| 1. |
- Arosio, Paolo, et al.
(author)
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Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation
- 2016
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Journal article (peer-reviewed)abstract
- It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.
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| 2. |
- Busse, Marta, et al.
(author)
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Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation
- 2007
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:45, s. 32802-32810
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Journal article (peer-reviewed)abstract
- The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyl-transferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2-Y419X mutant.
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| 3. |
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| 4. |
- Carlsson, Pernilla, et al.
(author)
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Heparan sulfate biosynthesis: Characterization of an NDST1 splice variant
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Other publication (pop. science, debate, etc.)abstract
- N-Deacetylase/N-sulfotransferases (NDSTs) are Golgi-located enzymes involved in the biosynthesis of heparan sulfate. They are bifunctional enzymes responsible for N-deacetylation of N-acetylglucosamine residues followed by N-sulfation of the generated free amino groups. In this paper we have identified and characterized a splice variant of NDST1 mRNA. The alternatively spliced mRNA transcript was shown to be present in varying amounts in different adult and embryonic mouse tissues. The protein resulting from translation of the spliced transcript (NDST1S) lacks the C-terminal half of fullength NDST and appears to be devoid of enzyme activity. As shown in HEK 293 cells overexpressing NDST1, a high expression of the splice variant resulted in reduced levels of NDST1. Unexpectedly, the level of N-sulfation was largely unaltered in heparan sulfate produced in NDST1S overexpressing cells while 6-O-sulfation was elevated and 2-O-sulfation was reduced. NDST1S shares the ability of NDST1 to interact with EXT2, one of the components of the heparan sulfate copolymerase. We speculate that NDST1S may alter the composition of the tentaive enzyme complex, the GAGosome, resulting in changes in the structure of heparan sulfate synthesized.
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| 5. |
- Carlsson, Pernilla, et al.
(author)
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Heparin/heparan sulfate biosynthesis : Processive formation of N-sulfated domains
- 2008
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:29, s. 20008-20014
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Journal article (peer-reviewed)abstract
- Heparan sulfate (HS) proteoglycans influence embryonic development as well as adult physiology through interactions with various proteins, including growth factors/morphogens and their receptors. The interactions depend on HS structure, which is largely determined during biosynthesis by Golgi enzymes. A key step is the initial generation of N-sulfated domains, primary sites for further polymer modification and ultimately for functional interactions with protein ligands. Such domains, generated through action of a bifunctional GlcNAc N-deacetylase/N-sulfotransferase (NDST) on a [GlcUA-GlcNAc](n) substrate, are of variable size due to regulatory mechanisms that remain poorly understood. We have studied the action of recombinant NDSTs on the [GlcUA-GlcNAc](n) precursor in the presence and absence of the sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). In the absence of PAPS, NDST catalyzes limited and seemingly random N-deacetylation of GlcNAc residues. By contrast, access to PAPS shifts the NDST toward generation of extended N-sulfated domains that are formed through coupled N-deacetylation/N-sulfation in an apparent processive mode. Variations in N-substitution pattern could be obtained by varying PAPS concentration or by experimentally segregating the N-deacetylation and N-sulfation steps. We speculate that similar mechanisms may apply also to the regulation of HS biosynthesis in the living cell.
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| 6. |
- Chen, Gefei, et al.
(author)
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Bri2 BRICHOS client specificity and chaperone activity are governed by assembly state
- 2017
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In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8
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Journal article (peer-reviewed)abstract
- . Protein misfolding and aggregation is increasingly being recognized as a cause of disease. In Alzheimer's disease the amyloid-beta peptide (A beta) misfolds into neurotoxic oligomers and assembles into amyloid fibrils. The Bri2 protein associated with Familial British and Danish dementias contains a BRICHOS domain, which reduces A beta fibrillization as well as neurotoxicity in vitro and in a Drosophila model, but also rescues proteins from irreversible nonfibrillar aggregation. How these different activities are mediated is not known. Here we show that Bri2 BRICHOS monomers potently prevent neuronal network toxicity of A beta, while dimers strongly suppress A beta fibril formation. The dimers assemble into high-molecular-weight oligomers with an apparent two-fold symmetry, which are efficient inhibitors of non-fibrillar protein aggregation. These results indicate that Bri2 BRICHOS affects qualitatively different aspects of protein misfolding and toxicity via different quaternary structures, suggesting a means to generate molecular chaperone diversity.
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| 7. |
- Cohen, Samuel I A, et al.
(author)
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A molecular chaperone breaks the catalytic cycle that generates toxic Aβ oligomers.
