| 1. |
- Gustafsson, Anna, et al.
(författare)
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Gas6 and the receptor tyrosine kinase Axl in clear cell renal cell carcinoma.
- 2009
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 4:10, s. e7575-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The molecular biology of renal cell carcinoma (RCC) is complex and not fully understood. We have recently found that the expression of the receptor tyrosine kinase Axl in the RCC tumors independently correlates with survival of the patients. PRINCIPAL FINDINGS: Here, we have investigated the role of Axl and its ligand Gas6, the vitamin-K dependent protein product of the growth arrest-specific gene 6, in clear cell RCC (ccRCC) derived cells. The Axl protein was highly expressed in ccRCC cells deficient in functional von Hippel-Lindau (VHL) protein, a tumor suppressor gene often inactivated in ccRCC. VHL reconstituted cells expressed decreased levels of Axl protein, but not Axl mRNA, suggesting VHL to regulate Axl expression. Gas6-mediated activation of Axl in ccRCC cells resulted in Axl phosphorylation, receptor down-regulation, decreased cell-viability and migratory capacity. No effects of the Gas6/Axl system could be detected on invasion. Moreover, in ccRCC tumor tissues, Axl was phosphorylated and Gas6 gamma-carboxylated, suggesting these molecules to be active in vivo. SIGNIFICANCE: These results provide novel information regarding the complex function of the Gas6/Axl system in ccRCC.
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| 2. |
- Ljunggren, Stefan, 1988-
(författare)
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Lipoproteomics : Environmental and Genetic Factors Affecting High-Density Lipoprotein (HDL)
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipoprotein particles act as lipid transporters in the blood stream, and measuring cholesterol content in specific subclasses of lipoprotein particles has long been, and still is, a frequently used tool to estimate the risk of cardiovascular disease (CVD). High-density lipoprotein (HDL) is a subclass of lipoproteins often regarded as providing protection against CVD via several functions including reverse cholesterol transport and anti-inflammatory capacities. However, the precise relationship between HDL cholesterol levels and health outcome is still unclear. Lately, new approaches to study HDL composition and function have therefore become more important.HDL function is to a large extent dependent on its proteome, containing more than 100 proteins. Investigating the proteome in individuals with altered gene expression for HDL-associated proteins or with known exposure to environmental contaminants may reveal new insights into how HDL metabolism is affected by various factors. This is of interest in order to better understand the role of HDL in CVD.Papers I and II focus on two different mutations in a structural HDL protein, apolipoprotein A-I (L202P and K131del), and one mutation in the scavenger receptor class B-1 (P297S), which is involved in selective lipid uptake of cholesterol mainly into hepatocytes and adrenal cells. The HDL proteome was analyzed using two-dimensional gel electrophoresis and mass spectrometry. The L202P mutation was identified in HDL of the heterozygote carriers together with a significant decrease of apolipoprotein E and increased zinc-alpha-2-glycoprotein. By contrast, the second apolipoprotein AI mutation (K131del) was associated with significantly elevated alpha-1-antitrypsin and transthyretin levels. Protein analyses of the scavenger receptor class B1 P297S heterozygotes showed a significant increase in HDL apoL-1 along with increased free apoE. The carriers showed no difference in antioxidative capability but a significant increase in apoA-I methionine oxidation.Papers III and IV focus on persistent organic pollutants that may influence HDL composition and function. These compounds accumulate in humans, and exposure has been linked to an increased risk of CVD. To provide a better understanding of the HDL system in relation to pollutants, a population living in a contaminated area was studied. Persistent organic pollutants in isolated HDL were quantified using high-resolution gas chromatography mass spectrometry and significantly increased levels were found in individuals with CVD as compared to healthy controls. Furthermore, there was a significant negative association between the pollutants and paraoxonase-1 anti-oxidant activity. Studying the proteome with nano-liquid chromatography tandem mass spectrometry led to the identification of 118 proteins in HDL, of which ten were significantly associated with the persistent organic pollutants.In summary, the present studies demonstrate protein pattern alterations in HDL associated with inherited genetic variants or pollutant exposure. The studies also provide a set of methods that are useful tools to further comprehend the complexity of lipoprotein metabolism and function. The results are important in order to improve our understanding of HDL in CVD and to explain an increased risk of CVD associated with exposure to organic pollutants.
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| 3. |
- Martuszewska, Danuta, et al.
(författare)
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Tensin3 is a negative regulator of cell migration and all four Tensin family members are downregulated in human kidney cancer.
