<p>Amyloidoses represent a heterogeneous group of diseases characterized by abnormal protein metabolism leading to extracellular deposition of fibrillar, proteinaceous amyloid in various tissues and organs of the body. To date more than 20 different proteins have been linked to diseases with amyloid depositions, of which Alzheimer’s disease and the prion-associated diseases are the most well known. Despite the origin of protein in the amyloid, the fibrils share some common biochemical and biophysical properties such as a diameter of 8-13 nm, a β-pleated sheet secondary structure packed in an ordered crystal-like way, Congo red and thioflavin binding with characteristic spectroscopic patterns and decoration of the fibrils with Serum amyloid P component and glycoseaminoglycans. </p><p>The plasma protein transthyretin (TTR) is associated with familial amyloidosis with polyneuropathy (FAP) and senile systemic amyloidosis (SSA). FAP is a lethal, autosomal inherited disorder caused by point mutations in the TTR-gene. More than 80 different mutations have been associated with amyloid formation and linked to FAP. The interpretation is that amino acid replacements at different sites of the polypeptide lead to reduced stability. Mutant TTR were constructed that have a strong tendency to self-aggregate under physiological conditions. The precipitates were shown to be amyloid by staining with thioflavin T and Congo red. As the mutants were sensitive to trypsin cleavage compared to plasma TTR, we suggest that the mutants represent amyloid precursors or that they may share structural properties with intermediates on a pathway leading to amyloid deposition. Monoclonal antibodies were generated that exclusively recognize the amyloidogenic folding of TTR providing direct biochemical evidence for a structural change in amyloidogenic intermediates. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and is proposed to be displaced at the initial phase of amyloid formation. Amyloidogenic intermediates of TTR were shown to induce a toxic, free radical dependent, response in cultured neuroblastoma cells. Morphological studies revealed a correlation between toxicity (apoptosis) and the presence of immature amyloid suggesting that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.</p><p>Serum amyloid P component (SAP) is a highly conserved plasma glycoprotein universally found associated with amyloid depositions independently of protein origin. SAP’s role in amyloid formation is contradictory since both inhibition and promotion of aggregation have been shown in the case of fibril formation from the Aβ peptide of Alzheimer’s disease. Amyloidogenic prefibrils of TTR were shown to bind SAP and no interference with aggregation was detected. SAP co-localize in patches with mutant TTR on the surface of neuroblastoma cells and prevent apoptosis induced by mutant TTR and Aβ peptide, while several other molecules known to decorate amyloid fibrils were without effect. </p>

<p>Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for mesenchymal cells and exert its effect by binding to two structurally related receptor tyrosine kinases, denoted α- and β-receptors. PDGF binding induces dimerization of its receptors, both homo-and heterodimerization, leading to their autophosphorylation on tyrosine residues and binding of downstream signaling molecules. This thesis describes autophosphorylation and binding of signal transduction molecules to homo- and heterodimeric PDGF receptor complexes.</p><p> Heterodimeric PDGF receptor complexes have been found to mediate a stronger mitogenic response than homodimeric receptor complexes. It was found that Tyr771 in the PDGF β-receptor was significantly less phosphorylated in the heterodimeric β-receptor compared to the homodimeric receptor, and this correlated with reduced binding of GTPase activating protein (GAP) for Ras and decreased activation of the Ras/Mitogen activated protein kinase pathway. </p><p> The mechanism behind the lowered phosphorylation of Tyr771 in the heterodimeric PDGF β-receptor was investigated. It was found that the SH2 domain-containing tyrosine phosphatase SHP-2 was responsible, at least in part, for the dephosphorylation of Tyr771 in the heterodimeric β-receptor. </p><p> PDGF-induced autophosphorylation of tyrosine residues in the receptors has been proposed to occur <i>in trans </i>between the receptor molecules in the dimers. We demonstrated by phosphopeptide mapping that all major autophosphorylation sites can be phosphorylated <i>in trans, </i>both in the PDGF α- and β-receptors. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen activated protein kinase revealed that the signaling capacities were retained. This illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signaling molecules.</p><p> Elucidating the mechanisms behind the unique signaling properties of the heterodimeric PDGF receptor complex, two heterodimer-specific autophosphorylation sites, Tyr692 and Tyr970, were identified and found to interact with the low molecular weight protein tyrosine phosphatase (LMW-PTP). Mutation of Tyr692 or Tyr970 to phenylalanine residues did not affect PDGF-induced mitogenicity, but the Tyr692 to phenylalanine mutation reduced the chemotactic response mediated by the heterodimeric PDGF receptor complex. A mechanism for the lowered chemotactic response was found to involve an increased RasGAP binding and a decreased SHP-2 binding to the heterodimeric β-receptor.</p>

