| 1. |
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| 2. |
- Andersson, Marlene, et al.
(författare)
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Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains
- 2014
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 12:8, s. e1001921-
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Tidskriftsartikel (refereegranskat)abstract
- Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive beta-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO(2)) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.
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| 3. |
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| 4. |
- Arkblad, Eva L, et al.
(författare)
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Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy
- 2006
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Ingår i: Neuromuscular disorders : NMD. - : Elsevier BV. - 0960-8966 .- 1873-2364. ; 16:12, s. 830-8
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Tidskriftsartikel (refereegranskat)abstract
- Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by decreased levels of survival motor neuron protein (SMN). In the majority of cases, this decrease is due to absence of the SMN1 gene. Multiplex ligation-dependent probe amplification (MLPA) is a modern quantitative molecular method. Applied in SMA cases, it improves diagnostics by simultaneously identifying the number of copies of several target sequences in the SMN1 gene and in nearby genes. Using MLPA in clinical diagnostics, we have identified a previously unreported, partial deletion of SMN1 (exons 1-6) in two apparently unrelated Swedish families. This mutation would not have been detected by conventional diagnostic methods. This paper illustrates the broad clinical and genetic spectrum of SMA and includes reports of MLPA results and clinical descriptions of a patient with homozygous absence of SMN1 and only one SMN2 (prenatal onset SMA type 1), an asymptomatic woman with five SMN2 (lacking SMN1) and representative patients with SMA types 1, 2 and 3.
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| 5. |
- Askarieh, Glareh, et al.
(författare)
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Self-assembly of spider silk proteins is controlled by a pH-sensitive relay
- 2010
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Ingår i: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 465:7295, s. 236-8
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Tidskriftsartikel (refereegranskat)abstract
- Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct.
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| 6. |
- Bergman, Annika, et al.
(författare)
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A high frequency of germline BRCA1/2 mutations in western Sweden detected with complementary screening techniques
- 2005
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Ingår i: Familial Cancer. - : Springer. - 1389-9600 .- 1573-7292. ; 4:2, s. 89-96
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Tidskriftsartikel (refereegranskat)abstract
- Dominant inheritance is presumed in 6-10% of breast and ovarian cancers. Mutations in BRCA1 and BRCA2 genes are the most commonly identified causative genes in such families. The frequency of mutation carriers with breast/ovarian cancer depends on the population studied, and display considerable variation that coincides with ethnic and geographical diversity. Mutation analyses were performed in 143 families registered at the Cancer Genetic Counseling Clinic of western Sweden. In a thorough mutation screening procedure, the entire BRCA1 and BRCA2 genes were analyzed using a combination of complementary mutation detection techniques. Mutations in either BRCA1 or BRCA2 were detected in 36% (52 out of 143) of all screened families. All families were clinically evaluated regarding age at diagnosis, type of cancer and number of cancer cases in the family. Among high-risk families, the mutation detection rate was 39% (46 out of 117). The detection rate observed among families with cases of ovarian cancer (42 out of 62, 68%), was substantially higher than in families with only breast cancer (10 out of 81, 12%). Age at ovarian cancer did not seem to have an effect on the detection rate. The analyses revealed 11 frameshift mutations, 4 nonsense mutations and 2 large deletions. Notably, the BRCA1 c.3171ins5 mutation accounted for 34 of 52 (65%) identified mutations. Seven mutations are novel: BRCA1c.409_410del; c.1912T>G; c.2228_2229del; c.3029delA; c.3433delA, a large deletion covering exons 1-3 of BRCA1and one BRCA2 mutation; BRCA2c.6287_6290del. We have shown that the founder mutation BRCA1 c.3171ins5 has a great influence on western Swedish breast/ovarian cancer families along with a high number of mutations unique for the region. In order to achieve a high mutation detection rate we suggest a combination of several detection techniques.
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| 7. |
- Eriksson, Daniel, et al.
(författare)
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Cytokine Autoantibody Screening in the Swedish Addison Registry Identifies Patients With Undiagnosed APS1
- 2018
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Ingår i: The Journal of clinical endocrinology and metabolism. - : The Endocrine Society. - 1945-7197 .- 0021-972X. ; 103:1, s. 179-186
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Tidskriftsartikel (refereegranskat)abstract
- Context: Autoimmune polyendocrine syndrome type 1 (APS1) is a monogenic disorder that features autoimmune Addison disease as a major component. Although APS1 accounts for only a small fraction of all patients with Addison disease, early identification of these individuals is vital to prevent the potentially lethal complications of APS1.Objective: To determine whether available serological and genetic markers are valuable screening tools for the identification of APS1 among patients diagnosed with Addison disease.Design: We systematically screened 677 patients with Addison disease enrolled in the Swedish Addison Registry for autoantibodies against interleukin-22 and interferon-α4. Autoantibody-positive patients were investigated for clinical manifestations of APS1, additional APS1-specific autoantibodies, and DNA sequence and copy number variations of AIRE.Results: In total, 17 patients (2.5%) displayed autoantibodies against interleukin-22 and/or interferon-α4, of which nine were known APS1 cases. Four patients previously undiagnosed with APS1 fulfilled clinical, genetic, and serological criteria. Hence, we identified four patients with undiagnosed APS1 with this screening procedure.Conclusion: We propose that patients with Addison disease should be routinely screened for cytokine autoantibodies. Clinical or serological support for APS1 should warrant DNA sequencing and copy number analysis of AIRE to enable early diagnosis and prevention of lethal complications.
