| 1. |
- Mark, Linda, et al.
(författare)
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Kaposi's sarcoma-associated herpes virus complement control protein: KCP - complement inhibition and more.
- 2007
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 44, s. 11-22
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Tidskriftsartikel (refereegranskat)abstract
- The complement system is an important part of innate immunity providing immediate protection against pathogens without a need for previous exposure, as well as priming the adaptive immune response through opsonisation, leukocyte recruitment and enhancing humoral immune responses. Its importance is not only shown through recurring fulminant infections in individuals with complement component deficiencies, but also through the many complement evasion strategies discovered for a wide range of infectious microbes (including acquisition of endogenous host complement inhibitors and expression of own homologues). Knowledge of these mechanisms at a molecular level may aid development of vaccines and novel therapeutic strategies. Here, we review the structure-function studies of the membrane-bound complement inhibitor KCP that is expressed on the surface of Kaposi's sarcoma-associated herpesvirus (KSHV) virions and infected cells. KCP accelerates the decay of classical C3 convertase and induces the degradation of activated complement factors C4b and C3b by serine proteinase, factor I. Molecular modeling and site-directed mutagenesis have identified sites on the surface of endogenous human inhibitors. KCP additionally enhances virion binding to permissive cells through a heparin/heparan sulfate-binding site located at the N-terminus of the protein.
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| 2. |
- Mark, Linda, et al.
(författare)
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KSHV complement control protein mimics human molecular mechanisms for inhibition of the complement system.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:43, s. 45093-45101
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Tidskriftsartikel (refereegranskat)abstract
- Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O.B., Blackbourn, D.J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.
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| 3. |
- Mark, Linda, et al.
(författare)
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The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1-2.
- 2006
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 43:10, s. 1665-1675
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Tidskriftsartikel (refereegranskat)abstract
- The Kaposi's, sarcoma-associated herpesvirus (KSHV) complement control protein (KCP) inhibits the human complement system, and is similar in structure and function to endogenous complement inhibitors. Other inhibitors such as C4d-binding protein and factor H, as well as the viral homologue vaccinia virus complement control protein are known to bind heparin and, for the two latter, also to glycosaminoglycans at the surface of cells. We report here that KCP also binds to heparin at physiological ionic strength. With help of site directed mutagenesis, positively charged amino acids in the two N-terminal complement control protein (CCP) domains 1-2 were found to be necessary for heparin binding. In silico molecular docking of heparin to KCP confirmed the experimental data, and further explored the heparin binding site. enabling us to present a model of the KCP-heparin interaction. Furthermore, the docking analysis also yielded insights of the KCP structure, by indicating that the angle between CCP domains 1-2 during the initial binding of heparin is more extended than in the model we have previously presented. We also found that KCP binds to heparan sulfate and weakly to glycosaminoglycans at the surface of cells. This might indicate that KCP at the Surface of viral particles aids in the primary attachment to the target cells, which is known to involve binding to heparan sulfate. Therefore. the present study contributes to the knowledge of heparin-protein interactions in general its well as to the understanding of the biology of KSHV. (c) 2005 Elsevier Ltd. All rights reserved.
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| 4. |
- Okroj, Marcin, et al.
(författare)
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Characterization of the complement inhibitory function of Rhesus rhadinovirus complement control protein (RCP).
- 2009
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 2008:Nov 6., s. 505-514
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Tidskriftsartikel (refereegranskat)abstract
- Rhesus Rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi's sarcoma-associated herpesvirus (KSHV). Both these viruses encode complement inhibitors: KSHV-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as complement inhibitor. Herein, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b- and C4b-degradation by factor I and decay-acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.
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| 5. |
- Bienaime, Frank, et al.
