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  • 51.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Protein engineering for biophysical studies of protein folding, stability and surface interactions2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The subject of this thesis can be split into two parts, one that is dealing with the stability and stabilization of proteins on its own merits and a second part that deals with the protein adsorption orientation on solid surfaces and how the stability of a protein influences the behavior at a solid/liquid interface. Thus, the common denominator and focus have been on protein stability. Molecular modeling and site-directed mutagenesis tools have been used to engineer a protein, human carbonic anhydrase II (HCA II), for these studies. The effects of these mutations on stability and surface interactions have then been studied by biophysical methods.

    Stabilization of HCA II by an engineered disulfide bridge: To find a way to stabilize the enzyme HCA II, homology modeling against the related and unusually stable carbonic anhydrase from Neisseria gonorrhoeae (NGCA) was performed. We were able to successfully utilize the homology modeling to graft a disulfide bridge from NGCA into the human enzyme. The disulfide bond was not formed spontaneously, but would only form after a prolonged exposure to oxidizing agents. However, formation of the disulfide bridge led to a dramatic stabilization of the native conformation.

    Accelerated formation of the disulfide bridge: It was found that it is the conformationally restrained localization of the introduced cysteines that is the reason that the protein is expressed in the reduced state and is not readily oxidized. However, upon exposure to low concentrations of denaturant, corresponding to the lower part of the denaturation curve for the first unfolding transition of the reduced state, there was a striking increase of the oxidation rate of correctly formed disulfide bridges. This provides a method for creating the oxidized disulfide variant of proteins, with engineered cysteines in the interior of proteins, which would otherwise not be formed within an acceptable time span.

    Refolding studies of stabilized variant of HCA II: The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition with suppression of the otherwise stable equilibrium. molten-globule intermediate, which normally is very prone to aggregation. Stopped-flow measurements also showed that the population of the transiently occurring molten globule was suppressed during refolding. This circumnavigation of misfolding traps and intermediates led to a markedly lowered tendency for aggregation and to significantly higher reactivation yields upon refolding of the fully denatured protein.

    Correlation between protein stability and surface induced denaturation: Negatively charged silica nanoparticles were used in order to determine the influence of protein stability on the denaturation rate upon adsorption. Various destabilized mutants were produced by site-directed mutagenesis of amino acids located in the interior of the protein. The silica nanoparticles induced a molten globule-like state in all of the variants. All protein variants initially adsorbed to the particles, and subsequently underwent conformational rearrangements in a stepwise manner. This study also showed that a decrease in the global stability of the protein is strongly correlated to increased rates of conformational change upon adsorption to the surface.

    Determination of protein adsorption orientation: By site-directed labeling fluorescent probes were specifically introduced on the surface of HCA II and it was shown, for the first time, that it is possible to specifically determine the orientation of an adsorbed protein in the native state to a surface (silica nanoparticles). By this approach it was possible to clearly demonstrate that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein and, furthermore, that the adsorption direction is strongly pH-dependent.

    Reduction of irreversible protein adsorption by protein stabilization: The strong correlation between decreased stability and increased rates of conformational changes of the protein upon adsorption to surfaces initiated yet another surface study. Three variants of HCA IT with lower, the same, and higher stability than the wild-type protein were monitored by surface plasmon resonance upon adsorption to and desorption from surfaces with fundamentally different properties. Regardless of the nature of the surface there were correlations between (i) the protein stability and kinetics of adsorption with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability, demonstrating that protein engineering for increased stability can be used to reduce irreversible protein adsorption.