- 2009
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:2
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The Tensin family of intracellular proteins (Tensin1, -2, -3 and -4) are thought to act as links between the extracellular matrix and the cytoskeleton, and thereby mediate signaling for cell shape and motility. Dysregulation of Tensin expression has previously been implicated in human cancer. Here, we have for the first time evaluated the significance of all four Tensins in a study of human renal cell carcinoma (RCC), as well as probed the biological function of Tensin3. PRINCIPAL FINDINGS: Expression of Tensin2 and Tensin3 at mRNA and protein levels was largely absent in a panel of diverse human cancer cell lines. Quantitative RT-PCR analysis revealed mRNA expression of all four Tensin genes to be significantly downregulated in human kidney tumors (50-100% reduction versus normal kidney cortex; P<0.001). Furthermore, the mRNA expressions of Tensins mostly correlated positively with each other and negatively with tumor grade, but not tumor size. Immunohistochemical analysis revealed Tensin3 to be present in the cytoplasm of tubular epithelium in normal human kidney sections, whilst expression was weaker or absent in 41% of kidney tumors. A subset of tumor sections showed a preferential plasma membrane expression of Tensin3, which in clear cell RCC patients was correlated with longer survival. Stable expression of Tensin3 in HEK 293 cells markedly inhibited both cell migration and matrix invasion, a function independent of putative phosphatase activity in Tensin3. Conversely, siRNA knockdown of endogenous Tensin3 in human cancer cells significantly increased their migration. CONCLUSIONS: Our findings indicate that the Tensins may represent a novel group of metastasis suppressors in the kidney, the loss of which leads to greater tumor cell motility and consequent metastasis. Moreover, tumorigenesis in the human kidney may be facilitated by a general downregulation of Tensins. Therefore, anti-metastatic therapies may benefit from restoring or preserving Tensin expression in primary tumors.
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| 4. |
- Trouw, Leendert, et al.
(författare)
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C4b-binding protein and factor h compensate for the loss of membrane-bound complement inhibitors to protect apoptotic cells against excessive complement attack
- 2007
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:39, s. 28540-28548
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Tidskriftsartikel (refereegranskat)abstract
- Apoptotic cells have been reported to down- regulate membrane-bound complement regulatory proteins ( m- C- Reg) and to activate complement. Nonetheless, most apoptotic cells do not undergo complement- mediated lysis. Therefore, we hypothesized that fluid phase complement inhibitors would bind to apoptotic cells and compensate functionally for the loss of m- C- Reg. We observed that m- C- Reg are down- regulated rapidly upon apoptosis but that complement activation follows only after a gap of several hours. Coinciding with, but independent from, complement activation, fluid phase complement inhibitors C4b- binding protein ( C4BP) and factorH( fH) bind to the cells. C4BP and fH do not entirely prevent complement activation but strongly limit C3 and C9 deposition. Late apoptotic cells, present in blood of healthy controls and systemic lupus erythematosus patients, are also positive for C4BP and fH. Upon culture, the percentage of late apoptotic cells increases, paralleled by increased C4BP binding. C4BP binds to dead cells mainly via phosphatidylserine, whereas fH binds via multiple interactions with CRP playing no major role for binding of C4BP or fH. In conclusion, during late apoptosis, cells acquire fluid phase complement inhibitors that compensate for the downregulation of m- C- Reg and protect against excessive complement activation and lysis.
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| 5. |
- Blom, Anna, et al.
(författare)
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Structural requirements for the complement regulatory activities of C4BP
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:29, s. 27136-27144
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.
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| 6. |
- Friedrich, Ute, et al.
(författare)
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Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:26, s. 24122-24128
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Tidskriftsartikel (refereegranskat)abstract
- Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC.
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| 7. |
- Villoutreix, Bruno O., et al.
(författare)
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Screening the molecular surface of human anticoagulant protein C: a search for interaction sites
- 2001
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Ingår i: Journal of Computer-Aided Molecular Design. - 1573-4951. ; 15:1, s. 13-27
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Tidskriftsartikel (refereegranskat)abstract
- Protein C (PC), a 62 kDa multi-modular zymogen, is activated to an anticoagulant serine protease (activated PC or APC) by thrombin bound to thrombomodulin on the surface of endothelial cells. PC/APC interacts with many proteins and the characterisation of these interactions is not trivial. However, molecular modelling methods help to study these complex biological processes and provide basis for rational experimental design and interpretation of the results. PC/APC consists of a Gla domain followed by two EGF modules and a serine protease domain. In this report, we present two structural models for full-length APC and two equivalent models for full-length PC, based on the X-ray structures of Gla-domainless APC and of known serine protease zymogens. The overall elongated shape of the models is further cross-validated using size exclusion chromatography which allows evaluation of the Stokes radius (rs for PC = 33.15 A; rs for APC = 34.19 A), frictional ratio and axial ratio. We then propose potential binding sites at the surface of PC/APC using surface hydrophobicity as a determinant of the preferred sites of intermolecular recognition. Most of the predicted binding sites are consistent with previously reported experimental data, while some clusters highlight new regions that should be involved in protein-protein interactions.
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| 8. |
- Webb, Joanna H., et al.
