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| 2. |
- Rahman, M. M., et al.
(författare)
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Protofibrillar and Fibrillar Amyloid-β Binding Proteins in Cerebrospinal Fluid
- 2018
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Ingår i: Journal of Alzheimer's Disease. - 1387-2877. ; 66:3, s. 1053-1064
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation and deposition of misfolded amyloid-β (Aβ) peptide in the brain is central to Alzheimer's disease (AD). Oligomeric, protofibrillar, and fibrillar forms of Aβ are believed to be neurotoxic and cause neurodegeneration in AD, but the toxicity mechanisms are not well understood and may involve Aβ-interacting molecular partners. In a previous study, we identified potential Aβ 42 protofibrillar-binding proteins in serum and cerebrospinal fluid (CSF) using an engineered version of Aβ 42 (Aβ 42 CC) that forms protofibrils, but not fibrils. Here we studied binding of proteins to Aβ 42 fibrils in AD and non-AD CSF and compared these with protofibrillar Aβ 42 CC-binding partners. Aβ 42 fibrils sequestered 2.4-fold more proteins than Aβ 42 CC protofibrils. Proteins with selective binding to fibrillar aggregates with low nanomolar affinity were identified. We also found that protofibrillar and fibrillar Aβ-binding proteins represent distinct functional categories. Aβ 42 CC protofibrils triggered interactions with proteins involved in catalytic activities, like transferases and oxidoreductases, while Aβ 42 fibrils were more likely involved in binding to proteoglycans, growth factors and neuron-Associated proteins, e.g., neurexin-1,-2, and-3. Interestingly, 10 brain-enriched proteins were identified among the fibril-binding proteins, while protofibril-extracted proteins had more general expression patterns. Both types of Aβ aggregates bound several extracellular proteins. Additionally, we list a set of CSF proteins that might have potential to discriminate between AD and non-AD CSF samples. The results may be of relevance both for biomarker studies and for studies of Aβ-related toxicity mechanisms. © 2018-IOS Press and the authors. All rights reserved.
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| 3. |
- Wolf-Watz, M, et al.
(författare)
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Solution properties of the free and DNA-bound Runt domain of AML1.
- 1999
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Ingår i: European Journal of Biochemistry. - 0014-2956 .- 1432-1033. ; 261:1, s. 251-60
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Tidskriftsartikel (refereegranskat)abstract
- The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.
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| 4. |
- Lendel, Christofer, et al.
(författare)
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A Hexameric Peptide Barrel as Building Block of Amyloid-β Protofibrils
- 2014
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Ingår i: Angewandte Chemie. - : Wiley. - 0044-8249 .- 1521-3757. ; 126:47, s. 12970-12974
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Tidskriftsartikel (refereegranskat)abstract
- Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer’s disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40.
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| 5. |
- Lendel, Christofer, 1976-, et al.
(författare)
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A Hexameric Peptide Barrel as Building Block of Amyloid-β Protofibrils
- 2014
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 53:47, s. 12756-12760
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Tidskriftsartikel (refereegranskat)abstract
- Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer’s disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40.
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| 6. |
- Lindborg, M., et al.
(författare)
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High-affinity binding to staphylococcal protein A by an engineered dimeric Affibody molecule
- 2013
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 26:10, s. 635-644
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are engineered binding proteins, in which the three-helix bundle motif of the Z domain derived from protein A is used as a scaffold for sequence variation. We used phage display to select Affibody binders to staphylococcal protein A itself. The best binder, called ZpA963, binds with similar affinity and kinetics to the five homologous E, D, A, B and C domains of protein A, and to a five-domain protein A construct with an average dissociation constant, K-D, of 20 nM. The structure of ZpA963 in complex with the Z domain shows that it interacts with a surface on Z that is identical in the five protein A domains, which explains the multi-domain affinity. This property allows for high-affinity binding by dimeric Affibody molecules that simultaneously engage two protein A domains in a complex. We studied two ZpA963 dimers in which the subunits were linked by a C-terminal disulfide in a symmetric dimer or head-to-tail in a fusion protein, respectively. The dimers both bind protein A with high affinity, very slow off-rates and with saturation-dependent kinetics that can be understood in terms of dimer binding to multiple sites. The head-to-tail (ZpA963)(2)htt dimer binds with an off-rate of k(off) 5 10(6) s(1) and an estimated K-D 16 pM. The results illustrate how dimers of selected monomer binding proteins can provide an efficient route for engineering of high-affinity binders to targets that contain multiple homologous domains or repeated structural units.
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| 7. |
- Wolf-Watz, M, et al.
(författare)
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Structure and backbone dynamics of Apo-CBFbeta in solution.
- 2001
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 40:38, s. 11423-11432
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Tidskriftsartikel (refereegranskat)abstract
- Runx proteins constitute a family of mammalian transcription factors that interact with DNA through their evolutionarily conserved Runt domain. CBFbeta, alternatively denoted PEBP2beta, is the non-DNA-binding heterodimer partner and acts to enhance the DNA binding affinity of Runx proteins. Runx proteins and CBFbeta are associated with a variety of biological functions and human diseases; they are, for example, together the most frequent targets for chromosomal rearrangements in acute human leukemias. We have determined the solution structure and characterized the backbone dynamics of C-terminally truncated fragments containing residues 1-141 of CBFbeta. The present apo-CBFbeta structure is very similar to that seen in a Runt-CBFbeta complex. An evaluation of backbone (15)N NMR relaxation parameters shows that CBFbeta is a rigid molecule with high order parameters throughout the backbone; the only regions displaying significant dynamics are a long loop and the C-terminal alpha-helix. A few residues display relaxation behavior indicating conformational exchange on microsecond to millisecond time scales, but only one of these is located at the Runt binding surface. Our structure and dynamics analysis of CBFbeta therefore suggests that the protein binds to Runt without large conformational changes or induced folding ("lock-and-key" interaction). The apo-CBFbeta structure presented here exhibits several significant differences with two other published NMR ensembles of very similar protein fragments. The differences are located in four regions outside of the central beta-barrel, whereas the beta-barrel itself is almost identical in the three NMR structures. The comparison illustrates that independently determined NMR structures may display rather large differences in backbone conformation in regions that appear to be well-defined in each of the calculated NMR ensembles.
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| 8. |
- Wärnmark, A, et al.
(författare)
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The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:49, s. 45939-45944
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Tidskriftsartikel (refereegranskat)abstract
- The N-terminal regions of the estrogen receptor ve (ER alpha -N) and beta (ER beta -N) were expressed and purified to homogeneity. Using NAM and circular dichroism spectroscopy, we conclude that both ER alpha -N and ER beta -N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ERa-N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ERa-N interacts with TBP, and suggest that structural changes are induced in ERa-N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ER beta -N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ERa-N and ER beta -N. The affinity of the ER alpha -N-TBP interaction was determined to be in the micromolar range (K-D = 10(-6) to 10(-5) m). Our results support models of TBP as a target protein for the N-terminal activation domain of ER alpha. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.
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