    Delarbeten
    1. Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond
    Öppna denna publikation i ny flik eller fönster >>Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond
    2002 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, nr 52, s. 15867-15875Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten−globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-46774 (URN)10.1021/bi020433+ (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
    2. Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations
    Öppna denna publikation i ny flik eller fönster >>Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations
    2005 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 9, s. 3487-3493Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-45492 (URN)10.1021/bi048610p (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
    3. Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase
    Öppna denna publikation i ny flik eller fönster >>Circumnavigating misfolding traps in the energy landscape through protein engineering: suppression of molten globule and aggregation in carbonic anhydrase
    2004 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 21, s. 6803-6807Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The native state of the enzyme human carbonic anhydrase (HCA II) has been stabilized by the introduction of a disulfide bond, the oxidized A23C/L203C mutant. This stabilized protein variant undergoes an apparent two-state unfolding process with suppression of the otherwise stable equilibrium, molten-globule intermediate, which is normally very prone to aggregation. Stopped-flow measurements also showed that lower amounts of the transiently occurring molten globule were formed during refolding. This led to a markedly lowered tendency for aggregation during equilibrium denaturing conditions and, more importantly, to significantly higher reactivation yields upon refolding of the fully denatured protein. Thus, a general strategy to circumvent aggregation during the refolding of proteins could be to stabilize the native state of a protein at the expense of partially folded intermediates, thereby shifting the unfolding behavior from a three-state process to a two-state one.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-45720 (URN)10.1021/bi049709z (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
    4. Adsorption of human carbonic anhydrase II variants to silica nanoparticles occur stepwise: binding is followed by successive conformational changes to a molten-globule-like state
    Öppna denna publikation i ny flik eller fönster >>Adsorption of human carbonic anhydrase II variants to silica nanoparticles occur stepwise: binding is followed by successive conformational changes to a molten-globule-like state
    2000 (Engelska)Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 16, nr 22, s. 8470-8479Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The surface adsorption behavior of protein variants of the enzyme human carbonic anhydrase II (HCA II) to silica nanoparticles has been investigated. Various destabilized mutants were produced by site-directed mutagenesis of amino acids located in the interior of the protein. The silica particles induced a molten-globule-like state in all of the variants. All protein variants initially adsorbed to the particles, and then underwent conformational rearrangements in a stepwise manner, as indicated by the loss of activity and the subsequent loss of tertiary structure. Activity, CD, and ANS fluorescence measurements showed that a decrease in the global stability of the protein is strongly correlated to increased rates of conformational change following particle adsorption. In contrast to unfolding processes induced by chemical denaturants or heat, in the transition to the molten-globule-like state induced by the silica particles, the active site region unfolds before the majority of the tertiary interactions are broken.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-47929 (URN)10.1021/la0002738 (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-13
    5. Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles
    Öppna denna publikation i ny flik eller fönster >>Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles
    2005 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, nr 5, s. 3536-3544Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Little is known about the direction and specificity of protein adsorption to solid surfaces, a knowledge that is of great importance in many biotechnological applications. To resolve the direction in which a protein with known structure and surface potentials binds to negatively charged silica nanoparticles, fluorescent probes were attached to different areas on the surface of the protein human carbonic anhydrase II. By this approach it was clearly demonstrated that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein. Furthermore, the adsorption direction is strongly pH-dependent. At pH 6.3, a histidine-rich area around position 10 is the dominating adsorption region. At higher pH values, when the histidines in this area are deprotonated, the protein is also adsorbed by a region close to position 37, which contains several lysines and arginines. Clearly the adsorption is directed by positively charged areas on the protein surface toward the negatively charged silica surface at conditions when specific binding occurs.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-30369 (URN)10.1529/biophysj.104.054809 (DOI)15916 (Lokalt ID)15916 (Arkivnummer)15916 (OAI)
    Tillgänglig från: 2009-10-09 Skapad: 2009-10-09 Senast uppdaterad: 2017-12-13
    6. Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability
    Öppna denna publikation i ny flik eller fönster >>Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability
    2005 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, nr 27, s. 25558-25564Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The influence of protein stability on the adsorption and desorption behavior to surfaces with fundamentally different properties (negatively charged, positively charged, hydrophilic, and hydrophobic) was examined by surface plasmon resonance measurements. Three engineered variants of human carbonic anhydrase II were used that have unchanged surface properties but large differences in stability. The orientation and conformational state of the adsorbed protein could be elucidated by taking all of the following properties of the protein variants into account: stability, unfolding, adsorption, and desorption behavior. Regardless of the nature of the surface, there were correlation between (i) the protein stability and kinetics of adsorption, with an increased amplitude of the first kinetic phase of adsorption with increasing stability; (ii) the protein stability and the extent of maximally adsorbed protein to the actual surface, with an increased amount of adsorbed protein with increasing stability; (iii) the protein stability and the amount of protein desorbed upon washing with buffer, with an increased elutability of the adsorbed protein with increased stability. All of the above correlations could be explained by the rate of denaturation and the conformational state of the adsorbed protein. In conclusion, protein engineering for increased stability can be used as a strategy to decrease irreversible adsorption on surfaces at a liquid-solid interface.

    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-50465 (URN)10.1074/jbc.M503665200 (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2017-12-12
  • 52.
    Karlsson, Martin
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Adsorption at the liquid-solid Interface - Influence of protein stability on conformational changes2007Ingår i: Encyclopedia of surfaces and colloid science / [ed] Ponisseril Somasundaran, Taylor & Francis, 2007, 2, Vol. 1Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Protein adsorption has large implications in a variety of fields and can be both a problem and an asset. Most often protein adsorption is accompanied by structural changes in the adsorbed protein. The degree and rate of these changes are dependent on the surface, conditions during adsorption and experimental set up as well as of intrinsic properties of the protein. The effect of conformational changes influences both practical applications and experimental results in studies of protein adsorption at the liquid/solid interface. The intrinsic property of the protein that is most instrumental for conformational changes upon adsorption is the stability of the protein. Hence, large efforts have been directed towards analysis of how both the nature of surfaces and conditions influence the stability of proteins upon adsorption. Less work has been focused on the reversed view, i.e. how the stability of proteins influences adsorption, the rate and degree of the subsequent conformational changes as well as the effects of these changes. However, the increasing use of proteins in a variety of medical and biotechnological applications requires a deeper knowledge of the importance and effects of stabilizing interactions in the protein structure. Engineered stabilized proteins that are less affected by surface interactions should be of potential use for various practical purposes.

  • 53.
    Karlsson, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Rationell enzymvaskning2011Patent (Övrig (populärvetenskap, debatt, mm))
  • 54.
    Klingstedt, Therése
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Kostareva, A
    Almazov Federal Centre of Heart, Blood and Endocrinology, St. Petersburg, Russian Federation.
    Sjöberg, G.
    Karolinska Institute, Stockholm, Sweden.
    Gudkova, A
    Almazov Federal Centre of Heart, Blood and Endocrinology, St. Petersburg, Russian Federation.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Sejersen, T
    Karolinska Institute, Stockholm, Sweden.
    Desmin L345P transgenic mice exhibit morphological and biochemical features of amyloidosis of two distinct types2010Ingår i: European Heart Journal, ISSN 0195-668X, E-ISSN 1522-9645, Vol. 31, nr Suppl. 1, s. 924-925Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    Background: Being a chief intermediate filament of the muscle tissue, desmin isimplicated in the sarcomeric organization, organelle positioning and mitochondrialfunction. Various desmin mutations have been reported as a possible cause forcardiomyopathies. Several reports on transgenic mice expressing mutant desminshowed deleterious effects of mutant desmin incorporated into filaments on cardiomyocyte function, but most importantly that accumulation of unfolded proteinaggregates plays an important pathogenic role in development of desminassociatedcardiomyopathies. Thus, in desmin transgenic mice with the L345Pmutation, which interferes in a dominant-negative manner with desmin polymerization,the accumulation of intracellular and extracellular amyloidogenic proteinaggregates was shown to be the key feature along with alteration of mitochondrialstructure and function. Therefore, the aim of this study was to characterizethe nature of amyloidogenic protein aggregates in L345P desmin transgenic miceon molecular and protein level.

    Material and methods: L345P desmin transgenic mice (DM) and WT mice 40weeks old were analyzed. Myocardial cryostat 10 micron sections were stainedwith conventional techniques (hematoxilin-eosin, Congo Red). A detailed amyloidcharacterization was carried out using novel optical probes called luminescentconjugated oligothiophenes and polythiophenes (LCOs and LCPs) that specificallystain various protein aggregates and give rise to conformation dependentemission spectra.