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| 8. |
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| 9. |
- Fredriksson, Camilla, et al.
(författare)
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Tissue Response to Subcutaneously Implanted Recombinant Spider Silk : An in Vivo Study
- 2009
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Ingår i: Materials. - Basel, Switzerland : MDPI AG. - 1996-1944. ; 2:4, s. 1908-1922
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Tidskriftsartikel (refereegranskat)abstract
- Spider silk is an interesting biomaterial for medical applications. Recently, a method for production of recombinant spider silk protein (4RepCT) that forms macroscopic fibres in physiological solution was developed. Herein, 4RepCT and MersilkTM (control) fibres were implanted subcutaneously in rats for seven days, without any negative systemic or local reactions. The tissue response, characterised by infiltration of macrophages and multinucleated cells, was similar with both fibres, while only the 4RepCT-fibres supported ingrowth of fibroblasts and newly formed capillaries. This in vivo study indicates that 4RepCT-fibres are well tolerated and could be used for medical applications, e.g., tissue engineering.
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| 10. |
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| 11. |
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| 12. |
- Hedhammar, My, Professor, 1975-, et al.
(författare)
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Sterilized recombinant spider silk fibers of low pyrogenicity
- 2010
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 11:4, s. 953-9
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Tidskriftsartikel (refereegranskat)abstract
- We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli , the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.
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| 13. |
- Jaudzems, Kristaps, et al.
(författare)
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pH-Dependent Dimerization of Spider Silk N-Terminal Domain Requires Relocation of a Wedged Tryptophan Side Chain
- 2012
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 422:4, s. 477-487
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Tidskriftsartikel (refereegranskat)abstract
- Formation of spider silk from its constituent proteins-spidroins-involves changes from soluble helical/coil conformations to insoluble beta-sheet aggregates. This conversion needs to be regulated to avoid precocious aggregation proximally in the silk gland while still allowing rapid silk assembly in the distal parts. Lowering of pH from about 7 to 6 is apparently important for silk formation. The spidroin N-terminal domain (NT) undergoes stable dimerization and structural changes in this pH region, but the underlying mechanisms are incompletely understood. Here, we determine the NMR and crystal structures of Euprosthenops australis NT mutated in the dimer interface (A72R). Also, the NMR structure of wild-type (wt) E. australis NT at pH 7.2 and 300 mM sodium chloride was determined. The wt NT and A72R structures are monomers and virtually identical, but they differ from the subunit structure of dimeric wt NT mainly by having a tryptophan (W10) buried between helix 1 and helix 3, while W10 is surface exposed in the dimer. Wedging of the W10 side chain in monomeric NT tilts helix 3 approximately 5-6 angstrom into a position that is incompatible with that of the observed dimer structure. The structural differences between monomeric and dimeric NT domains explain the tryptophan fluorescence patterns of NT at pH 7 and pH 6 and indicate that the biological function of NT depends on conversion between the two conformations.
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| 14. |
- Johansson, Jan, et al.
(författare)
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A novel anti-amyloid chaperone
- 2010
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Ingår i: Amyloid. - 1350-6129 .- 1744-2818. ; 17, s. 103-104
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 15. |
- Johansson, Jan, et al.
(författare)
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Method of producing polymers of spider silk proteins
- 2012
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Patent (övrigt vetenskapligt/konstnärligt)abstract
- Amethodofproducingpolymersofan isolatedspidersilkprotein consistingoffrom 170 to 600 amino acid residues involves providing a solutionofsaidspidersilkprotein in a liquid medium at pH 6.4 or higher and/or an ion composition that prevents polymerisationofthespidersilkprotein. The propertiesofthe liquid medium are adjusted to a pHof6.3 or lower and an ion composition that allows polymerisationofthespidersilkprotein. Thespidersilkprotein is allowed to form solidpolymersin the liquid medium, and the resulting solidspidersilkproteinpolymersare isolated from the liquid medium. The resultingpolymersare useful as fibers, films, foams, nets or meshes.
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| 16. |
- Johansson, Jan, et al.