- 2015
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In: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9985 .- 1545-9993. ; 22:3, s. 207-213
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Journal article (peer-reviewed)abstract
- Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.
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| 8. |
- Göransson, Anna-Lena
(author)
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The Alzheimer Aβ Peptide : Identification of Properties Distinctive for Toxic Prefibrillar Species
- 2012
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Licentiate thesis (other academic/artistic)abstract
- Proteins must have specific conformations to function correctly inside cells. However, sometimes they adopt the wrong conformation, causing dysfunction and disease. A number of amyloid diseases are caused by misfolded proteins that form amyloid fibrils. One such disease is Alzheimer’s disease (AD). The protein involved in this deadly disease is the amyloid β (Aβ) peptide. The formation of soluble prefibrillar oligomeric Aβ species has been recognized as an important factor in the development of AD. The aim of work described in this thesis was to investigate which properties of these oligomeric species can be linked to toxicity. We approached this task by comparing the aggregation behavior and biophysical properties of aggregates formed by variants of the Aβ peptide that have been shown to differ in neurotoxicity when expressed in the central nervous system (CNS) of Drosophila melanogaster. A combined set involving different fluorescent probes was used in parallell with transmission electron microscopy. The toxicity of species formed during the aggregation process was examined by exposing human SH-SY5Y neuroblastoma cells to Aβ aggregates. We deduced that there is a correlation between cell toxicity and the propensity of the Aβ peptide to form small prefibrillar assemblies at an early stage of aggregation in vitro. Moreover, these prefibrillar species were characterized by their ability to be recognized by pentamer formyl thiophene acetic acid (p-FTAA) and the presence of exposed hydrophobic patches. We also found that larger aggregates did not induce cell death.
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| 9. |
- Hermansson, Erik, et al.
(author)
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The chaperone domain BRICHOS prevents CNS toxicity of amyloid-beta peptide in Drosophila melanogaster
- 2014
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In: Disease Models and Mechanisms. - : The Company of Biologists. - 1754-8411 .- 1754-8403. ; 7:6, s. 659-665
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Journal article (peer-reviewed)abstract
- Aggregation of the amyloid-beta peptide (A beta) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer's disease (AD). Mutations of the BRICHOS chaperone domain are associated with amyloid disease and recent in vitro data show that BRICHOS efficiently delays A beta 42 oligomerization and fibril formation. We have generated transgenic Drosophila melanogaster flies that express the A beta 42 peptide and the BRICHOS domain in the central nervous system (CNS). Co-expression of A beta 42 and BRICHOS resulted in delayed A beta 42 aggregation and dramatic improvements of both lifespan and locomotor function compared with flies expressing A beta 42 alone. Moreover, BRICHOS increased the ratio of soluble: insoluble A beta 42 and bound to deposits of A beta 42 in the fly brain. Our results show that the BRICHOS domain efficiently reduces the neurotoxic effects of A beta 42, although significant A beta 42 aggregation is taking place. We propose that BRICHOS-based approaches should be explored with an aim towards the future prevention and treatment of AD.
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| 10. |
- Johansson, Jan, et al.
(author)
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A novel anti-amyloid chaperone
- 2010
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In: Amyloid. - 1350-6129 .- 1744-2818. ; 17, s. 103-104
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Conference paper (other academic/artistic)
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| 11. |
- Johansson, Jan, et al.
(author)
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BRICHOS binds to a designed amyloid-forming beta-protein and reduces proteasomal inhibition and aggresome formation
- 2016
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 473, s. 167-178
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Journal article (peer-reviewed)abstract
- The BRICHOS domain is associated with proliferative, degenerative and amyloid diseases, and it has been shown to inhibit fibril formation and toxicity of the Alzheimer's disease-associated amyloid beta-peptide. ProSP-C (prosurfactant protein C) BRICHOS binds to stretches of hydrophobic amino acid residues, which are unfolded or in beta-strand conformation, suggesting that it may have broad anti-amyloid activity. We have studied the effect of the proSP-C BRICHOS domain on the designed amyloidogenic beta-sheet proteins beta 17 and beta 23. beta 17 expressed in the secretory pathway of HEK (human embryonic kidney)-293 cells forms intracellular inclusions, whereas beta 23 is rapidly degraded. Co-expression of BRICHOS leads to a reduction in beta 17 inclusion size and increased levels of soluble beta 17 and beta 23. Furthermore, BRICHOS interacts with the beta-proteins intracellularly, reduces their ubiquitination and decreases aggresome formation and proteasomal inhibition. Collectively, these data suggest that BRICHOS is capable of delaying the aggregation process and toxicity of amyloidogenic proteins in a generic manner.
