Inflammasomes are multiprotein complexes that play a critical role in the regulation of inflammation and the inflammatory responses against pathogens. NLRP3 (NOD-, LRR-and pyrin domain-containing protein 3) is among the molecules involved in the homonymous and well-studied NLRP3 inflammasome that accounts for the release of the pro-inflammatory cytokines interleukin (IL) 1-β and 18. Two signals (priming and activation) that include molecular and cellular events lead to the activation of the complex; the main events involved during the activation phase are ionic fluxes, mitochondrial dysfunction, and lysosomal damage. Calcium mobilisation belongs to the signalling events of ionic fluxes associated with the complex assembly initiation. Although no consensus has been established regarding the ionic Ca2+ fluxes and the exact mechanisms contributing to NLRP3 activation, several sources agree that Ca2+ mobilisation homeostasis is essential for the canonical function of the NLRP3 inflammasome, and other cellular processes associated with it. This systematic review aimed to determine the factors and mechanisms related to Ca2+ mobilisation contributing to inflammasome activation, examine NLRP3-associated pathologies, and propose potential therapeutic targets. The literature sources found were evaluated using the CASP tool. The obtained information revealed an intertwined relation of Ca2+ flux with the calcium-sensing receptor, reactive oxygen species (ROS) generation, lysosome rupture, Ca2+-permeable channels and K+ efflux contributing to NLRP3 inflammasome activation. The summarised knowledge in this review has led to the proposal of future studies through references to different NLRP3-related diseases such as Alzheimer’s and diabetes type II, while potential therapeutic targets were also discussed.

Inflammation is a defensive process that allows immune cells to be mobilized to help with infection removal and tissue regeneration. Inflammasomes are multiprotein oligomers in the cytoplasm and components of the innate immune system that have a role in inflammation. Glycogen synthase kinase 3 (GSK3) is a critical molecule involved in a wide range of inflammatory reactions. It has been reported to suppress the production of pro-inflammatory mediators in response to LPS when it is inhibited. The aim of this project was to study the effect of GSK3 inhibition in a concentration-dependent manner on the production of IL-6 as well as ASC-speck formation in THP1 ASC GFP cells stimulated with LPS and activated using nigericin. Using the cell culture supernatant ELISA was performed to quantify the IL-6 protein secreted by THP-1 macrophages. Using reverse-transcribed cDNA, qPCR was performed to measure the IL-6 gene expression. Finally, live-cell imaging was done to visualize the ASC-speck formation. It was found that upon stimulation of THP-1 cells a remarkable increase in the production of IL-6 was observed, however, the inhibitor did not suppress the production of IL-6 as hypothesized. This could be primarily due to the presence of another NF-κB pathway which is not mediated by GSK3 and therefore could not be inhibited using the GKS3 inhibitor. Future studies could decrease the LPS concentration to see if the uninhibited pathway can be observed at lower stimulation. Another probable solution could be lowering the FBS percentage to avoid potential inhibition.

Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation. Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation. We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils. The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.

<p>Conformational changes in human proteins can induce several types of diseases. The nature of the conformational changes is largely unknown, but some lead to amyloid fibril formation. Amyloid fibrils accumulate in the extra-cellular space of tissues resulting in disruption of organ function. Transthyretin (TTR) is a plasma protein involved in three amyloid diseases, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The latter disease involves conformational changes in the wild-type structure of the protein, whereas the others are caused by a gene mutation. </p><p>Our goal is to increase the knowledge of why and how some proteins aggregate into amyloid fibrils by solving and analyzing structures of different TTR variants of which some can form amyloid fibrils, whereas others cannot. The crystal structures of wild-type TTR and many of its disease-causing mutants have previously been determined, and observed structural discrepancies between mutant and wild type were claimed to be of importance for amyloid formation. We performed a comparative analysis of all, at that point, known structures of TTR. As a reference for our study, we determined a 1.5 Å resolution structure of human wild-type TTR. We found that the previously reported structural differences between wild type and mutant TTR were insignificant and did not provide clues to the mechanism for amyloid formation.</p><p>We showed the double mutant TTR-Ala108Tyr/Leu110Glu to be less amyloidogenic than wild-type transthyretin. Since the structure of few non-amyloidogenic mutants are known, we solved its structure in two space groups, C2 and P21212, where the latter was consistent with most of the structures of transthyretin. Only the highly amyloidogenic mutant ATTR-Leu55Pro has previously been solved in C2. The packing of molecules in our C2 crystal was close-to-identical to the ATTR-Leu55Pro crystal structure, ruling out the described ATTR-Leu55Pro packing interactions as significant for amyloidosis. The C2 structure displayed a large shift in residues Leu55-Leu58, a structural change previously found only in amyloidogenic TTR variants. Combined with previous data, this suggests that transthyretin in solution contains a mixture of molecules with different conformations. This metastability of transthyretin provides insight to why some proteins aggregate into amyloid fibrils.</p><p>The natural ligand thyroxine has been shown to stabilize TTR. Small molecules, based on thyroxine, with the potential to serve as inhibitors for amyloid fibril formation are under development. Iodine is a component of thyroxine and we found that TTR also bound free iodide ions. Taking advantage of the anomalous scattering of iodide, we solved the iodide-bound TTR structure using the single-wavelength anomalous dispersion method. In addition, we determined the TTR-chloride structure. Both chloride and iodide stabilized transthyretin where iodide stabilized better. From the thyroxine-TTR structure, three halogen-binding pockets have been identified in each TTR monomer. We found three bound iodides per TTR monomer, two of which were in the thyroxine-binding channel. This indicates that only two of the three halogen-binding pockets in the thyroid-hormone binding channel are optimal for halogen binding. Our results might be useful for the continuing design of small molecule ligands, which in the end can lead to inhibitors for amyloid diseases.</p>