    Results: The most prominent feature of DM mice myocardium was misfoldedprotein depositions in perivascular space and between muscle fibers. Analysisof samples from DM mouse stained with LCO or LCP revealed the presence ofaggregates emitting light with two different emission spectra. Since the spectralproperties of the LCOs or LCPs are dependent on their conformation, the appearanceof two dissimilar emission spectra indicates that the probes might bindto two different types of amyloid aggregates within the tissue. Interestingly, aggregateswith emission spectra similar to one of the two types found in the DMmouse could also be found in WT mice, but in a much lower extent, suggesting asporadic cardiac amyloid pathology in C57 Bl/6 mice at 40 weeks, probably, as anative aging attribute.

    Conclusions: The L345P desmin mutation causes focal amyloid protein depositionin heart muscle of two distinct types. White first one can be a natural attributeof C57 Bl/6 mice detected with age, another one can be specifically responsiblefor the development of desmin-related cardiomyopathy.

  • 55.
    Klingstedt, Therése
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Luminescent conjugated poly- and oligo-thiophenes: optical ligands for spectral assignment of a plethora of protein aggregates2012Ingår i: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 40, s. 704-710Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The deposition of protein aggregates in various parts of our body gives rise to several devastating diseases, and the development of probes for the selective detection of aggregated proteins is crucial to advance our understanding of the pathogenesis underlying these diseases. LCPs (luminescent conjugated polythiophenes) are fluorescent probes that bind selectively to protein aggregates. The conjugated thiophene backbone is flexible and offers a connection between the conformation and the emission properties, hence binding of LCPs gives the molecule a spectral fingerprint. The present review covers the utilization of LCPs to study the heterogeneity of protein aggregates. It emphasizes specifically the introduction of well-defined probes called LCOs (luminescent conjugated oligothiophenes) and reports how these molecules can be used for real-time in vivo imaging of cerebral plaques as well as for spectral discrimination of protein aggregates and detection of early species in the fibrillation pathway of amyloid beta-peptide.

  • 56. Knudsen, J.F.
    et al.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Sokol, G.H.
    Cantilena, R
    The cyclooxygenase-2 inhibitor celecoxib is a potent inhibitor of human carbonic anhydrase II2004Ingår i: Inflammation, ISSN 0360-3997, E-ISSN 1573-2576, Vol. 28, nr 5, s. 285-290Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cyclooxygenase-2 (COX-2) is up-regulated in stromal and inflammatory cells. The inducible COX-2 isoform is expressed during inflammation, in some cancers, and in brain tissue after global and focal ischemia. Tissue acidosis is a dominant factor in inflammation, and contributes to pain and hyperalgesia. Recently, compelling epidemiological and clinical evidence has documented the COX-independent effects of some COX-2 inhibitors (i.e., celecoxib, valdecoxib, and rofecoxib), among these effects are carbonic anhydrase (CA) inhibition. Carbonic anhydrases are zinc metalloenzymes expressed in various cell types, including those of the kidney, where they act as general acid-base catalysts. The kidneys are also known to express the highest concentration of COX-2 messenger ribonucleic acid. Celecoxib, like the prototypic CA inhibitor acetazolamide, is structurally characterized by an unsubstituted sulfonamide moiety. In the present study, we report that celecoxib exhibits the characteristics of a potent CA inhibitor, showing inhibitory human carbonic anhydrase II (hCAII) activity in the nanomolar range. Valdecoxib was relatively less potent. Rofecoxib, which lacks the unsubstituted sulfonamide moiety characteristic of CA inhibitors, showed no significant hCAII inhibitory activity. The current study corroborates our earlier report of structure-activity relationships as predictors of such metabolic events as hyperchloremia, acidosis, and changes in calcium and phosphate disposition, and clinical manifestations associated with CA inhibition reported with celecoxib. These data showing inhibition of hCAII by the unsubstituted sulfonamides celecoxib and valdecoxib, but not by rofecoxib, may have important implications for the elucidation of the mechanisms of action as well as the side effects associated with COX-2 inhibitors. © 2004 Springer Science+Business Media, Inc.

  • 57. Lindgren, M.
    et al.
    Eaton, S.
    Eaton, G
    Jonsson, Bengt-Harald
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Svensson, Magdalena
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Electron Spin Echo Decay at a Probe of Aminoxyl Environment in Spin-labeled Mutants of Carbonic Anhydrase1997Ingår i: Journal of the Chemical Society. Perkin transactions II, ISSN 0300-9580, s. 2549-2554Artikel i tidskrift (Refereegranskat)
  • 58.
    Lindgren, M.
    et al.
    Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
    Glimsdal, E.
    Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
    Åslund, Andreas
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Simon, Rozalyn
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska fakulteten.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Luminescence and two-photon absorption cross section of novel oligomeric luminescent conjugated polythiophenes for diagnostics of amyloid fibrils2010Ingår i: Nonlinear Optics Quantum Optics, ISSN 1543-0537, Vol. 40, nr 1-4, s. 241-251Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Here we present the TPA cross section and quantum efficiencies of a series of novel oligomeric luminescent conjugated polythiophenes used for detection and spectral diagnostics of amyloid protein aggregates of the amyloid-beta peptide associated with Alzheimers disease. Specifically, these probes consist of pentameric or heptameric thiophenes derivatives with carboxylic substituents attached onto various thiophene rings. The probes absorbs over a broad range approx. 400-500 nm with quantum efficiency of approx. 20% in at neutral pH conditions, and also showed TPA cross sections of 5-50 GM in the range 700-840 nm, in the same order of magnitude as commonly used fluorescein derivatives. Importantly, the multiphoton excitation capabilities of LCPs provided excellent performance when compared to imaging using conventional "single photon" excitation. It is also demonstrated their utilization in both one-and two-photon excitation laser scanning microscope spectral imaging for diagnostics of Alzheimer disease pathology in ex vivo histological sections.