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| 12. |
- Johansson, Jan, et al.
(author)
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Control of amyloid assembly by autoregulation
- 2012
-
In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 447, s. 185-192
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Research review (peer-reviewed)abstract
- The assembly of proteins into amyloid fibrils can be an element of both protein aggregation diseases and a functional unit in healthy biological pathways. In both cases, it must be kept under tight control to prevent undesired aggregation. In normophysiology, proteins can self-chaperone amyloidogenic segments by restricting their conformational flexibility in an overall stabilizing protein fold. However, some aggregation-prone segments cannot be controlled in this manner and require additional regulatory elements to limit fibrillation. The present review summarizes different molecular mechanisms that proteins use to control their own assembly into fibrils, such as the inclusion of a chaperoning domain or a blocking segment in the proform, the controlled release of an amyloidogenic region from the folded protein, or the adjustment of fibrillation propensity according to pH. Autoregulatory elements can control disease-related as well as functional fibrillar protein assemblies and distinguish a group of self-regulating amyloids across a wide range of biological functions and organisms.
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| 13. |
- Knight, Stefan D., et al.
(author)
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The BRICHOS Domain, Amyloid Fibril Formation, and Their Relationship
- 2013
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:43, s. 7523-7531
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Journal article (peer-reviewed)abstract
- Amyloid diseases are defined by tissue deposition of insoluble, fibrillar beta-sheet polymers of specific proteins, but it appears that toxic oligomeric species rather than the fibrils are the main cause of tissue degeneration. Many proteins can form amyloid-like fibrils in vitro, but only similar to 30 proteins have been found to cause mammalian amyloid disease, suggesting that physiological mechanisms that protect against amyloid formation exist. The transmembrane region of lung surfactant protein C precursor (proSP-C) forms amyloid-like fibrils in vitro, and SP-C amyloid has been found in lung tissue from patients with interstitial lung disease (ILD). ProSP-C contains a BRICHOS domain, in which many ILD-associated mutations are localized, and the BRICHOS domain can prevent SP-C from forming amyloid-like fibrils. Recent data suggest that recombinant BRICHOS domains from proSP-C and Bri2 (associated with familial dementia and amyloid formation) interact with peptides with a strong propensity to form beta-sheet structures, including amyloid beta-peptide associated with Alzheimer's disease. Such interactions efficiently delay formation of fibrils and oligomers. The BRICHOS domain is defined at the sequence level and is found in similar to 10 distantly related proprotein families. These have widely different or unknown functions, but several of the proteins are associated with human disease. Structural modeling of various BRICHOS domains, based on the X-ray structure of the proSP-C BRICHOS domain, identifies a conserved region that is structurally complementary to the beta-sheet- and/or amyloid-prone regions in the BRICHOS domain-containing proproteins. These observations make the BRICHOS domain the first example of a chaperone-like domain with specificity for beta-prone regions.
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| 14. |
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| 15. |
- Lee, Hunsang, et al.
(author)
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Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells
- 2014
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In: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 450:4, s. 1587-1592
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Journal article (peer-reviewed)abstract
- Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6(102), SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C-in, form. MRP6(102) and SP-C(Leu/Val) are inserted into the membrane in C-out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
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| 16. |
- Nerelius, Charlotte, et al.
(author)
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Conformational preferences of non-polar amino acid residues: An additional factor in amyloid formation
- 2010
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In: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 402, s. 515-518
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Journal article (peer-reviewed)abstract
- Amyloid consists of beta-sheet polymers and is associated with disease and with functional assemblies Amyloid-forming proteins differ widely in native structures and sequences. We describe here how conformational preferences of non-polar amino acid residues can affect amyloid formation The most nonpolar residues promote either beta-strands (Val, Ile, Phe, and Cys. VIFC) or alpha-helices (Leu, Ala, and Met, LAM), while the most polar residues promote only alpha-helices. For 12 proteins associated with disease, the localizations of the amyloid core regions are known Eleven of these contain segments that are biased for VIFC, but essentially lack segments that are biased for LAM For the amyloid beta-peptide associated with Alzheimer's disease and an amyloidogenic fragment of the prion protein, observed effects of mutations support that VIFC bias favors formation of beta-sheet aggregates and arnyloid, while LAM bias prevents it VIFC and LAM profiles combine information on secondary structure propensities and polarity, and add a simple criterion to the prediction of amyloidogenic regions (C) 2010 Elsevier Inc All rights reserved
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| 17. |
- Noborn, Fredrik, et al.
