| 21. |
- Sjölander, Daniel, et al.
(författare)
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Establishing the fluorescent amyloid ligand h-FTAA for studying human tissues with systemic and localized amyloid
- 2016
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Ingår i: Amyloid. - : Informa UK Limited. - 1350-6129 .- 1744-2818. ; 23:2, s. 98-108
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Tidskriftsartikel (refereegranskat)abstract
- Rapid and accurate detection of amyloid deposits in routine surgical pathology settings are of great importance. The use of fluorescence microscopy in combination with appropriate amyloid specific dyes is very promising in this regard. Here we report that a luminescent conjugated oligothiophene, h-FTAA, rapidly and with high sensitivity and selectivity detects amyloid deposits in verified clinical samples from systemic amyloidosis patients with AA, AL and ATTR types; as well as in tissues laden with localized amyloidosis of AANF, AIAPP and ASem1 type. The probe h-FTAA emitted yellow red fluorescence on binding to amyloid deposits, whereas no apparent staining was observed in surrounding tissue. The only functional structure stained with h-FTAA showing the amyloidotypic fluorescence spectrum was Paneth cell granules in intestine. Screening of 114 amyloid containing tissues derived from 107 verified (Congo red birefringence and/or immunohistochemistry) amyloidosis patients revealed complete correlation between h-FTAA and Congo red fluorescence (107/107, 100% sensitivity). The majority of Congo red negative control cases (27 of 32, 85% specificity) were negative with h-FTAA. Small Congo red negative aggregates in kidney, liver, pancreas and duodenum were found by h-FTAA fluorescence in five control patients aged 72-83 years suffering from diverse diseases. The clinical significance of these false-positive lesions is currently not known. Because h-FTAA fluorescence is one magnitude brighter than Congo red and as the staining is performed four magnitudes lower than the concentration of dye, we believe that these inclusions are beyond detection by Congo red. We conclude that h-FTAA is a fluorescent hypersensitive, rapid and powerful tool for identifying amyloid deposits in tissue sections. Use of h-FTAA can be exploited as a rapid complementary technique for accurate detection of amyloid in routine surgical pathology settings. Our results also implicate the potential of the technique for detection of prodromal amyloidosis as well as for discovery of new amyloid-like protein aggregates in humans.
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| 22. |
- Sjölander, Daniel, et al.
(författare)
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Evaluation of the fluorescent amyloid ligand h-FTAA in human tissues with systemic and localized amyloid
- 2014
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Rapid and accurate detection of amyloid deposits in routine surgical pathology settings are of great importance. The use of fluorescence microscopy in combination with appropriate amyloid specific dyes is very promising in this regard. Most systemic amyloidosis are progressive and lethal. Disease specific therapy depends on the identification of the offending proteins. Here we report that a luminescent conjugated oligothiophene, h-FTAA, rapidly and with high sensitivity and selectivity detects amyloid deposits in verified clinical samples from systemic amyloidosis patients with AA, AL, and ATTR types; as well as in tissues laden with localized amyloidosis of AANF, AIAPP and ASem1 type. The probe h-FTAA emitted yellow red fluorescence on binding to amyloid deposits, whereas no apparent staining was observed in surrounding tissue. Screening of 114 amyloid containing tissues derived from §07 verified (Congo red birefringence and immunohistochemistry) amyloidosis patients revealed complete correlation between h-FTAA and Congo red fluorescence. We conclude that h-FTAA is a fluorescent hypersensitive, rapid and powerful tool for identifying amyloid deposits in tissue sections. H-FTAA staining can be utilized as a rapid complementary technique for accurate detection of amyloid in routine surgical pathology settings. It was also revealed that within 5 of 15 age matched Congo red negative control samples h-FTAA detects microdeposits of amyloid-like protein aggregates in liver and kidney. The results emphasize the potential of the dye for detection of prodromal amyloidosis as well as for discovery of novel amyloid-like protein aggregates in humans.
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| 23. |
- Solé-Domènech, Santiago, et al.
