST1 protein levels and affect HS structure is described. III) “Heparan sulfate biosynthesis in zebrafish: Five NDST genes with distinct expression patterns during embryonic development”, in which five zebrafish NDSTs were cloned and shown to be expressed in a temporally and spatially regulated manner.