<p>In my thesis, the cell cycle analysis of archaea and hyperthermophilic organisms is presented for the first time. Crenarchaea from the genus <i>Sulfolobus</i> were used as a model system. Plow cytometry and light microscopy were applied to investigate the timing and coordination of different cell cycle events. Furthermore, DNA content, nucleoid structure, and nucleoid distribution at different stages during the cell cycle were studied. The <i>Sulfolobus</i> cell cycle was characterized as having a short pre-replication and a long post-replication period. The presence of a low proportion of cells with segregated genomes in the exponentially growing population suggested 3 considerable time delay between termination of chromosome replication and completion of nucleoid partition, reminiscent of the G₂period in eukaryotic cells.</p><p>The first available collection of conditional-lethal mutants of any archaeon or hyperthemophile was used to elucidate the coordination of cell cycle events. The studies showed that chromosome replication, nucleoid partition and cell division in <i>Sulfolobus</i> <i>acidocaldarius</i>, which are normally tightly coordinated during cellular growth, could be separately inhibited or uncoupled by mutation.</p><p>The <i>ftsZ</i> gene, which is involved in cell division in bacteria and euryarchaea, was isolated from the halophilic archaeon <i>Haloferax mediterranei</i>. Transcriptional start sites were mapped, and putative translation initiation elements were identified. In both the upstream and downstream regions of the <i>ftsZ</i> gene, open reading frames were found to be conserved within the genus <i>Haloferax</i>. Furthermore, at the 3' end of the <i>ftsZ</i> gene, the homologs of the bacterial <i>secE</i> and <i>nusG</i> genes are conserved in almost all euryarchaea analyzed so far. The studies also demonstrated the functional conservation of the FtsZ protein in different archaeal species, as well as between euryarchaea and bacteria.</p>

<p>Integrins are heterodimeric cell surface receptors, composed of an α- and a β-subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and cooperate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins α2β1 and α11β1 were studied. In addition, the impact of β1 cytoplasmic tyrosines in collagen-induced signalling was analyzed.</p><p>The integrin α11β1 is the latest identified collagen-binding integrin. In this study, tissue distribution of α11 mRNA and protein during embryonal development was explored, and the first α11β1-mediated cellular functions were established. Both α11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. α11 protein was also detected in cornea keratinocytes. α11β1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, α11β1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated α11β1-mediated contraction and directed migration.</p><p>Expression of β1Y783,795F in β1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected α2β1-mediated functions in response to collagen. The β1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar collagen, but not in collagen gels. PDGF-BB stimulated contraction via αvβ3, which also induced activation of Cas in collagen gels. The YY-FF mutation also abolished β1A-dependent downregulation of β3.</p><p>In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins αvβ3 mediated contraction. Clustering of β1-integrins by antibodies and PDGF-BB stimulated αvβ3-mediated contraction in an ERK-dependent way. Expression of α2β1, but not α11β1, prevented αvβ3-mediated contraction. Contraction by α2β1 and α11β1 was ERK-independent.</p>

<p>Transforming growth factor-β (TGF-β) superfamily members are multi-functional regulators of cell fate. These factors signal by binding to a limited number of highly conserved transmembrane type I and type II serine/threonine kinase receptors. These receptors initiate signals into the cell via the Smad proteins. Up to date, 8 different mammalian Smads are reported and are divided into three subgroups; receptor regulated Smads (R-Smads), common mediator Smads (Co-Smads) and inhibitory Smads (I-Smads). This thesis investigates the function and regulation of TGF-β/Smad signaling through identification and characterization of Smad interacting proteins.</p><p>I-Smads, i.e. Smad6 and Smad7, are potent antagonists of the TGF-β superfamily signaling. We found that Smad7, but not Smad6, inhibits TGF-β1-induced growth inhibition and expression of immediate early response genes. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-β receptor activation. Moreover, we found that the MH2 domain is important for nuclear export.</p><p>To investigate further the role of inhibitory Smads, we have identified AMSH as a Smad6 interacting protein using a yeast two-hybrid screening method. AMSH was previously discovered as the associated molecule with the SH3 domain of STAM. AMSH interacts with I-Smads, but not with R- and Co-Smads upon receptor activation and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. AMSH was found to prevent Smad6 from binding to activated type I receptors and/or activated R-Smads. Smad anchor for receptor activation (SARA) is critical for Smad2 and Smad3 activation by TGF-β receptors. The present studies show that the localization of SARA in early endosomes is regulated through its FYVE domain. We have found that the FYVE domain of SARA is sufficient and necessary for the early endosomal localization, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-β/Smad signaling.</p><p>Both Notch and BMP signaling pathways are important for vascular development. We have found that Herp2, which is originally known as one of the Notch target genes, is synergistically induced upon activation of Notch and BMP signaling pathways in endothelial cells (ECs). The critical elements for synergistical activation of Herp2 gene by BMP and Notch pathway were identified. Furthermore, the Notch intracellular domain interacts with Smad5 upon BMP receptor and this interaction becomes stronger in the presence of pCAF. Interestingly, Herp2 was found to antagonize BMP receptor- or Id-mediated EC migration.</p>