(författare)
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Localization of cholesterol, amyloid and glia in Alzheimer's disease transgenic mouse brain tissue using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and immunofluorescence imaging
- 2013
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Ingår i: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 0001-6322 .- 1432-0533. ; 125:1, s. 145-157
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Tidskriftsartikel (refereegranskat)abstract
- The spatial distributions of lipids, amyloid-beta deposits, markers of neurons and glial cells were imaged, at submicrometer lateral resolution, in brain structures of a mouse model of Alzheimer's disease using a new methodology that combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) and confocal fluorescence microscopy. The technology, which enabled us to simultaneously image the lipid and glial cell distributions in Tg2576 mouse brain structures, revealed micrometer-sized cholesterol accumulations in hippocampal regions undergoing amyloid-beta deposition. Such cholesterol granules were either associated with individual amyloid deposits or spread over entire regions undergoing amyloidogenesis. Subsequent immunohistochemical analysis of the same brain regions showed increased microglial and astrocytic immunoreactivity associated with the amyloid deposits, as expected from previous studies, but did not reveal any particular astrocytic or microglial feature correlated with cholesterol granulation. However, dystrophic neurites as well as presynaptic vesicles presented a distribution similar to that of cholesterol granules in regions undergoing amyloid-beta accumulation, thus indicating that these neuronal endpoints may retain cholesterol in areas with lesions. In conclusion, the present study provides evidence for an altered cholesterol distribution near amyloid deposits that would have been missed by several other lipid analysis methods, and opens for the possibility to study in detail the putative liaison between lipid environment and protein structure and function in Alzheimer's disease.
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| 24. |
- Syrén, Per-Olof, et al.
(författare)
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Design, structure and plasma binding of ancestral β-CoV scaffold antigens
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pandemic caused by Severe acute respiratory syndrome coronavirus 2 has had devastating consequences on global health and economy. Despite the success of vaccination campaigns emerging variants are of concern and novel viruses with the potential to drive future pandemics are circulating in nature. Development of vaccines can be challenging, as key viral protein antigens can be unstable or aggregate. In this study, we present the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interacted with plasma of patients recovered from COVID-19 but did not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yielded high resolution structures (2.6-2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. This captured closed state is stabilised by an intricate hydrogen‑bonding network mediated by well-resolved loops, both within and across monomers, tethering the N‑terminal domain and RBD together, which determines their relative spatial orientation. Finally, we show how AnSAs are potent scaffolds by replacing the ancestral RBD with the Wuhan wild-type sequence, which restored ACE2 binding and increased the interaction with convalescent plasma. In contrast to rational antigen design depending on prior structural knowledge, our work highlights how stable and potentially interesting antigens can be generated using exclusively available sequence information.
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| 25. |
- Abrahamsson, Maria, et al.
(författare)
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Steric influence on the excited-state lifetimes of ruthenium complexes with bipyridyl-alkanylene-pyridyl ligands.
- 2008
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Ingår i: Inorganic Chemistry. - : ACS. - 0020-1669 .- 1520-510X. ; 47:9, s. 3540-3548
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Tidskriftsartikel (refereegranskat)abstract
- The structural effect on the metal-to-ligand charge transfer (MLCT) excited-state lifetime has been investigated in bis-tridentate Ru(II)-polypyridyl complexes based on the terpyridine-like ligands [6-(2,2'-bipyridyl)](2-pyridyl)methane (1) and 2-[6-(2,2'-bipyridyl)]-2-(2-pyridyl)propane (2). A homoleptic ([Ru(2)(2)](2+)) and a heteroleptic complex ([Ru(ttpy)(2)](2+)) based on the new ligand 2 have been prepared and their photophysical and structural properties studied experimentally and theoretically and compared to the results for the previously reported [Ru(1)(2)](2+). The excited-state lifetime of the homoleptic Ru-II complex with the isopropylene-bridged ligand 2 was found to be 50 times shorter than that of the corresponding homoleptic Ru-II complex of ligand 1, containing a methylene bridge. A comparison of the ground-state geometries of the two homoleptic complexes shows that steric interactions involving the isopropylene bridges make the coordination to the central Ru-II ion less octahedral in [Ru(2)(2)](2+) than in [Ru(1)(2))(2+). Calculations indicate that the structural differences in these complexes influence their ligand field splittings as well as the relative stabilities of the triplet metal-to-ligand charge transfer ((MLCT)-M-3) and metal-centered ((MC)-M-3) excited states. The large difference in measured excited-state lifetimes for the two homoleptic Ru-II complexes is attributed to a strong influence of steric interactions on the ligand field strength, which in turn affects the activation barriers for thermal conversion from (MLCT)-M-3 states to short-lived (MC)-M-3 states.