(författare)
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Mutations in components of complement influence the outcome of Factor I-associated atypical hemolytic uremic syndrome
- 2010
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Ingår i: Kidney International. - : Elsevier BV. - 1523-1755 .- 0085-2538. ; 77:4, s. 339-349
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Tidskriftsartikel (refereegranskat)abstract
- Genetic studies have shown that mutations of complement inhibitors such as membrane cofactor protein, Factors H, I, or B and C3 predispose patients to atypical hemolytic uremic syndrome (aHUS). Factor I is a circulating serine protease that inhibits complement by degrading C3b and up to now only a few mutations in the CFI gene have been characterized. In a large cohort of 202 patients with aHUS, we identified 23 patients carrying exonic mutations in CFI. Their overall clinical outcome was unfavorable, as half died or developed end-stage renal disease after their first syndrome episode. Eight patients with CFI mutations carried at least one additional known genetic risk factor for aHUS, such as a mutation in MCP, CFH, C3 or CFB; a compound heterozygous second mutation in CFI; or mutations in both the MCP and CFH genes. Five patients exhibited homozygous deletion of the Factor H-related protein 1 (CFHR-1) gene. Ten patients with aHUS had one mutation in their CFI gene (Factor I-aHUS), resulting in a quantitative or functional Factor I deficiency. Patients with a complete deletion of the CFHR-1 gene had a significantly higher risk of a bad prognosis compared with those with one Factor I mutation as their unique vulnerability feature. Our results emphasize the necessity of genetic screening for all susceptibility factors in patients with aHUS. Kidney International (2010) 77, 339-349; doi: 10.1038/ki.2009.472; published online 16 December 2009
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| 6. |
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| 7. |
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| 8. |
- Blom, Anna M, et al.
(författare)
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A Novel Non-Synonymous Polymorphism (p.Arg240His) in C4b-Binding Protein Is Associated with Atypical Hemolytic Uremic Syndrome and Leads to Impaired Alternative Pathway Cofactor Activity.
- 2008
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Ingår i: Journal of Immunology. - 1550-6606. ; 180:9, s. 6385-6391
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Tidskriftsartikel (refereegranskat)abstract
- Atypical hemolytic uremic syndrome (aHUS) is a disorder characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Mutations, polymorphisms, and copy number variation in complement factors and inhibitors are associated with aHUS. In this study, we report the first functional non-synonymous polymorphism in the complement inhibitor C4b-binding protein (C4BP) alpha-chain (c.719G>A; p.Arg240His), which is associated with aHUS. This heterozygous change was found in 6/166 aHUS patients compared with 5/542 normal (chi2 = 6.021; p = 0.014), which was replicated in a second cohort of aHUS patients in which we found 5/170 carriers. The polymorphism does not decrease expression efficiency of C4BP. p.Arg240His is equally efficient as the wild type in binding and supporting degradation of C4BP but its ability to bind C3b and act as cofactor to its degradation both in fluid phase and on surfaces is impaired. This observation supports the hypothesis that dysregulation of the alternative pathway of complement is pivotal for aHUS. Three of the patients carry also mutations in membrane cofactor protein and factor H strengthening the hypothesis that individuals may carry multiple susceptibility factors with an additive effect on the risk of developing aHUS.
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| 9. |
- Calzavarini, Sara, et al.
(författare)
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Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation
- 2013
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Ingår i: Thrombosis and Haemostasis. - 0340-6245. ; 110:1, s. 31-38
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Tidskriftsartikel (refereegranskat)abstract
- Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients.
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| 10. |
- Dahlbäck, Björn, et al.
(författare)
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Regulation of Blood Coagulation by the Protein C Anticoagulant Pathway. Novel Insights Into Structure-Function Relationships and Molecular Recognition.