<p>Basement membranes are thin extracellular sheets that surround muscle, fat and peripheral nerve cells and underlay epithelial and endothelial cells. Laminins are one of the main protein families of these matrices. Integrins and dystroglycan are receptors for laminins, connecting cells to basement membranes. Each laminin consists of three different chains, (α, β, γ). Laminin-1 (α1β1γ1) was the first laminin to be found and is the most frequently studied. Despite this, it was unclear where its α1 chain was expressed. A restricted distribution of the α1 chain in the adult epithelial basement membranes was demonstrated in the present study. In contrast, dystroglycan was found to have a much broader distribution. Dystroglycan is an important receptor for α2-laminins in muscle, but binds also α1-laminins. The more ubiquitous α5-laminins were also shown to bind dystroglycan, but with distinctly lower affinity than α1- and α2- laminins. </p><p>The biological roles of different laminin isoforms have been investigated. Differences were found in the capacity of various tested laminins to promote epithelial cell adhesion. The α5-laminins were potent adhesive substrates, a property shown to be dependent on α3 and α6 integrins. Each receptor alone could promote efficient epithelial cell adhesion to α5-laminins. Yet, only α6 integrin and in particular the α6A cytoplasmic splice variant could be linked to laminin-mediated activation of a mitogen-activated protein kinase (MAP kinase) pathway. Attachment to either α1- or α5-laminins activated extracellular-signal regulated kinase (ERK) in cells expressing the integrin α6A variant, but not in cells expressing α6B. A new role for dystroglycan as a suppressor of this activation was demonstrated. Dystroglycan antibodies, or recombinant fragments with high affinity for dystroglycan, decreased ERK activation induced by integrin α6 antibodies. Integrin αvβ3 was identified as a novel co-receptor for α5-laminin trimers. Cell attachment to α5-laminins was found to facilitate growth factor induced cell proliferation. This proliferation could be blocked by antibodies against integrin αvβ3 or by an inhibitor of the MEK/ERK pathway. Therefore, integrin αvβ3 binding to α5-laminins could be of biological significance.</p>

<p>The formation and maintenance of tissues in multicellular animals are crucially dependent on cellular interactions with the extracellular matrix (ECM). Two different studies on such interactions are presented herein.</p><p>Studies on expression of laminins in normal and dystrophic skeletal muscle, clarified a much debated issue regarding discrepancies seen for laminin α1-chain expression between human and mouse tissues. Lack of laminin α1-chain expression was verified in both mouse and human skeletal muscle. Furthermore, the earlier discrepancies seen for laminin α1-chain expression was explained by showing that an antibody-reagent, commonly used in human studies, recognised the laminin α5-chain rather than the laminin α1-chain</p><p>The integrin α11-chain (forming α11β1 integrin) is the latest addition to the integrin receptor family, and belongs to the I domain-containing group of integrin α-chains. Previous studies had shown that α11β1 is a collagen receptor. In the present study, the <i>in vitro</i> and <i>in vivo</i> functions of the α11-chain were further characterised. Distribution studies on embryonic human and mouse tissues showed that the α11-chain was expressed on mesenchymal cells in the developing tendon, perichondrium, intervertebral disc, and cornea. The interactions of α11β1 integrin with collagen type I and IV were studied <i>in vitro</i>. The α11β1 bound to these collagens in a manner similar to integrin α2β1 (with collagen type I being the preferred ligand for α11β1). Furthermore, α11β1 was shown to mediate migration on collagen type I coated surfaces, and to mediate contraction of collagen type I gels. The <i>in vivo</i> functions of the α11-chain were investigated by the generation of integrin α11-chain null-mice, using gene targeted disruption of the itga11 in embryonic stem cells. Two independent lines of mice lacking α11 protein were generated. Phenotypic analysis of these mice indicated a role for α11β1 in the formation of the musculoskeletal system.</p>