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| 26. |
- Aguilar-Calvo, Patricia, et al.
(författare)
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Generation of novel neuroinvasive prions following intravenous challenge
- 2018
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Ingår i: Brain Pathology. - : WILEY. - 1015-6305 .- 1750-3639. ; 28:6, s. 999-1011
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Tidskriftsartikel (refereegranskat)abstract
- Prions typically spread into the central nervous system (CNS), likely via peripheral nerves. Yet prion conformers differ in their capacity to penetrate the CNS; certain fibrillar prions replicate persistently in lymphoid tissues with no CNS entry, leading to chronic silent carriers. Subclinical carriers of variant Creutzfeldt-Jakob (vCJD) prions in the United Kingdom have been estimated at 1:2000, and vCJD prions have been transmitted through blood transfusion, however, the circulating prion conformers that neuroinvade remain unclear. Here we investigate how prion conformation impacts brain entry of transfused prions by challenging mice intravenously to subfibrillar and fibrillar strains. We show that most strains infiltrated the brain and caused terminal disease, however, the fibrillar prions showed reduced CNS entry in a strain-dependent manner. Strikingly, the highly fibrillar mCWD prion strain replicated in the spleen and emerged in the brain as a novel strain, indicating that a new neuroinvasive prion had been generated from a previously non-neuroinvasive strain. The new strain showed altered plaque morphology, brain regions targeted and biochemical properties and these properties were maintained upon intracerebral passage. Intracerebral passage of prion-infected spleen re-created the new strain. Splenic prions resembled the new strain biochemically and intracerebral passage of prion-infected spleen re-created the new strain, collectively suggesting splenic prion replication as a potential source. Taken together, these results indicate that intravenous exposure to prion-contaminated blood or blood products may generate novel neuroinvasive prion conformers and disease phenotypes, potentially arising from prion replication in non-neural tissues or from conformer selection.
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| 27. |
- Almstedt, Karin, 1980-, et al.
(författare)
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Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
- 2009
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Ingår i: Prion. - Austin : Landes Bioscience Journals. - 1933-6896 .- 1933-690X. ; 3:4, s. 224-235
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Tidskriftsartikel (refereegranskat)abstract
- Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly α-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90-231 and 121-231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non-thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.
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| 28. |
- Almstedt, Karin, 1980-
(författare)
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Protein Misfolding in Human Diseases
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories:Loss-of-function diseasesGain-of-toxic-function diseasesInfectious misfolding diseases Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein. The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease.Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.
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| 29. |
- Almstedt, Karin, et al.
(författare)
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Small-Molecule Suppression of Misfolding of Mutated Human Carbonic Anhydrase II Linked to Marble Brain Disease
- 2009
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 48:23, s. 5358-5364
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Tidskriftsartikel (refereegranskat)abstract
- Carbonic anhydrase II deficiency syndrome or Marble brain disease (MBD) is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. Here we report a small-molecule stabilization study of the exceptionally destabilized HCA II mutant H107Y employing inhibitors based on p-aminobenzoyisulfonamide compounds and 1,3,4-thiadiazolylsulfonamides as well as amino acid activators. Protein stability assays showed a significant stabilization by the aromatic sulfonamide inhibitors when present at 10 mu M concentration, providing shifts of the midpoint of thermal denaturation between 10 degrees C and 16 degrees C and increasing the free energies of denaturation 0.5-3.0 kcal/mol as deduced from GuHCl denaturation. This study could be used as a starting point for the design of small-molecule folding modulators and possibly autoactivatable molecules for suppression of misfolding of destabilized HCA II mutants.
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| 30. |
- Almstedt, Karin, 1980-, et al.
(författare)
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Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease.
- 2008
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Ingår i: Biochemistry. - Washington : ACS. - 0006-2960 .- 1520-4995. ; 47:5, s. 1288-1298
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Tidskriftsartikel (refereegranskat)abstract
- Marble brain disease (MBD) also known as Guibaud−Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded β-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619−633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.
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