<p>Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed that numerous endocytic regulatory proteins, among them ASAP1 and Dab2, interact with CIN85 via their PxxxPR motifs. The SH3 domains of CIN85 are able to cluster and exchange its effectors at subsequent stages of EGFR endocytosis, thus participating in the control of receptor internalization, recycling and degradation in the lysosome. We proposed that CIN85 functions as a scaffold molecule implicated in control of multiple steps in downregulation of RTKs. </p><p>Furthermore, we identified two novel Cbl- and ubiquitin-interacting adaptor proteins named Sts-1 and Sts-2 (Suppressors of T-cell receptor signaling). Ligand-induced and Cbl-mediated recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization and subsequent block of receptor degradation followed by prolonged mitogenic signaling pathways. Our results indicate that Sts-1 and Sts-2 represent a new class of negative regulators of Cbl functions in receptor endocytosis. </p><p>In conclusion, this thesis describes novel mechanisms by which Cbl, coupled to its effectors, orchestrates trafficking of RTKs. Detailed understanding of how these processes are controlled under physiological as well as under pathological conditions may be important for future therapeutic approaches.</p>

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for mesenchymal cells and exert its effect by binding to two structurally related receptor tyrosine kinases, denoted α- and β-receptors. PDGF binding induces dimerization of its receptors, both homo-and heterodimerization, leading to their autophosphorylation on tyrosine residues and binding of downstream signaling molecules. This thesis describes autophosphorylation and binding of signal transduction molecules to homo- and heterodimeric PDGF receptor complexes. Heterodimeric PDGF receptor complexes have been found to mediate a stronger mitogenic response than homodimeric receptor complexes. It was found that Tyr771 in the PDGF β-receptor was significantly less phosphorylated in the heterodimeric β-receptor compared to the homodimeric receptor, and this correlated with reduced binding of GTPase activating protein (GAP) for Ras and decreased activation of the Ras/Mitogen activated protein kinase pathway. The mechanism behind the lowered phosphorylation of Tyr771 in the heterodimeric PDGF β-receptor was investigated. It was found that the SH2 domain-containing tyrosine phosphatase SHP-2 was responsible, at least in part, for the dephosphorylation of Tyr771 in the heterodimeric β-receptor. PDGF-induced autophosphorylation of tyrosine residues in the receptors has been proposed to occur in trans between the receptor molecules in the dimers. We demonstrated by phosphopeptide mapping that all major autophosphorylation sites can be phosphorylated in trans, both in the PDGF α- and β-receptors. Analyses of the abilities of heterodimeric receptor complexes of one kinase-active and one kinase-inactive receptor to mediate mitogenicity, chemotaxis and activation of mitogen activated protein kinase revealed that the signaling capacities were retained. This illustrates a functional co-operation between the two receptor molecules in the dimer, where one receptor provides a functional kinase and the other acts as a substrate and provides docking sites for downstream signaling molecules. Elucidating the mechanisms behind the unique signaling properties of the heterodimeric PDGF receptor complex, two heterodimer-specific autophosphorylation sites, Tyr692 and Tyr970, were identified and found to interact with the low molecular weight protein tyrosine phosphatase (LMW-PTP). Mutation of Tyr692 or Tyr970 to phenylalanine residues did not affect PDGF-induced mitogenicity, but the Tyr692 to phenylalanine mutation reduced the chemotactic response mediated by the heterodimeric PDGF receptor complex. A mechanism for the lowered chemotactic response was found to involve an increased RasGAP binding and a decreased SHP-2 binding to the heterodimeric β-receptor.