- 2005
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1524-4636. ; 25:7, s. 1311-1320
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Forskningsöversikt (refereegranskat)abstract
- The protein C system provides important control of blood coagulation by regulating the activities of factor VIIIa (FVIIIa) and factor Va(FVa), cofactors in the activation of factor X and prothrombin, respectively. The system comprises membrane-bound and circulating proteins that assemble into multi-molecular complexes on cell surfaces. Vitamin K-dependent protein C, the key component of the system, circulates in blood as zymogen to an anticoagulant serine protease. It is efficiently activated on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor (EPCR) further stimulates the protein C activation. Activated protein C (APC) together with its cofactor protein S inhibits coagulation by degrading FVIIIa and FVa on the surface of negatively charged phospholipid membranes. Efficient FVIIIa degradation by APC requires not only protein S but also intact FV, which like thrombin is a Janus-faced protein with both procoagulant and anticoagulant potential. In addition to its anticoagulant properties, APC has antiinflammatory and antiapoptotic functions, which are exerted when APC binds to EPCR and proteolytic cleaves protease-activated receptor 1 (PAR-1). The protein C system is physiologically important, and genetic defects affecting the system are the most common risk factors of venous thrombosis. The proteins of the protein C system are composed of multiple domains and the 3-dimensional structures of several of the proteins are known. The molecular recognition of the protein C system is progressively being unraveled, giving us new insights into this fascinating and intricate molecular scenario at the atomic level.
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| 11. |
- Dahlbäck, Björn, et al.
(författare)
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The anticoagulant protein C pathway.
- 2005
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Ingår i: FEBS Letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 579:15, s. 3310-3316
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Forskningsöversikt (refereegranskat)
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| 12. |
- 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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| 13. |
- Golec, Ewelina, et al.
(författare)
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Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets
- 2022
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences (PNAS). - 0027-8424 .- 1091-6490. ; 119:24
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Tidskriftsartikel (refereegranskat)abstract
- Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of beta-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse beta-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non-GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.
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| 14. |
- Hafizi, Sassan, et al.
(författare)
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Tensin2 reduces intracellular phosphatidylinositol 3,4,5-trisphosphate levels at the plasma membrane
- 2010
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 399:3, s. 396-401
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Tidskriftsartikel (refereegranskat)abstract
- Tensins are proposed cytoskeleton-regulating proteins. However, Tensin2 additionally inhibits Akt signalling and cell survival. Structural modelling of the Tensin2 phosphatase (PTPase) domain revealed an active site-like pocket receptive towards phosphoinositides. Tensin2-expressing HEK293 cells displayed negligible levels of plasma membrane phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P-3) under confocal microscopy. However, mock-transfected cells, and Tensin2 cells harbouring a putative phosphatase-inactivating mutation, exhibited significant PtdIns(3,4,5)P-3 levels, which decreased upon phosphatidylinositol 3-kinase inhibition with LY294002. In contrast, wtTensin3, mock and mutant cells were identical in membrane PtdIns(3,4,5)P-3 and Akt phosphorylation. In vitro lipid PTPase activity was however undetectable in isolated recombinant PTPase domains of both Tensins, indicating a possible loss of structural stability when expressed in isolation. In summary, we provide evidence that Tensin2, in addition to regulating cytoskeletal dynamics, influences phosphoinositide-Akt signalling through its PTPase domain.
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| 15. |
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| 16. |
- Jarva, Hanna, et al.
(författare)
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Molecular Characterization of the Interaction between Porins of Neisseria gonorrhoeae and C4b-Binding Protein.
- 2007
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Ingår i: Journal of Immunology. - 1550-6606. ; 179:1, s. 540-547
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Tidskriftsartikel (refereegranskat)abstract
- Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitors C4b-binding protein (CUP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCPI) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to PorlA strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to PorlA strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to PorlA gonococci. C4BP binding to Por1-B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.
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| 17. |
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| 18. |
- Knobe, Karin, et al.
(författare)
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Functional Analysis of the Factor IX Epidermal Growth Factor-Like Domain Mutation Ile66Thr Associated with Mild Hemophilia B.
- 2006
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Ingår i: Pathophysiology of Haemostasis and Thrombosis. - : S. Karger AG. - 1424-8832 .- 1424-8840. ; 35:5, s. 370-375
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Tidskriftsartikel (refereegranskat)abstract
- he present study focused on the functional role of the mutation Ile66Thr located in the N-terminal epidermal growth factor-like domain of coagulation factor IX (FIX). This mutation causes mild hemophilia B with approximately 25% FIX coagulant activity and FIX antigen levels of around 90% of normal. In the 3-dimensional structure of porcine FIXa and in the subsequent 3-dimensional model of human FIXa that we have previously developed, residue 66 is exposed to the solvent and can be replaced by many amino acids, including Thr, without affecting the major folding/stability of the molecule. This is consistent with the basically normal antigen levels observed. We found that the FIX Ile66Thr mutant was activated to a normal extent by FVIIa/TF and FXIa. However, the ability of FIX Ile66Thr to activate FX was impaired in both the presence and absence of FVIIIa, indicating that Ile66 is not directly involved in the binding of FIX to FVIIIa.