(author)
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Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein
- 2011
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:14, s. 5584-5589
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Journal article (peer-reviewed)abstract
- Transthyretin (TTR) is a homotetrameric protein that transports thyroxine and retinol. Tetramer destabilization and misfolding of the released monomers result in TTR aggregation, leading to its deposition as amyloid primarily in the heart and peripheral nervous system. Over 100 mutations of TTR have been linked to familial forms of TTR amyloidosis. Considerable effort has been devoted to the study of TTR aggregation of these mutants, although the majority of TTR-related amyloidosis is represented by sporadic cases due to the aggregation and deposition of the otherwise stable wild-type (WT) protein. Heparan sulfate (HS) has been found as a pertinent component in a number of amyloid deposits, suggesting its participation in amyloidogenesis. This study aimed to investigate possible roles of HS in TTR aggregation. Examination of heart tissue from an elderly cardiomyopathic patient revealed substantial accumulation of HS associated with the TTR amyloid deposits. Studies demonstrated that heparin/HS promoted TTR fibrillization through selective interaction with a basic motif of TTR. The importance of HS for TTR fibrillization was illustrated in a cell model; TTR incubated with WT Chinese hamster ovary cells resulted in fibrillization of the protein, but not with HS-deficient cells (pgsD-677). The effect of heparin on TTR fibril formation was further demonstrated in a Drosophila model that overexpresses TTR. Heparin was colocalized with TTR deposits in the head of the flies reared on heparin-supplemented medium, whereas no heparin was detected in the nontreated flies. Heparin of low molecular weight (Klexane) did not demonstrate this effect.
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| 18. |
- Oskarsson, Marie E., et al.
(author)
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The BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells
- 2018
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:12, s. E2752-E2761
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Journal article (peer-reviewed)abstract
- Aggregation of islet amyloid polypeptide (IAPP) into amyloid fibrils in islets of Langerhans is associated with type 2 diabetes, and formation of toxic IAPP species is believed to contribute to the loss of insulin-producing beta cells. The BRICHOS domain of integral membrane protein 2B (Bri2), a transmembrane protein expressed in several peripheral tissues and in the brain, has recently been shown to prevent fibril formation and toxicity of Aβ42, an amyloid-forming peptide in Alzheimer disease. In this study, we demonstrate expression of Bri2 in human islets and in the human beta-cell line EndoC-βH1. Bri2 colocalizes with IAPP intracellularly and is present in amyloid deposits in patients with type 2 diabetes. The BRICHOS domain of Bri2 effectively inhibits fibril formation in vitro and instead redirects IAPP into formation of amorphous aggregates. Reduction of endogenous Bri2 in EndoC-βH1 cells with siRNA increases sensitivity to metabolic stress leading to cell death while a concomitant overexpression of Bri2 BRICHOS is protective. Also, coexpression of IAPP and Bri2 BRICHOS in lateral ventral neurons of Drosophila melanogaster results in an increased cell survival. IAPP is considered to be the most amyloidogenic peptide known, and described findings identify Bri2, or in particular its BRICHOS domain, as an important potential endogenous inhibitor of IAPP aggregation and toxicity, with the potential to be a possible target for the treatment of type 2 diabetes.
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| 19. |
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| 20. |
- Presto, Jenny, et al.
(author)
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Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation
- 2008
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:12, s. 4751-4756
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Journal article (peer-reviewed)abstract
- Heparan sulfate (HS) proteoglycans influence embryonic development and adult physiology through interactions with protein ligands. The interactions depend on HS structure, which is determined largely during biosynthesis by Golgi enzymes. How biosynthesis is regulated is more or less unknown. During polymerization of the HS chain, carried out by a complex of the exostosin proteins EXT1 and EXT2, the first modification enzyme, glucosaminyl N-deacetylase/N-sulfotransferase (NDST), introduces N-sulfate groups into the growing polymer. Unexpectedly, we found that the level of expression of EXT1 and EXT2 affected the amount of NDST1 present in the cell, which, in turn, greatly influenced HS structure. Whereas overexpression of EXT2 in HEK 293 cells enhanced NDST1 expression, increased NDST1 N-glycosylation, and resulted in elevated HS sulfation, overexpression of EXT1 had opposite effects. Accordingly, heart tissue from transgenic mice overexpressing EXT2 showed increased NDST activity. Immunoprecipitaion experiments suggested an interaction between EXT2 and NDST1. We speculate that NDST1 competes with EXT1 for binding to EXT2. Increased NDST activity in fibroblasts with a gene trap mutation in EXT1 supports this notion. These results support a model in which the enzymes of HS biosynthesis form a complex, or a GAGosome.
