No description available.

Abstract Collagen XIII belongs to the subgroup of collagenous transmembrane proteins. It has a wide tissue distribution and has been localized to many sites of cell-matrix and cell-cell interaction in tissues. Biochemical and in silico analyses of collagen XIII and other collagenous transmembrane proteins revealed that the biosynthesis of this structurally varied group is characterized by a coiled-coil motif following the transmembrane domain, and these trimerization domains appear to be associated with each of the collagenous domains. The collagen XIII trimer was shown to have an interchain disulfide bond at the junction of the NC1 and COL1 domains, and several other collagenous transmembrane proteins have a pair of cysteines in the same location. Furthermore, furin cleavage at the NC1 domain can be expected in most of the proteins. Mice heterozygous for the Col13a1del transgene, encoding a mutant collagen XIII, developed clonal mature B-cell lineage lymphomas originating in the mesenteric lymph node (MLN). The incidence of disease in conventionally reared mice was 2-fold higher than for mice raised in a specific pathogen-free facility. The lymphomas often associated with large populations of macrophages and T cells. Lymphomas expressed little if any collagen XIII, suggesting that the effect of the mutation was B-cell extrinsic and likely to be associated with collagen XIII-positive tissues drained by the MLN. Studies of the small intestines of transgenic mice showed highly abnormal subepithelial basement membranes (BM), associated with heightened expression of genes involved in immune responses. These findings suggest that collagen XIII-dependent maintenance of the intestinal BM is a critical determinant of cancer susceptibility. Collagen XIII exhibited a wide tissue distribution at the protein level, and the most intense expression was found in lung. Tissues contained 1-4 collagen XIII polypeptides, their size ranging between 78 and 102 kDa. Collagen XIII staining was detected in a restricted set of blood vessels in the liver, pancreas, adrenal gland, epididymis and brain. Moreover, Col13a1del transgene expression in the absence of endogenous collagen XIII proved to be deleterious for mouse embryonal development, leading to early fetal mortality.

No description available.

The nuclear envelope forms the interface between the nucleus and the cytoplasm. The nuclear envelope consists of the two concentric lipid membranes, the nuclear pores and the nuclear lamina. The inner nuclear membrane contains hundreds of unique transmembrane proteins showing high tissue diversity. Mutations of some proteins in the nuclear envelope give rise to a broad spectrum of diseases called envelopathies or laminopathies. In this thesis, I aimed to study the functional organization of the nuclear envelope by identifying and characterizing interactions between the nuclear envelope proteins. For this, we developed a novel method called the Membrane Protein Crosslink Immuno-Precipitation, which enable identification of protein-protein interactions in the nuclear envelope in live cells. We identified several novel interactions of the inner nuclear membrane protein, Samp1, and studied the interaction between the Samp1 and the nuclear GTPase, Ran in detail. Samp1 can bind to Ran and is thus the first known transmembrane Ran binding protein and Samp1 might provide a local binding site for Ran in the inner nuclear membrane. We found that Samp1 also binds to the inner nuclear membrane protein, Emerin and Ran can regulate the Samp1-Emerin interaction in the nuclear envelope. During mitosis, Samp1 distributes in the mitotic spindle. Therefore, we investigated a possible functional role of Samp1 in the mitotic machinery. Samp1 depletion resulted in aneuploid phenotypes, metaphase prolongation and decreased distribution of γ-tubulin and β-tubulin in the mitotic spindle. We found that Samp1 can bind to γ-tubulin, which is essential for the microtubule nucleation and hence for the spindle stability. The new interesting features of Samp1 provide insights on the unforeseen functions of the nuclear envelope proteins. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Unfolded protein response (UPR) is a complex cellular mechanism induced upon ER stress caused by various environmental factors. Single spanning signal transducers of UPR were reported to recognise also lipid-induced ER stress. Studies of these transducers, namely PERK and IRE1 uncovered that they can sense change in membrane properties and activate themselves by clustering. Moreover, signal transducer IRE1 retained ability to sense changes in the membrane properties with TMD exchanged for a polyLeu α-helix. It was thus unclear what mechanism drives lipid-induced UPR via IRE1. We employed model membrane system in form of LUVs, where properties of membranes can be readily altered by specific lipid composition. As a simplified model of the UPR signal transducers in the ER, synthetic transmembrane peptides with polyLeu core were used. Dynamic light scattering (DLS) has been used for qualitative and semi-quantitative analysis of LUVs. Clustering of synthetic peptides was determined by time resolved anisotropy of fluorescence. DLS results demonstrate successful formation of vesicles with a desired size in all planned composition. On the contrary to the studies in living cells, the presence of cholesterol or palmitic acid in model membranes did not induce the aggregation of transmembrane peptides....