(författare)
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Localization of a hydrophobic binding site for anticoagulant protein S on the beta -chain of complement regulator C4b-binding protein
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:6, s. 4330-4337
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) is a plasma glycoprotein involved in regulation of the complement system. C4BP consists of seven alpha-chains and one unique beta-chain, all constructed of repeating complement control protein (CCP) modules. The beta-chain, made up of three CCPs, binds tightly to vitamin K-dependent protein S, a cofactor to anticoagulant activated protein C. When bound to C4BP, protein S loses its activated protein C cofactor function. In this study, we have mutated potentially important amino acids located at the surface of CCP1 of the beta-chain to probe the protein S-C4BP interaction. The substitutions were designed after analysis of a homology-based three-dimensional structure of the beta-chain and were L27T/F45Q, I16S/V18S, V31T/I33N, I16S/V18S/V31T/I33N, L38S/V39S, and K41E/K42E. The mutants were expressed in a prokaryotic system, purified using an N-terminal His-tag, refolded using an oxido-shuffling system, and tested in several assays for their ability to bind protein S. Our data define Ile(16), Val(18), Val(31), and Ile(33) as crucial for protein S binding, with secondary effects from Leu(38) and Val(39). In addition, Lys(41) and Lys(42) contribute slightly to the interaction. Our results further confirm that surface hydrophobicity analysis may be used to identify ligand recognition sites.
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| 9. |
- Hafizi, Sassan, et al.
(författare)
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Individual domains of Tensin2 exhibit distinct subcellular localisations and migratory effects.
- 2010
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Ingår i: International Journal of Biochemistry and Cell Biology. - : Elsevier BV. - 1878-5875 .- 1357-2725. ; 42, s. 52-61
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Tidskriftsartikel (refereegranskat)abstract
- Tensins are large intracellular proteins believed to link the extracellular matrix to the cytoskeleton via integrins. Tensins are multidomain proteins consisting of homologous C1, PTPase, C2, SH2 and PTB domains. Full-length Tensin proteins can undergo cleavage inside cells, thus yielding domains in isolation that may have discrete subcellular localisations and downstream effects. We expressed different isoforms of Tensin2 and their individual domains as recombinant green fluorescent protein (GFP)-fusion constructs in DU145 human prostate cancer cells. Under fluorescence confocal microscopy, the isolated domains of Tensin2 all displayed discrete distributions throughout the cytoplasm and the nucleus. In particular, partial constructs containing the C1 domain localised preferentially to the nucleus, including the isolated C1 domain and the PTPase domain. In contrast, all three full-length isoforms of Tensin2 were present exclusively in discrete punctate bodies throughout the cytoplasm. This punctate staining showed colocalisation with the tumour suppressor protein DLC-1 as well as with actin (phalloidin). Furthermore, DU145 cells transiently expressing partial Tensin2 constructs containing the PTB domain showed an increased haptotactic migration. In addition, stimulation of renal carcinoma cells stably expressing Tensin2 by the survival factor Gas6 caused phosphorylation of its receptor Axl, but no effect on Tensin2, which was already maximally phosphorylated at time 0. In conclusion, our results indicate that differential proteolytic cleavage of Tensin2 can liberate domains with discrete localisations and functions, which has implications for the role of Tensins in cancer cell survival and motility.
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| 10. |
- Norström, Eva, et al.
(författare)
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Importance of protein S and phospholipid for activated protein C-mediated cleavages in factor Va.
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:27, s. 24904-24911
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Tidskriftsartikel (refereegranskat)abstract
- The procoagulant function of activated factor V (FVa) is inhibited by activated protein C (APC) through proteolytic cleavages at Arg(306), Arg(506), and Arg(679). The effect of APC is potentiated by negatively charged phospholipid membranes and the APC cofactor protein S. Protein S has been reported to selectively stimulate cleavage at Arg(306), an effect hypothesized to be related to reorientation of the active site of APC closer to the phospholipid membrane. To investigate the importance of protein S and phospholipid in the APC-mediated cleavages of individual sites, recombinant FV variants FV(R306Q/R679Q) and FV(R506Q/R679Q) ( can be cleaved only at Arg(506) and Arg306(,) respectively) were created. The cleavage rate was determined for each cleavage site in the presence of varied protein S concentrations and phospholipid compositions. In contrast to results on record, we found that protein S stimulated both APC cleavages in a phospholipid composition-dependent manner. Thus, on vesicles containing both phosphatidylserine and phosphatidylethanolamine, protein S increased the rate of Arg(306) cleavage 27-fold and that of Arg(506) cleavage 5-fold. Half-maximal stimulation was obtained at similar to30 nM protein S for both cleavages. In conclusion, we demonstrate that APC-mediated cleavages at both Arg(306) and Arg(506) in FVa are stimulated by protein S in a phospholipid composition-dependent manner. These results provide new insights into the mechanism of APC cofactor activity of protein S and the importance of phospholipid composition.
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