In my thesis, the cell cycle analysis of archaea and hyperthermophilic organisms is presented for the first time. Crenarchaea from the genus Sulfolobus were used as a model system. Plow cytometry and light microscopy were applied to investigate the timing and coordination of different cell cycle events. Furthermore, DNA content, nucleoid structure, and nucleoid distribution at different stages during the cell cycle were studied. The Sulfolobus cell cycle was characterized as having a short pre-replication and a long post-replication period. The presence of a low proportion of cells with segregated genomes in the exponentially growing population suggested 3 considerable time delay between termination of chromosome replication and completion of nucleoid partition, reminiscent of the G2 period in eukaryotic cells. The first available collection of conditional-lethal mutants of any archaeon or hyperthemophile was used to elucidate the coordination of cell cycle events. The studies showed that chromosome replication, nucleoid partition and cell division in Sulfolobus acidocaldarius, which are normally tightly coordinated during cellular growth, could be separately inhibited or uncoupled by mutation. The ftsZ gene, which is involved in cell division in bacteria and euryarchaea, was isolated from the halophilic archaeon Haloferax mediterranei. Transcriptional start sites were mapped, and putative translation initiation elements were identified. In both the upstream and downstream regions of the ftsZ gene, open reading frames were found to be conserved within the genus Haloferax. Furthermore, at the 3' end of the ftsZ gene, the homologs of the bacterial secE and nusG genes are conserved in almost all euryarchaea analyzed so far. The studies also demonstrated the functional conservation of the FtsZ protein in different archaeal species, as well as between euryarchaea and bacteria.

Integrins are heterodimeric cell surface receptors, composed of an α- and a β-subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and cooperate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins α2β1 and α11β1 were studied. In addition, the impact of β1 cytoplasmic tyrosines in collagen-induced signalling was analyzed. The integrin α11β1 is the latest identified collagen-binding integrin. In this study, tissue distribution of α11 mRNA and protein during embryonal development was explored, and the first α11β1-mediated cellular functions were established. Both α11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. α11 protein was also detected in cornea keratinocytes. α11β1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, α11β1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated α11β1-mediated contraction and directed migration. Expression of β1Y783,795F in β1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected α2β1-mediated functions in response to collagen. The β1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar collagen, but not in collagen gels. PDGF-BB stimulated contraction via αvβ3, which also induced activation of Cas in collagen gels. The YY-FF mutation also abolished β1A-dependent downregulation of β3. In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins αvβ3 mediated contraction. Clustering of β1-integrins by antibodies and PDGF-BB stimulated αvβ3-mediated contraction in an ERK-dependent way. Expression of α2β1, but not α11β1, prevented αvβ3-mediated contraction. Contraction by α2β1 and α11β1 was ERK-independent.

Transforming growth factor-β (TGF-β) superfamily members are multi-functional regulators of cell fate. These factors signal by binding to a limited number of highly conserved transmembrane type I and type II serine/threonine kinase receptors. These receptors initiate signals into the cell via the Smad proteins. Up to date, 8 different mammalian Smads are reported and are divided into three subgroups; receptor regulated Smads (R-Smads), common mediator Smads (Co-Smads) and inhibitory Smads (I-Smads). This thesis investigates the function and regulation of TGF-β/Smad signaling through identification and characterization of Smad interacting proteins. I-Smads, i.e. Smad6 and Smad7, are potent antagonists of the TGF-β superfamily signaling. We found that Smad7, but not Smad6, inhibits TGF-β1-induced growth inhibition and expression of immediate early response genes. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-β receptor activation. Moreover, we found that the MH2 domain is important for nuclear export. To investigate further the role of inhibitory Smads, we have identified AMSH as a Smad6 interacting protein using a yeast two-hybrid screening method. AMSH was previously discovered as the associated molecule with the SH3 domain of STAM. AMSH interacts with I-Smads, but not with R- and Co-Smads upon receptor activation and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. AMSH was found to prevent Smad6 from binding to activated type I receptors and/or activated R-Smads. Smad anchor for receptor activation (SARA) is critical for Smad2 and Smad3 activation by TGF-β receptors. The present studies show that the localization of SARA in early endosomes is regulated through its FYVE domain. We have found that the FYVE domain of SARA is sufficient and necessary for the early endosomal localization, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-β/Smad signaling. Both Notch and BMP signaling pathways are important for vascular development. We have found that Herp2, which is originally known as one of the Notch target genes, is synergistically induced upon activation of Notch and BMP signaling pathways in endothelial cells (ECs). The critical elements for synergistical activation of Herp2 gene by BMP and Notch pathway were identified. Furthermore, the Notch intracellular domain interacts with Smad5 upon BMP receptor and this interaction becomes stronger in the presence of pCAF. Interestingly, Herp2 was found to antagonize BMP receptor- or Id-mediated EC migration.