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| 21. |
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| 22. |
- Presto, Jenny, 1973-
(author)
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N-Sulfation and Polymerization in Heparan Sulfate Biosynthesis
- 2006
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Doctoral thesis (other academic/artistic)abstract
- Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is N-sulfated by the enzyme N-deacetylase N-sulfotransferase (NDST). N-Sulfation occurs in domains where further modifications (including O-sulfations) take place, giving the chain a complex sulfation pattern.In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that N-deacetylation is the rate-limiting step in HS N-sulfation and that two different NDST molecules can work on the same GlcN unit.By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for N-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on N-deacetylase activity. We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones..
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| 23. |
- Saenz, Alejandra, et al.
(author)
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Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment
- 2015
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:28, s. 17628-17641
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Journal article (peer-reviewed)abstract
- Surfactant protein C (SP-C) is a novel amyloid protein found in the lung tissue of patients suffering from interstitial lung disease (ILD) due to mutations in the gene of the precursor protein pro-SP-C. SP-C is a small alpha-helical hydrophobic protein with an unusually high content of valine residues. SP-C is prone to convert into beta-sheet aggregates, forming amyloid fibrils. Nature's way of solving this folding problem is to include a BRICHOS domain in pro-SP-C, which functions as a chaperone for SP-C during biosynthesis. Mutations in the pro-SP-C BRICHOS domain or linker region lead to amyloid formation of the SP-C protein and ILD. In this study, we used an in vitro transcription/translation system to study translocon-mediated folding of the WT pro-SP-C poly-Val and a designed poly-Leu transmembrane (TM) segment in the endoplasmic reticulum (ER) membrane. Furthermore, to understand how the pro-SP-C BRICHOS domain present in the ER lumen can interact with the TM segment of pro-SP-C, we studied the membrane insertion properties of the recombinant form of the pro-SP-C BRICHOS domain and two ILD-associated mutants. The results show that the co-translational folding of the WT pro-SP-C TM segment is inefficient, that the BRICHOS domain inserts into superficial parts of fluid membranes, and that BRICHOS membrane insertion is promoted by poly-Val peptides present in the membrane. In contrast, one BRICHOS and one non-BRICHOS ILD-associated mutant could not insert into membranes. These findings support a chaperone function of the BRICHOS domain, possibly together with the linker region, during pro-SP-C biosynthesis in the ER.
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| 24. |
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| 25. |
- Willander, Hanna, et al.
(author)
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BRICHOS domain associated with lung fibrosis, dementia and cancer - a chaperone that prevents amyloid fibril formation?
- 2011
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In: FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 278, s. 3893-3904
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Research review (peer-reviewed)abstract
- The BRICHOS domain was initially defined from sequence alignments of the Bri protein associated with familial dementia, chondromodulin associated with chondrosarcoma and surfactant protein C precursor (proSP-C) associated with respiratory distress syndrome and interstitial lung disease (ILD). Today BRICHOS has been found in 12 protein families. Mutations in the Bri2 and proSP-C genes result in familial dementia and ILD, respectively, and both these conditions are associated with amyloid formation. Amyloid is of great medical relevance as it is found in several major incurable diseases, like Alzheimer's and Parkinson's disease and diabetes mellitus. Work on recombinant BRICHOS domains and transfected cells indicate that BRICHOS is a chaperone domain that, during biosynthesis, binds to precursor protein regions with high beta-sheet propensities, thereby preventing them from amyloid formation. Regions prone to form beta-sheets are present in all BRICHOS-containing precursor proteins and are probably eventually released by proteolytic cleavage, generating different peptides with largely unknown bioactivities. Recombinant BRICHOS domains from Bri2 and proSP-C have been found to efficiently prevent SP-C, the amyloid beta-peptide associated with Alzheimer's disease, and medin, found in aortic amyloid, from forming amyloid fibrils. The data collected so far on BRICHOS raise several interesting topics for further research: (a) amyloid formation is a potential threat for many more proteins than the ones recognized so far in amyloid diseases; (b) amyloid formation of widely different peptides involves intermediate(s) that are recognized by the BRICHOS domain, suggesting that they have distinct structural similarities; and (c) the BRICHOS domain might be harnessed in therapeutic strategies against amyloid diseases.
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