  • 59.
    Lindgren, Mikael
    et al.
    Norwegian University Science and Technology.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Amyloid oligomers: spectroscopic characterization of amyloidogenic protein states2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr 6, s. 1380-1388Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    It is assumed that protein fibrils manifested in amyloidosis result from an aggregation reaction involving small misfolded protein sequences being in an oligomeric or prefibrillar state. This review covers recent optical spectroscopic studies of amyloid protein misfolding, oligomerization and amyloid fibril growth. Although amyloid fibrils have been studied using established protein-characterization techniques throughout the years, their oligomeric precursor states require sensitive detection in real-time. Here, fluorescent staining is commonly performed using thioflavin T and other small fluorescent molecules such as 4-(dicyanovinyl)- julolidine and 1-amino-8-naphtalene sulphonate that have high affinity to hydrophobic patches. Thus, populated oligomeric intermediates and related prefibrillar structures have been reported for several human amyloidogenic systems, including amyloid-beta protein, prion protein, transthyretin, alpha-synuclein, apolipoprotein C-II and insulin. To obtain information on the progression of the intermediate states, these were monitored in terms of fluorescence parameters, such as anisotropy, and quantum efficiency changes upon protein binding. Recently, new antibody stains have allowed precise monitoring of the oligomer size and distributions using multicolor labelling and single molecule detection. Moreover, a pentameric thiophene derivative (p-FTAA) was reported to indicate early precursors during A-beta(1-40) fibrillation, and was also demonstrated in real-time visualization of cerebral protein aggregates in transgenic AD mouse models by multiphoton microscopy. Conclusively, molecular probes and optical spectroscopy are now entering a phase enabling the in vivo interrogation of the role of oligomers in amyloidosis. Such techniques used in parallel with in vitro experiments, of increasing detail, will probably couple structure to pathogenesis in the near future.

  • 60. Lindgren, Mikael
    et al.
    Jonsson, Per
    Sörgjerd, Karin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Fluorescence molecular probes for sensitive point detection of amyloid fibrils and protofibrils2005Ingår i: SPIE,2005, 2005Konferensbidrag (Refereegranskat)
  • 61.
    Lindgren, Mikael
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Stabo-Eeg, Frantz
    Norwegian University of Science and Technology.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Inganäs, Olle
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Biosensing and -imaging with enantiomeric luminescent conjugated polythiophenes using single - and multiphoton excitation2006Ingår i: Proceedings of International Symposium on Biophotonics, Nanophotonics and Metamaterials, IEEE , 2006, s. 226-226Konferensbidrag (Refereegranskat)
  • 62.
    Lindgren, Mikael
    et al.
    Department of Physics, Norwegian University of Science and Technology, 7491 Trondheim, Norway.
    Sörgjerd, Karin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy2005Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, nr 6, s. 4200-4212Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300–500 kD) within 2 h that matured after 20 h into larger spherical clusters (30–50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300–500 kD) with an apparent dissociation constant of 1.6 mM, which was slightly better than for ThT (6.8 mM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482nm wavelength when bound to amyloid fibrils.

  • 63.
    Lindqvist Appell, Malin
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet.
    Wennerstrand, Patricia
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Peterson, Curt
    Linköpings universitet, Institutionen för medicin och hälsa, Klinisk farmakologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Onkologiska kliniken US.
    Hertervig, Erik
    Department of Gastroenterology, Lund University.
    Mårtensson, Lars-Göran
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene2010Ingår i: Pharmacogenetics and genomics, ISSN 1744-6880, Vol. 20, nr 11, s. 700-707Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND: The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. METHODS AND RESULTS: In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III-X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. CONCLUSIONS: We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

  • 64.
    Linhult, M.
    et al.
    Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden.
    Gulich, S.
    Gülich, S., Dept. of Molec. Biophys./Biochem., Yale University, New Haven, CT, United States.
    Graslund, T.
    Gräslund, T., Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden.
    Simon, A.
    Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden.
    Karlsson, Martin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Sjoberg, A.
    Sjöberg, A., Affibody AB, Bromma, Sweden.
    Nord, K.
    Affibody AB, Bromma, Sweden.
    Hober, S.
    Department of Biotechnology, Royal Institute of Technology, KTH, Stockholm, Sweden, Department of Biotechnology, KTH, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.
    Improving the Tolerance of a Protein A Analogue to Repeated Alkaline Exposures Using a Bypass Mutagenesis Approach2004Ingår i: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 55, nr 2, s. 407-416Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Staphylococcal protein A (SPA) is a cell surface protein expressed by Staphylococcus aureus. It consists of five repetitive domains. The five SPA-domains show individual interaction to the Fc-fragment as well as certain Fab-fragments of immunoglobulin G (IgG) from most mammalian species. Due to the high affinity and selectivity of SPA, it has a widespread use as an affinity ligand for capture and purification of antibodies. One of the problems with proteinaceous affinity ligands in large-scale purification is their sensitivity to alkaline conditions. SPA however, is considered relatively stable to alkaline treatment. Nevertheless, it is desirable to further improve the stability in order to enable an SPA-based affinity medium to withstand even longer exposure to the harsh conditions associated with cleaning-in-place (CIP) procedures. For this purpose, a protein engineering strategy, which was used earlier for stabilization and consists of replacing the asparagine residues, is employed. Since Z in its "nonengineered" form already has a significant tolerance to alkaline treatment, small changes in stability due to the mutations are difficult to assess. Hence, in order to enable detection of improvements regarding the alkaline resistance of the Z domain, we chose to use a bypass mutagenesis strategy using a mutated variant Z(F30A) as a surrogate framework. Z(F30A) has earlier been shown to possess an affinity to IgG that is similar to the wild-type but also demonstrates decreased structural stability. Since the contribution of the different asparagine residues to the deactivation rate of a ligand is dependent on the environment and also the structural flexibility of the particular region, it is important to consider all sensitive amino acids one by one. The parental Z-domain contains eight asparagine residues, each with a different impact on the alkaline stability of the domain. By exchanging asparagine 23 for a threonine, we were able to increase the stability of the Z(F30A) domain in alkaline conditions. Also, when grafting the N23T mutation to the Z scaffold, we were able to detect an increased tolerance to alkaline treatment compared to the native Z molecule. © 2004 Wiley-Liss, Inc.