(författare)
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Monomeric N-terminal spider silk protein domain and uses thereof
- 2013
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Patent (övrigt vetenskapligt/konstnärligt)abstract
- A novel protein is comprising a moiety of 100-160 amino acid residues having at least 80% identity with the N-terminal moiety of a spider silk protein, wherein the amino acid residue corresponding to position 72 in SEQ ID NO 1 is not Ala or Gly. The protein moiety is useful in a fusion protein for enhancing the solubility of another moiety in the fusion protein, which is a desired protein or polypeptide. Novel methods are provided for producing such a fusion protein, and optionally a desired protein or polypeptide from said fusion protein.
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| 17. |
- Johansson, Jan, et al.
(författare)
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Production of proteins and polypeptides
- 2016
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Patent (övrigt vetenskapligt/konstnärligt)abstract
- The present invention relates to the field of production of proteins and polypeptides, and more specifically to production of spider silk proteins (spidroins) and other, non-spidroin proteins and polypeptides. The present invention provides a method of producing a desired non-spidroin protein/polypeptide. There is also provided novel fusion protein intermediates for production of the desired proteins and polypeptides as well as polynucleic acid molecules encoding these intermediates.
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| 18. |
- Johansson, Jan, et al.
(författare)
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Protein
- 2017
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Patent (övrigt vetenskapligt/konstnärligt)abstract
- The present invention provides a fusion protein comprising: i) a solubility-enhancing moiety which is derived from the N-terminal (NT) fragment of a spider silk protein; and ii) a C-type lectin polypeptide. The present invention also provides a truncated SP-A polypeptide which lacks the N-terminal domain of full SP-A and is capable of trimerisation, and to the use of such a truncated SP-A polypeptide in treating or preventing a disease.
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| 19. |
- Kronqvist, Nina, et al.
(författare)
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Efficient protein production inspired by how spiders make silk
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT star) that is pH insensitive, stabilized and hypersoluble compared to wildtype NT. NT star-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT star enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT star also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
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| 20. |
- Kronqvist, Nina, et al.
(författare)
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Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation
- 2014
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 5:1, s. 3254-
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.
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| 21. |
- Landreh, M., et al.
(författare)
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A pH-dependent dimer lock in spider silk protein
- 2010
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Ingår i: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 404:2, s. 328-336
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Tidskriftsartikel (refereegranskat)abstract
- Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below--but not above--6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.
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| 22. |
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| 23. |
- Nerelius, Charlotte, et al.
(författare)
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Anti-Amyloid Activity of the C-Terminal Domain of proSP-C against Amyloid beta-Peptide and Medin
- 2009
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:17, s. 3778-3786
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid fibrils are found in similar to 25 different diseases, including Alzheimer's disease. Lung surfactant protein C (SP-C) forms fibrils in association with pulmonary disease. It was recently found that the C-terminal domain of proSP-C (CTC), which is localized to the endoplasmic reticulum (ER) lumen, protects the transmembrane (TM) part of (pro)SP-C from aggregation into amyloid until it has a folded into an alpha-helix. CTC appears to have a more general anti-amyloid effect by also acting on TM regions of other proteins. Here we investigate interactions of CTC with the amyloid beta-peptide (A beta) associated with Alzheimer's disease and medin, a peptide that forms fibrils in the most common form of human amyloid. CTC prevents fibril formation in A beta and medin and forms a complex with A beta oligomers, as judged by size-exclusion chromatography and electrospray ionization mass spectrometry. These data suggest that CTC functions as a chaperone that acts preferentially against unfolded TM segments and structural motifs found during amyloid fibril formation, a mechanism that may be exploited in forming a basis for future anti-amyloid therapy.
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| 24. |
- Nerelius, Charlotte, et al.
(författare)
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Preventing Amyloid Formation by Catching Unfolded Transmembrane Segments
- 2009
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 389, s. 227-229
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Tidskriftsartikel (refereegranskat)abstract
- A subset of protein misfolding diseases, including, for example, Alzheimer's disease, is associated with the formation of highly insoluble amyloid fibrils with a beta-sheet structure. The amyloidogenic human lung surfactant protein C (SP-C) is generated from SP-C precursor, which has a C-terminal domain (CTC) that prevents SP-C amyloid fibril formation. Analysis of the substrate specificity of CTC reveals that it binds to all amino acid residues that promote membrane insertion, provided that they are in a nonhelical conformation. In line with this unexpectedly general substrate specificity, the anti-amyloid function of CTC extends to a transmembrane segment other than that of (pro)SP-C, namely, the amyloid beta-peptide associated with Alzheimer's disease. These findings indicate that CTC is the first known chaperone to be directed towards nonhelical transmembrane segments and that it may be employed for the development of new diagnostics or anti-amyloid therapies. (C) 2009 Elsevier Ltd. All rights reserved.