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| 19. |
- Knobe, Karin, et al.
(författare)
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Probing the activation of protein C by the thrombin-thrombomodulin complex using structural analysis, site-directed mutagenesis, and computer modeling
- 1999
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Ingår i: Proteins. - 0887-3585. ; 35:2, s. 218-234
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Tidskriftsartikel (refereegranskat)abstract
- Protein C (PC) is activated to an essential anticoagulant enzyme (activated PC or APC) by thrombin (T) bound to thrombomodulin (TM), a membrane receptor present on the surface of endothelial cells. The understanding of this complex biological system is in part limited due to the lack of integration of experimental and structural data. In the work presented here, we analyze the PC-T-TM pathway in the context of both types of information. First, structural analysis of the serine protease domain of PC suggests that a positively charged cluster of amino acids could be involved in the activation process. To investigate the importance of these basic amino acids, two recombinant PC mutants were constructed using computer-guided site-directed mutagenesis. The double mutant had the K62[217]N/K63[218]D substitution and in the single mutant, K86[241] was changed to S. Both mutants were activated by free thrombin at rates equivalent to that of wild-type PC (wt-PC) and they demonstrated similar calcium-dependent inhibition of their activation. The K86[241]S mutant and wt-PC were activated by thrombin bound to soluble TM at a similar rate. In contrast, the K62[217]N/ K63[218]D mutant was activated by the T-TM complex at a 10-fold lower catalytic efficiency due to a lowering in k(cat) and increase in Km. Molecular models for PC and thrombin bound to a segment of TM were developed. The experimental results and the modeling data both indicate that electrostatic interactions are of crucial importance to orient PC onto the T-TM complex. A key electropositive region centered around loops 37[191] and 60[214] of PC is defined. PC loop 37[191] is located 7-8 A from the TM epidermal growth factor (EGF) 4 while the loop 60[214] is about 10 A away from TM EGF4. Both loops are far from thrombin. A key function of TM could be to create an additional binding site for PC. The Gla domain of PC points toward the membrane and away from thrombin or the EGF modules of TM during the activation process.
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| 20. |
- Lindström, Lisa, et al.
(författare)
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Therapeutic Targeting of Nuclear Gamma-Tubulin in RB1-negative Tumors.
- 2015
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Ingår i: Molecular Cancer Research. - 1557-3125. ; 13:7, s. 1073-1082
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Tidskriftsartikel (refereegranskat)abstract
- In addition to its cytosolic function, gamma-tubulin is a chromatin-associated protein. Reduced levels of nuclear gamma-tubulin increase the activity of E2 promoter-binding factors (E2F) and raise the levels of retinoblastoma (RB1) tumor suppressor protein. In tumor cells lacking RB1 expression, decreased gamma-tubulin levels induce cell death. Consequently, impairment of the nuclear activity of gamma-tubulin has been suggested as a strategy for targeted chemotherapy of RB1-deficient tumors; thus, tubulin inhibitors were tested to identify compounds that interfere with gamma-tubulin. Interestingly, citral increased E2F activity but impaired microtubule dynamics while citral analogs, like citral dimethyl acetal (CDA), increased E2F activity without affecting microtubules. The cytotoxic effect of CDA on tumor cells was attenuated by increased expression of either RB1 or gamma-tubulin, and increased by reduced levels of either RB1 or gamma-tubulin. Mechanistic study, in silico and in vitro, demonstrated that CDA prevents GTP binding to gamma-tubulin and suggested that the FDA approved drug dimethyl fumarate is also a gamma-tubulin inhibitor. Finally, in vivo growth of xenograft tumors carrying defects in the RB1 signaling pathway were inhibited by CDA treatment. These results demonstrate that inhibition of gamma-tubulin has the potential to specifically target tumor cells and may aid in the design of safer and more efficient chemotherapeutic regimes.