  • 65.
    Lundqvist, Martin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Andrésen, Cecilia
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Christensson, Sara
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Johansson, Sara
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Karlsson, Martin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Broo, Klas
    Department of Occupational and Environmental Medicine, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.
    Jonsson, Bengt-Harald
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Proteolytic cleavage reveals interaction patterns between silica nanoparticles and two variants of human carbonic anhydrase2005Ingår i: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 21, nr 25, s. 11903-11906Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    To characterize the sites on the protein surface that are involved in the adsorption to silica nanoparticles and the subsequent rearrangements of the protein/nanoparticle interaction, a novel approach has been used. After incubation of protein with silica nanoparticles for 2 or 16 h, the protein was cleaved with trypsin and the peptide fragments were analyzed with mass spectrometry. The nanoparticle surface area was in 16-fold excess over available protein surface to minimize the probability that the initial binding would be affected by other protein molecules. When the fragment patterns obtained in the presence and absence of silica nanoparticles were compared, we were able to characterize the protein fragments that interact with the surface. This approach has allowed us to identify the initial binding sites on the protein structure and the rearrangement of the binding sites that occur upon prolonged incubation with the surface.

  • 66.
    Maas, Coen
    et al.
    Univ Med Ctr Utrecht, Dept Clin Chem & Hematol, NL-3508 GA Utrecht, Netherlands.
    Govers-Riemslag, Jos W. P.
    Maastricht Univ, Lab Clin Thrombosis & Haemostasis, Dept Internal Med, Maastricht, Netherlands.
    Bouma, Barend
    Univ Med Ctr Utrecht, Dept Clin Chem & Hematol, NL-3508 GA Utrecht, Netherlands.
    Schiks, Bettina
    Univ Med Ctr Utrecht, Dept Clin Chem & Hematol, NL-3508 GA Utrecht, Netherlands.
    Hazenberg, Bouke P. C.
    Univ Med Ctr Groningen, Dept Rheumatol & Clin Immunol, Groningen, Netherlands.
    Lokhorst, Henk M.
    Univ Med Ctr Utrecht, Dept Clin Chem & Hematol, NL-3508 GA Utrecht, Netherlands.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    ten Cate, Hugo
    Maastricht Univ, Lab Clin Thrombosis & Haemostasis, Dept Internal Med, Maastricht, Netherlands.
    de Groot, Philip G.
    Univ Med Ctr Utrecht, Dept Clin Chem & Hematol, NL-3508 GA Utrecht, Netherlands.
    Bouma, Bonno N.
    Scripps Res Inst, Dept Mol & Expt Med, La Jolla, CA USA.
    Gebbink, Martijn F. B. G.
    Univ Med Ctr Utrecht, Dept Clin Chem & Hematol, NL-3508 GA Utrecht, Netherlands.
    Misfolded proteins activate Factor XII in humans, leading to kallikrein formation without initiating coagulation2008Ingår i: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 118, nr 9, s. 3208-3218Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    When blood is exposed to negatively charged surface materials such as glass, an enzymatic cascade known as the contact system becomes activated. This cascade is initiated by autoactivation of Factor XII and leads to both coagulation (via Factor XI) and an inflammatory response (via the kallikrein-kinin system). However, while Factor XII is important for coagulation in vitro, it is not important for physiological hemostasis, so the physiological role of the contact system remains elusive. Using patient blood samples and isolated proteins, we identified a novel class of Factor XII activators. Factor XII was activated by misfolded protein aggregates that formed by denaturation or by surface adsorption, which specifically led to the activation of the kallikreinkinin system without inducing coagulation. Consistent with this, we found that Factor XII, but not Factor XI, was activated and kallikrein was formed in blood from patients with systemic amyloidosis, a disease marked by the accumulation and deposition of misfolded plasma proteins. These results show that the kallikrein-kinin system can be activated by Factor XII, in a process separate from the coagulation cascade, and point to a protective role for Factor XII following activation by misfolded protein aggregates.

  • 67.
    Magnusson, Karin
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Simon, Rozalyn
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Joshi-Barr, Shivanjali
    University of Calif San Diego.
    Sigurdson, Christina
    University of Calif San Diego.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Conformation-sensitive probes for strain-specific characterization of prion aggregates in PRION, vol 6, issue , pp 47-472012Ingår i: PRION, Landes Bioscience , 2012, Vol. 6, s. 47-47Konferensbidrag (Refereegranskat)
    Abstract [en]

    n/a

  • 68.
    Mahajan, Vineet
    et al.
    Medical University of Graz.
    Klingstedt, Therése
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Simon, Rozalyn
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Thueringer, Andrea
    Medical University of Graz.
    Kashofer, Karl
    Medical University of Graz.
    Haybaeck, Johannes
    Medical University of Graz.
    Denk, Helmut
    Medical University of Graz.
    Abuja, Peter M
    Medical University of Graz.
    Zatloukal, Kurt
    Medical University of Graz.
    Cross beta-Sheet Conformation of Keratin 8 Is a Specific Feature of Mallory-Denk Bodies Compared With Other Hepatocyte Inclusions2011Ingår i: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 141, nr 3, s. 1080-U428Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    BACKGROUND andamp; AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steato-hepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross beta-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross beta-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, alpha(1)-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross beta-sheet conformation than K18, whereas p62 never had cross beta-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly alpha-helical to cross beta-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8(-/-) mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.