<p>RNase P is an essential ribonuclease responsible for removal of the 5’ leader of tRNA precursors. Bacterial RNase P consists of an RNA subunit and a small basic protein. The catalytic activity is associated with the RNA subunit, i.e. bacterial RNase P RNA is a ribozyme. The protein subunit is, however, essential for activity in vivo. RNase P RNA, as well as the holoenzyme, requires the presence of divalent metal ions for activity. The aim of this thesis was to increase our understanding of the catalytic mechanism of RNase P RNA mediated cleavage. The importance of the nucleotides close to the cleavage site and the roles of divalent metal ions in RNase P RNA-catalyzed reaction were investigated. Escherichia coli RNase P RNA (M1 RNA) was used as a model system.</p><p>It was shown that different metal ions have differential effects on cleavage site recognition. Cleavage activity was rescued by mixing metal ions that do not promote cleavage activity by themselves. This suggests that efficient and correct cleavage is the result of metal ion cooperativity in the RNase P RNA-mediated cleavage reaction. The results suggested that one of the metal ions involved in this cooperativity is positioned in the vicinity of a well-known interaction between RNase P RNA and its substrate. Based on my studies on how different metal ions bind to RNA and influence its activity we raise the interesting possibility that the activity of biocatalysts that depend on RNA for activity are up- or downregulated depending on the intracellular concentrations of the bulk biological metal ions Mg²⁺ and Ca²⁺.</p><p>The nucleotides upstream of the cleavage site in the substrate were found to influence the cleavage efficiency. This was not exclusively due to intermolecular base pairing within the substrate but also dependent on the identities of the nucleotides at position –2 and –1. The strength of the base pair at position –1/+73 was demonstrated to affect cleavage efficiency. These observations are in keeping with previous suggestion that the nucleotides close to the cleavage site are important for RNase P cleavage. We conclude that the residue at -1 is a positive determinant for cleavage by RNase P. Hence, my studies extend our understanding of the RNase P cleavage site recognition process.</p>

<p>Microorganisms that degrade lignocellulose play an important role in maintaining the global carbon cycle. Under cellulolytic conditions, the fungus <i>Phanerochaete chrysosporium</i> produces an extracellular flavocytochrome, cellobiose dehydrogenase (CDH), with a proposed role in lignocellulose degradation. CDH consists of 755 amino acids including a C-terminal flavodehydrogenase linked by a peptide hinge to an N-terminal <i>b</i>-type cytochrome. The enzyme catalyses the oxidation of cellobiose to cellobiono-1,5-lactone, followed by transfer of electrons to an electron acceptor, either directly by the flavodehydrogenase domain, or via the cytochrome domain. This thesis presents a structural study on the individual domains of <i>P. chrysosporium</i> cellobiose dehydrogenase.</p><p>The crystal structure of the cytochrome was determined at 1.9 Å resolution. It folds as a β-sandwich with the topology of the antibody Fab V(H) domain, and the haem iron is ligated by Met65 and His163. This is only the second example of a <i>b</i>-type cytochrome with this ligation. The haem propionates are surface exposed to facilitate interdomain electron transfer.</p><p>The structure of a cytochrome Met65His mutant was determined at 1.9 Å resolution. In the mutant, the iron is ligated by the histidyl δ and ε nitrogens, rather than the usual N-ε/N-εligation. This is the first example of a <i>bis</i>-His N-ε/N-δ coordinated protoporphyrin IX iron. The structure of the flavoprotein domain was determined at 1.5 Å resolution. It is partitioned into an FAD-binding subdomain of α/β-type and a substrate-binding subdomain consisting of a seven-stranded β-sheet and six α-helices. Furthermore, the structure of the flavoprotein with the inhibitor cellobiono-1,5-lactam at 1.8 Å resolution lends support to a hydride-transfer mechanism for the reductive-half reaction of CDH although a radical mechanism cannot be excluded.</p>