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| 25. |
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| 26. |
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| 27. |
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| 28. |
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| 29. |
- Rising, Anna, et al.
(författare)
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Systemic AA amyloidosis in the red fox (Vulpes vulpes)
- 2017
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Ingår i: Protein Science. - : WILEY. - 0961-8368 .- 1469-896X. ; 26:11, s. 2312-2318
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full-length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter-species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model.
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| 30. |
- Rönnberg, Henrik, et al.
(författare)
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Surfactant Protein C in Canine Pulmonary Fibrosis
- 2009
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Ingår i: Journal of Veterinary Internal Medicine. - : Wiley. - 0891-6640 .- 1939-1676. ; 23, s. 1170-1174
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundCanine pulmonary fibrosis (CPF) occurs most commonly in West Highland White Terriers. The differing incidences of CPF among dog breeds suggest that genetic factors contribute to its pathophysiology. Pulmonary fibrosis in humans is associated with mutations in the gene coding for lung surfactant protein C (SP-C) (SFTPC).Hypothesis/ObjectivesTo investigate the histopathologic changes and SP-C composition and genetic structure in dogs with CPF.AnimalsFive dogs with PF, 2 dogs with other lung diseases, and 3 healthy dogs.MethodsLung tissue from dogs with clinically suspected CPF and 5 control cases was analyzed histopathologically. Bronchoalveolar lavage fluid (BALF) collected postmortem from 3 terriers with histopathologically confirmed pulmonary fibrosis and the 5 controls were analyzed by Western blots, and the exons of SFTPC were sequenced for 2 dogs with PF and 1 dog with other lung disease.ResultsSP-C could not be detected in BALF of 1 dog with PF, although SP-B was present. A mutation was detected in SFTPC exon 5 of this dog. From 2 dogs with PF and in all 5 control dogs SP-B and SP-C were detected in BALF.ConclusionsTaken together, the results indicate that canine and human lung fibrosis share histopathologic features and that analysis of SP-C and its gene in a larger set of dogs with PF is warranted.
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| 31. |
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| 32. |
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| 33. |
- Willander, Hanna, et al.
(författare)
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High-resolution structure of a BRICHOS domain and its implications for anti-amyloid chaperone activity on lung surfactant protein C
- 2012
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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. - 0027-8424 .- 1091-6490. ; 109:7, s. 2325-2329
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Tidskriftsartikel (refereegranskat)abstract
- BRICHOS domains are encoded in >30 human genes, which are associated with cancer, neurodegeneration, and interstitial lung disease (ILD). The BRICHOS domain from lung surfactant protein C proprotein (proSP-C) is required for membrane insertion of SP-C and has anti-amyloid activity in vitro. Here, we report the 2.1 angstrom crystal structure of the human proSP-C BRICHOS domain, which, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry, reveals how BRICHOS domains may mediate chaperone activity. Observation of amyloid deposits composed of mature SP-C in lung tissue samples from ILD patients with mutations in the BRICHOS domain or in its peptide-binding linker region supports the in vivo relevance of the proposed mechanism. The results indicate that ILD mutations interfering with proSP-C BRICHOS activity cause amyloid disease secondary to intramolecular chaperone malfunction.
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| 34. |
- Willander, Hanna, et al.
(författare)
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The Brichos domain of prosurfactant protein C can hold and fold a transmembrane segment
- 2009
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18, s. 1175-1182
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Tidskriftsartikel (refereegranskat)abstract
- Prosurfactant protein C (proSP-C) is a 197-residue integral membrane protein, in which the C-terminal domain (CTC, positions 59-197) is localized in the endoplasmic reticulum (ER) lumen and contains a Brichos domain (positions 94-197). Mature SP-C corresponds largely to the transmembrane (TM) region of proSP-C. CTC binds to SP-C, provided that it is in nonhelical conformation, and can prevent formation of intracellular amyloid-like inclusions of proSP-C that harbor mutations linked to interstitial lung disease (ILD). Herein it is shown that expression of proSP-C (1-58), that is, the N-terminal propeptide and the TM region, in HEK293 cells results in virtually no detectable protein, while coexpression of CTC in trans yields SDS-soluble monomeric proSP-C (1-58). Recombinant human (rh) CTC binds to cellulose-bound peptides derived from the nonpolar TM region, but not the polar cytosolic part, of proSP-C, and requires >= 5-residues for maximal binding. Binding of rhCTC to a nonhelical peptide derived from SP-C results in a-helix formation provided that it contains a long TM segment. Finally, rhCTC and rhCTC Brichos domain shows very similar substrate specificities, but rhCTC(L188Q), a mutation linked to ILD is unable to bind all peptides analyzed. These data indicate that the Brichos domain of proSP-C is a chaperone that induces alpha-helix formation of an aggregation-prone TM region.
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