  • 69.
    Margalith, Ilan
    et al.
    University of Zurich Hospital, Switzerland .
    Suter, Carlo
    University of Zurich Hospital, Switzerland .
    Ballmer, Boris
    University of Zurich Hospital, Switzerland .
    Schwarz, Petra
    University of Zurich Hospital, Switzerland .
    Tiberi, Cinzia
    University of Zurich Hospital, Switzerland .
    Sonati, Tiziana
    University of Zurich Hospital, Switzerland .
    Falsig, Jeppe
    University of Zurich Hospital, Switzerland .
    Nyström, Sofie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Åslund, Andreas
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Yam, Alice
    Novartis Diagnost, USA .
    Whitters, Eric
    Novartis Diagnost, USA .
    Hornemann, Simone
    University of Zurich Hospital, Switzerland .
    Aguzzi, Adriano
    University of Zurich Hospital, Switzerland .
    Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 23, s. 18872-18887Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23-231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23-231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits.

  • 70.
    Maria Psonka-Antonczyk, Katarzyna
    et al.
    Norwegian University of Science and Technology.
    Duboisset, Julien
    Norwegian University of Science and Technology.
    Torger Stokke, Bjorn
    Norwegian University of Science and Technology.
    Zako, Tamotsu
    Riken Institute Phys and Chemistry Research.
    Kobayashi, Takahiro
    Riken Institute Phys and Chemistry Research.
    Maeda, Mizuo
    Riken Institute Phys and Chemistry Research.
    Nyström, Sofie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Mason, Jeffrey
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Proteinkemi. Linköpings universitet, Tekniska fakulteten.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Lindgren, Mikael
    Norwegian University of Science and Technology.
    Nanoscopic and Photonic Ultrastructural Characterization of Two Distinct Insulin Amyloid States2012Ingår i: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, ISSN 1661-6596, Vol. 13, nr 2, s. 1461-1480Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.

  • 71.
    Mishra, Rajesh
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Olofsson, Linus
    Karlsson, Martin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Nicholls, Ian A.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    A conformationally isoformic thermophilic protein with high kinetic unfolding barriers2008Ingår i: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 65, nr 5, s. 827-839Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH. © 2008 Birkhaueser.

  • 72.
    Mishra, Rajesh
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Sjölander, Daniel
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Proteinkemi. Linköpings universitet, Tekniska fakulteten.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red2011Ingår i: MOLECULAR BIOSYSTEMS, ISSN 1742-206X, Vol. 7, nr 4, s. 1232-1240Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 degrees C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 degrees C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 degrees C) were detected. Nile red was also successfully employed in detecting A beta 1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stokes shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stokes shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of A beta 1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.

  • 73.
    Mishra, Rajesh
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Sörgjerd, Karin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Nyström, Sofie
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Nordigården, Amanda
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Chiu, Yu-Jui
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Lysozyme Amyloidogenesis Is Accelerated by Specific Nicking and Fragmentation but Decelerated by Intact Protein Binding and Conversion2007Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 366, nr 3, s. 1029-1044Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We have revisited the well-studied heat and acidic amyloid fibril formation pathway (pH 1.6, 65 °C) of hen egg-white lysozyme (HEWL) to map the barriers of the misfolding and amyloidogenesis pathways. A comprehensive kinetic mechanism is presented where all steps involving protein hydrolysis, fragmentation, assembly and conversion into amyloid fibrils are accounted for. Amyloid fibril formation of lysozyme has multiple kinetic barriers. First, HEWL unfolds within minutes, followed by irreversible steps of partial acid hydrolysis affording a large amount of nicked HEWL, the 49-101 amyloidogenic fragment and a variety of other species over 5-40 h. Fragmentation forming the 49-101 fragment is a requirement for efficient amyloid fibril formation, indicating that it forms the rate-determining nucleus. Nicked full-length HEWL is recruited efficiently into amyloid fibrils in the fibril growth phase or using mature fibrils as seeds, which abolished the lag phase completely. Mature amyloid fibrils of HEWL are composed mainly of nicked HEWL in the early equilibrium phase but go through a "fibril shaving" process, affording fibrils composed of the 49-101 fragment and 53-101 fragment during more extensive maturation (incubation for longer than ten days). Seeding of the amyloid fibril formation process using sonicated mature amyloid fibrils accelerates the fibril formation process efficiently, however, addition of intact full-length lysozyme at the end of the lag phase slows the rate of amyloidogenesis. The intact full-length protein, in contrast to nicked lysozyme, slows fibril formation due to its slow conversion into the amyloid fold, probably due to inclusion of the non-amyloidogenic 1-48/102-129 portion of HEWL in the fibrils, which can function as a "molecular bumper" stalling further growth. © 2006 Elsevier Ltd. All rights reserved.

  • 74.
    Nicklagård, Erik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Diabetes typ 3?: Molekylärfysiologiska länkar och samband från den samlade litteraturen2011Självständigt arbete på grundnivå (kandidatexamen), 10,5 poäng / 16 hpStudentuppsats (Examensarbete)
    Abstract [sv]

    Alzheimers sjukdom (AD) är den vanligaste formen av demens och kännetecknas av intracellulärt neurofibrillärt trassel (NFT) bestående av proteinet tau och extracellulära plack, uppbyggda av peptiden amyloid beta (Aβ). En växande skara studier har börjat peka mot att AD är en hjärnspecifik typ av diabetes. Insulinresistens följt av hyperinsulinemi och hyperglykemi är kännetecken för diabetes mellitus typ 2 (DMT2) och har visat sig vara en riskfaktor för AD. Insulin, ett hormon som kontrollerar glukoshomeostasen i perifera nervsystemet (PNS) och är viktigt för minne och inlärning, transporteras över blod-hjärnbarriären i en mättnadsbar transportmekanism och dess koncentration i centrala nervsystemet (CNS) minskar vid DMT2 och AD. Insulin-like growth factor 1 (IGF-1), ett neuronskyddande protein som minskar ogynnsam β-sekretasklyvning av amyloid precursor protein (APP) i amyloidkaskadhypotesen, minskar i koncentration i hjärnan när mycket insulin transporteras in i CNS. γ-sekretas ökar sin aktivitet på APP vid höga halter kolesterol som är vanligt vid DMT2, Aβ fungerar då som en negativ inhibitor till HMG-Coa reduktas (HMGR), enzymet som bildar kolesterol och kan därmed reglera kolesterolhalterna. Regleringssystem för Aβ i blod-hjärnbarriären (BBB) som p-GP, LRP-1 och RAGE rubbas vid DMT2. Aβ och insulin delar samma degraderingssystem, insulin degrading enzyme (IDE), som reglerar halterna Aβ och insulin. Dessutom har Aβ oligomerer visat sig kunna bryta ned insulinreceptorer (IR). Vidare har läkemedel mot diabetes visat sig lindra demens hos AD patienter. I den här rapporten gås de molekylärfysiologiska sambanden igenom i detalj. Slutligen finns det fog för ett samband mellan metabolt syndrom, en riskfaktor för DMT2, och AD.