<p>Epithelial cell polarisation is a prerequisite for the branching morphogenesis in several organs. Differential screening techniques were used to identify genes, which are upregulated during induction of epithelium in early kidney development. This investigation revealed two separate genes, Nuclear localising protein 1 (Nulp1), a previously undescribed gene with sequence characteristics of the basic helix-loop-helix transcription factor family, and glycoprotein 210 (gp210, POM210), an integral membrane protein constituent of the nuclear pore complex (NPC). Of these, gp210 was found to be upreglated during conversion of mesenchyme to epithelium. </p><p>The nuclear envelope, which demarcates the nuclear region in the eukaryotic cell, consists of an inner and an outer membrane that are fused at the locations for NPCs. These large macromolecular assemblages are tube like structures connecting the cytoplasmic and nuclear compartments of the cell. NPCs serve as the only conduits for exchange of molecular information between these cellular rooms. Electron microscopy techniques have revealed detailed information about the NPC architecture. A number of proteins (nucleoporins) have been characterised and embodied as components of the NPC structure. Active, energy dependent nucleocytoplasmic transport of RNAs and proteins is mediated by a group of soluble receptor proteins, collectively termed karyopherins. </p><p>Gp210 has been suggested to be important for nuclear pore formation. Nevertheless, our analyses showed a limited expression pattern of gp210, with its mRNA and protein largely confined to epithelial cells in the mouse embryo. Furthermore, in several cell lines, gp210 was undetectable. The expression pattern of gp210 was not synchronised with some other nucleoporins, indicating NPC heterogeneity. Characterisation of the structure of the human gp210 gene, including its promoter region, gave insight about possible cell-type specific gene regulatory mechanisms. </p><p>Regulation of molecular traffic between the nucleus and the cytoplasm leads to transcriptional control. Cell specific configuration of the NPC structure, due to diffential expression of gp210, could be involved in this control. Gp210 could be of importance for the development of epithelial cell polarisation.</p>

<p>x</p> / <p>Platelet-derived growth factors (PDGF), a family of potent mitogens and chemoattractants for cells of mesenchymal origin, elicit their biological effects through the binding of two related receptor tyrosine kinases, denoted α- and β-receptors. The binding of PDGF to the receptors causes receptor dimerization and autophosphorylation on tyrosine residues. Src homology 2 (SH2) domain-containing proteins then bind the phosphorylated receptors, mediating further propagation of the signal. This thesis describes how the interaction between the PDGF receptors and some of their downstream targets can modify the cellular response to PDGF.</p><p>The tyrosine phosphatase SHP-2 has been implicated in activation of the Ras/MAPK pathway downstream of several receptor tyrosine kinases. We found that SHP-2 binds to phosphorylated Y763 in the PDGF β-receptor, in addition to the already reported binding to Y1009. Cells expressing PDGF β-receptors with Y763 and Y1009 mutated to phenylalanine exhibited decreased Ras-GTP loading and reduced activation of Erk2 in response to PDGF. Whereas these cells did not show any change in the mitogenic response to PDGF, the PDGF-induced chemotaxis was significantly reduced in cells expressing mutant compared to wild-type receptor.</p><p>The phosphorylation of Y771 of the PDGF β-receptor had been shown to be significantly lower in the αβ-heterodimeric receptor compared to in the ββ-homodimer, causing reduced binding of RasGAP to the heterodimer and increased Ras/MAPK activation. We could demonstrate that the reduced phosphorylation of Y771 is due to dephosphorylation by tyrosine phosphatases, including SHP-2.</p><p>SHP-2 had been shown to associate with the docking protein Gab1 after growth factor stimulation. We showed that the adaptor protein Grb2 was required for PDGF mediated phosphorylation of Gab1, and that phosphorylated Gab1, Grb2 and SHP-2 create a complex upon PDGF stimulation. Using a cell system with an inducible Gab1 expression, we further demonstrated that Gab1 increased SHP-2 activity in response to PDGF, without affecting the interaction between SHP-2 and the b-receptor. Induction of Gab1 correlated with an increase in both PDGF-induced Erk and p38 MAPK activation, whereas Akt activation was unaffected. The latter finding was in line with our observation that PDGF had no effect on the interaction between Gab1 and p85 of PI3’-kinase. The increase in MAPK activity after Gab1 induction and PDGF treatment did not correlate with an increase in PDGF-induced mitogenicity; instead these cells displayed more pronounced actin reorganization in response to PDGF.</p><p>In conclusion, our data indicate that SHP-2 regulates the PDGF response both through direct dephosphorylation of the receptor and through its interaction with Gab1. PDGF stimulated activation of SHP-2 seems to be correlated not only with mitogenesis, but also with reorganization of the actin cytoskeleton and cell migration.</p>