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| 21. |
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| 22. |
- Mohlin, Frida, et al.
(författare)
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Functional characterization of two novel non-synonymous alterations in CD46 and a Q950H change in factor H found in atypical hemolytic uremic syndrome patients.
- 2015
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; 65:2, s. 367-376
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Tidskriftsartikel (refereegranskat)abstract
- Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation, characterized by hemolytic anemia, thrombocytopenia and acute renal failure. Mutations in complement inhibitors are major risk factors for development of aHUS. The three aHUS patients reported in this study had several previously identified alterations in complement inhibitors; e.g. risk haplotypes in CD46 and factor H but we also identified two novel heterozygous non-synonymous CD46 alterations (p.E142Q and p.G259V). Presence of G259V caused decreased expression of the recombinant mutant CD46 compared to wild type (WT). Western blot analysis showed that the majority of the expressed G259V protein was in the precursor form, suggesting that it is processed less efficiently than WT. Low CD46 expression on the surface of the patient's neutrophils confirmed the in vitro results. Further, G259V had a substantially impaired ability to act as a cofactor to factor I, in the degradation of both C3b and C4b. The E142Q mutant showed neither decreased expression nor impaired function. Two of the patients also had a heterozygous non-synonymous alteration in factor H (p.Q950H), reported previously in aHUS but not functionally tested. This variant showed moderately impaired function in hemolytic assays, both using patient sera and recombinant proteins. The recombinant Q950H also showed a somewhat decreased expression compared to WT but the complement inhibitory function in fluid phase was normal. Taken together, we report a novel CD46 alteration showing both a decreased protein expression and substantially impaired cofactor function (G259V) and another without an effect on expression or cofactor function (E142Q). Moreover, mild consequences of a previously reported aHUS associated rare variant in factor H (Q950H) was also revealed, underlining the clear need for functional characterization of each new aHUS associated mutation.
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| 23. |
- Nilsson, Sara, et al.
(författare)
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Analysis of binding sites on complement factor I that are required for its activity.
- 2010
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 285, s. 6235-6245
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Tidskriftsartikel (refereegranskat)abstract
- The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1 and membrane cofactor protein. FI is a serine protease composed of two chains; the light chain comprises the serine protease domain, while the heavy chain contains several domains: the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. In order to understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well-expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (Km) of all FI mutants towards a small substrate was not altered while some mutants showed increased maximum initial velocity (Vmax). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used whereas only some mutations in the CD5 and LDLr1/2 domains had similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.
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| 24. |
- Nilsson, Sara, et al.
(författare)
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Genetic, molecular and functional analyses of complement factor I deficiency.
- 2009
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Ingår i: European Journal of Immunology. - : Wiley. - 1521-4141 .- 0014-2980. ; 39:1, s. 310-323
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Tidskriftsartikel (refereegranskat)abstract
- Complete deficiency of complement inhibitor factor I (FI) results in secondary complement deficiency due to uncontrolled spontaneous alternative pathway activation leading to susceptibility to infections. Current genetic examination of two patients with near complete FI deficiency and three patients with no detectable serum FI and also close family members revealed homozygous or compound heterozygous mutations in several domains of FI. These mutations were introduced into recombinant FI and the resulting proteins were purified for functional studies, while transient transfection was used to analyze expression and secretion. The G170V mutation resulted in a protein that was not expressed, whereas the mutations Q232K, C237Y, S250L, I339M and H400L affected secretion. Furthermore, the C237Y and the S250L mutants did not degrade C4b and C3b as efficiently as the WT. The truncated Q336x mutant could be expressed, in vitro, but was not functional because it lacks the serine protease domain. Furthermore, this truncated FI was not detected in serum of the patient. Structural investigations using molecular modeling were performed to predict the potential impact the mutations have on FI structure. This is the first study that investigates, at the functional level, the consequences of molecular defects identified in patients with full FI deficiency.
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| 25. |
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