  • 75.
    Nicklagård, Erik
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Quantification of Alzheimer DiseaseAmyloid β Peptide 43 in Human BrainWith a Newly Developed Enzyme-LinkedImmunosorbent Assay (ELISA)2011Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [en]

    A 20 weeks project at Karolinska Institutet (KI), Huddinge, Sweden is in this master thesis summarized. Alzheimer’s disease is the most common form of dementia in the world. One of the pathological hallmarks seen in AD patients consists of amyloid plaques assembled of beta amyloid (Aβ) peptide aggregates. A lot of research has been done on Aβ40 and Aβ42 but not on the longer variant with 43 residues. An earlier study by Welander et al, quantified the Aβ43 peptide from amyloid plaque cores with high-performance liquid chromatography coupled to mass-spectrometry (HPLC-MS/MS)1. Here, I present the initial development of an enzyme-linked immunosorbent assay (ELISA) with the goal to quantify Aβ43 peptides in soluble fractions of human brain tissue. An ELISA method with the possibility to quantify Aβ43 peptides from cerebral spinal fluid might have the prospect to serve as a diagnostic tool for AD in the future.

    Commercial ELISA kits coated with antibodies against all Aβ species was not suitable for detecting Aβ43 in soluble brain tissue from human AD patients. This is due to the high amount of Aβ40 (and in some extent Aβ42) in the samples, which will bind to the same epitope as Aβ43 on the capturing antibody. These shorter Aβ species will be in excess and bind to the capturing antibody thereby ousting Aβ43 from binding in. A better way for quantifying Aβ43 with ELISA might instead be to coat a polystyrene plate with α-Aβ43 antibodies, which are c-terminal specific to Aβ43. This will abolish the competition between the different Aβ species and function as an immunoprecipitation of unwanted species. This yielded adequate quantification of Aβ43 (2.64 pM) from tris-buffer saline (TBS) fractions from a human brain sample from AD.

  • 76.
    Nilsson, Peter
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Luminescent conjugated polymers: Illuminating the dark matters of biology and pathology2008Ingår i: Advanced Materials, ISSN 0935-9648, E-ISSN 1521-4095, Vol. 20, nr 13, s. 2639-2645Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Novel technologies for studying biological events are of great interest and luminescent conjugated polymers (LCPs), a material normally used in organic electronics, have proven useful for the detection of a wide range of disease-related biological processes. The conformation-sensitive optical properties of LCPs provide the ability to study the biochemical activity of biological events on the basis of a structure-function relation, rather than a molecular basis. In this Research News article, the LCP technique and its usefulness for studying protein aggregation diseases are highlighted. We also discuss the much-needed illuminating insights of the underlying pathological events regarding protein aggregation diseases. In addition, essential future basic research advances that relate to further development of LCPs as molecular probes are presented. We also confer the intriguing prospect of employing amyloid fibrils, that is, a symmetric stable nanomaterial normally associated with the dark side of horrific pathology, as a scaffold for functional polymer-protein hybrid materials. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA.

  • 77.
    Nilsson, Peter
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Ahlgren, Fredrik
    Division of Cell Biology Linköpings universitet.
    Herland, Anna
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik.
    Schnell, Edrun A
    The Norwegian University of Science and Technology.
    Lindgren, Mikael
    The Norwegian University of Science and Technology.
    Westermark, Gunilla
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Inganäs, Olle
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik.
    Conjugated Polyelectrolytes - Conformation - Sensitive Optical Probes for staining and Characterization of Amyloid Deposits2006Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 7, s. 1096-1104Artikel i tidskrift (Refereegranskat)
    Abstract [en]

      

  • 78.
    Nilsson, Peter
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Herland, Anna
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Inganäs, Olle
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik. Linköpings universitet, Tekniska högskolan.
    Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation2005Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 10, s. 3718-3724Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The in vivo deposition of amyloid fibrils is a hallmark of many devastating diseases known as the amyloidoses. Amyloid formation in vitro may also complicate production of proteins in the biotechnology industry. Simple, sensitive, and versatile tools that detect the fibrillar conformation of amyloidogenic proteins are thus of great importance. We have developed a negatively charged conjugated polyelectrolyte that displays different characteristic optical changes, detected visually or by absorption and emission, depending on whether the protein with which it forms a complex is in its native state or amyloid fibril conformation. This simple, rapid, and novel methodology was applied here to two amyloidogenic proteins, insulin and lysozyme, and its validity for detection of their fibrillar conformation was verified by currently used methods such as circular dichroism, transmission electron microscopy, and Congo red absorption.