Wortmannin is a potent inhibitor of phosphatidylinositol 3-kinase (PI3K) and membrane trafficking in many cells. To test the hypothesis that cystic fibrosis transmembrane conductance regulator (CFTR) traffics into and out of the plasma membrane during cAMP-stimulated epithelial Cl- secretion, we have studied the effects of wortmannin on forskolin-stimulated Cl- secretion by the human colonic cell line T84. At the PI3K inhibitory concentration of 100 nM, wortmannin did not affect significantly forskolin-stimulated Cl- secretion measured as short-circuit current (I(SC)). However, 500 nM wortmannin significantly inhibited forskolin-stimulated I(SC). cAMP activation of apical membrane CFTR Cl- channels in α-toxin-permeabilized monolayers was not reduced by 500 nM wortmannin, suggesting that inhibition of other transporters accounts for the observed reduction in T84 Cl- secretion. Forskolin inhibits apical endocytosis of horseradish peroxidase (HRP), but wortmannin did not alter forskolin inhibition of apical HRP endocytosis. In the absence of forskolin, wortmannin stimulated HRP endocytosis significantly. We conclude that, in T84 cells, apical fluid phase endocytosis is not dependent on PI3K activity and that CFTR does not recycle through a PI3K-dependent and wortmannin-sensitive membrane compartment.

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13146号 / 論農博第2852号 / 新制||農||1056(附属図書館) / 学位論文||H30||N5093(農学部図書室) / (主査)教授 植田 和光, 教授 加納 健司, 教授 三芳 秀人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Hormonally-triggered regulatory hierarchies play a major role in organismal development. Disruption of a single member of such a hierarchy can lead to irregular development and disease. Therefore, knowledge of the members involved and the mechanisms controlling signaling through such pathways is of great importance in understanding how resulting developmental defects occur. Type II transmembrane serine proteases (TTSPs) make up a family of cell surface-associated proteases that play important roles in the development and homeostasis of a number of mammalian tissues. Aberrant expression of TTSPs is linked to several human disorders, including deafness, heart and respiratory disease and cancer. However, the mechanism by which these proteases function remains unknown. The ecdysone-responsive Stubble TTSP of Drosophila serves as a good model in which to study the functional mechanism of the TTSP family. The Stubble protease interacts with the intracellular Rho1 (RhoA) pathway to control epithelial development in imaginal discs. The Rho1 signaling pathway regulates cellular behavior via control of gene expression and actin cytoskeletal dynamics. However, the mechanism by which the Stubble protease interacts with the Rho1 pathway to control epithelial development, in particular leg imaginal disc morphogenesis, has yet to be elucidated. The Stubble protein consists of several conserved domains. One approach to a better understanding of the mechanism of action of Stubble in regulating Rho1 signaling is to define which of the conserved domains within the protease are required for proper function. Sequence analysis of twelve recessive Stubble mutant alleles has revealed that the proteolytic domain is essential for proper function. Alleles containing mutations which disrupt regions of the protease domain necessary for protease activation or substrate binding, as well as those with deletions or truncations that remove some portion of the proteolytic domain, result in defective epithelial development in vivo. In contrast, mutations in other regions of the Stubble protein, including the disulfide-knotted and cytoplasmic domains, were not observed. Another important step for defining the connection between Stubble and Rho1 signaling is to identify a Stubble target that acts as an upstream regulator of the Rho1 pathway. We performed a genetic screen in which 97 of the 147 Drosophila non-olfactory and non-gustatory G-protein-coupled receptors (GPCRs), a family of proteins that has been shown to be protease-activated and to activate Rho1 signaling, were tested for interactions with a mutant allele of Stubble. We found 4 genomic regions uncovering a total of 7 GPCRs that interact genetically when in heterozygous combination with a Stubble mutant. Further analysis of these genes is necessary to determine if any of these GPCRs is targeted by Stubble during activation of the Rho1 pathway.

No description available.