  • 79.
    Nilsson, Peter
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Åslund, Andreas
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi.
    Berg, Ina
    Nyström, Sofie
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Konradsson, Peter
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi.
    Herland, Anna
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik.
    Inganäs, Olle
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biomolekylär och Organisk Elektronik.
    Stabo-Eeg, Frantz
    Lindgren, Mikael
    Westermark, Gunilla
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Avdelningen för medicinsk cellbiologi.
    Lannfelt, Lars
    Nilsson, Lars N G
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Imaging distinct conformational states of amyloid-β fibrils in Alzheimer's disease using novel luminescent probes2007Ingår i: ACS Chemical Biology, ISSN 1554-8929, Vol. 2, nr 8, s. 553-560Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Using luminescent conjugated polyelectrolyte probes (LCPs), we demonstrate the possibility to distinguish amyloid-β 1-42 peptide (Aβ1-42) fibril conformations, by analyzing in vitro generated amyloid fibrils of Aβ1-42 formed under quiescent and agitated conditions. LCPs were then shown to resolve such conformational heterogeneity of amyloid deposits in vivo. A diversity of amyloid deposits depending upon morphology and anatomic location was illustrated with LCPs in frozen ex vivo brain sections from a transgenic mouse model (tg-APPswe) of Alzheimer's disease. Comparative LCP fluorescence showed that compact-core plaques of amyloid β precursor protein transgenic mice were composed of rigid dense amyloid. A more abundant form of amyloid plaque displayed morphology of a compact center with a protruding diffuse exterior. Surprisingly, the compact center of these plaques showed disordered conformations of the fibrils, and the exterior was composed of rigid amyloid protruding from the disordered center. This type of plaque appears to grow from more loosely assembled regions toward solidified amyloid tentacles. This work demonstrates how application of LCPs can prove helpful to monitor aggregate structure of in vivo formed amyloid deposits such as architecture, maturity, and origin.

  • 80.
    Nystrom, Sofie
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Nelson, Erin
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Reitan, Nina
    Norwegian University of Science and Technology.
    Ellingsen, Pal
    Norwegian University of Science and Technology.
    Brorsson, Ann-Christin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik. Linköpings universitet, Tekniska högskolan.
    Mason, Jeffrey
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    Johansson, Leif
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Halvledarmaterial. Linköpings universitet, Tekniska högskolan.
    Sluzny, Chanan
    Appl Spectral Imaging, Migdal Haemeq.
    Handrick, Susann
    Charite.
    Prokop, Stefan
    Charite.
    Wegenast-Braun, Bettina
    German Centre Neurodegenerat Disease.
    Hornemann, Simone
    University of Zurich Hospital.
    Kågedal, Katarina
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Experimentell patologi. Linköpings universitet, Hälsouniversitetet.
    Lindgren, Mikael
    Norwegian University of Science and Technology.
    Heppner, Frank
    Charite.
    Jucker, Mathias
    German Centre Neurodegenerat Disease.
    Aguzzi, Adriano
    University of Zurich Hospital.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Monitoring amyloid formation and maturation in vitro and in vivo using LCO fluorescence in PRION, vol 6, issue , pp 13-132012Ingår i: PRION, Landes Bioscience , 2012, Vol. 6, s. 13-13Konferensbidrag (Refereegranskat)
    Abstract [en]

    n/a

  • 81.
    Nyström, Sofie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Early events in disease associated protein misfolding2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The scope of this thesis is to unravel some of the mysteries concerning events takingplace early in the amyloid cascade. In vitro studies of early misfolded states ofamyloidogenic proteins are important since the use of recombinant proteins allow us to monitor slight changes in environmental conditions as well as in amino acid composition and thereby illuminate the problem at near atomic resolution.

    Human prion protein (HuPrP) (associated with e.g. Creutzfeldt-Jakob disease) andthe Aβ1-42 peptide (associated with Alzheimer’s disease) recombinantly expressed in Escherichia coli have been used as model systems for these studies.

    A new protocol for amyloid fibril formation of human prion protein under native conditions was developed. This revealed an unusual pathway of conformational conversion from early formed disordered aggregates that later matured into amyloidfibrils.

    The polymorphism 129M/V in HuPrP has a large impact on susceptibility both to sporadic and infectious prion diseases. Some features of this polymorphism havebeen elucidated, employing a mutational study in position 129 (M, A, L, V, P, M, W,E, and K). These investigations have rendered new knowledge about the impact ofsize, charge and β-carbon branching in position 129 upon early intermolecular interactions and the effects of fibril seeding.

    Investigations of the interactions between different assembly forms of HuPrP and components of the innate immune system revealed that both native, oligomeric and fibrillar forms of HuPrP activate both the classical and alternative pathways of the Complement System. Most efficient activation is achieved upon binding of oligomeric HuPrP to the complement component C1q.

    We have developed a system for recombinant expression of human A,1-42. The monomeric peptides are assembled into various sized soluble oligomers (trimer, hexamer, nonamer, dodecamer). The oligomeric forms were stable in 8 M urea, 6 MGuHCl and SDS suggesting that these were covalently cross-linked. Some mechanistic features in the assembly process have been investigated and we have shown that cupric ions facilitates formation of stable oligomers in our system.

    Delarbeten
    1. Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
    Öppna denna publikation i ny flik eller fönster >>Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates
    2009 (Engelska)Ingår i: Prion, ISSN 1933-6896, Vol. 3, nr 4, s. 224-235Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    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.

    Ort, förlag, år, upplaga, sidor
    Austin: Landes Bioscience Journals, 2009
    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-21064 (URN)10.4161/pri.3.4.10112 (DOI)000280061100009 ()
    Tillgänglig från: 2009-09-28 Skapad: 2009-09-28 Senast uppdaterad: 2019-11-08
    2. Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway
    Öppna denna publikation i ny flik eller fönster >>Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway
    2012 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 31, s. 25975-25984Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met129 (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90–231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, Tm = 65–66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3–4 °C. The most destabilizing substitution was M129P, which lowered the Tm by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.

    Ort, förlag, år, upplaga, sidor
    American Society for Biochemistry and Molecular Biology, 2012
    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-53174 (URN)10.1074/jbc.M112.372136 (DOI)000306916300025 ()
    Anmärkning

    funding agencies|EU-FP7 Health Programme Project LUPAS||Swedish Research Council||Knut and Alice Wallenberg Foundation||Swedish Foundation for Strategic Research||Linkoping University Center for Neuroscience||

    Tillgänglig från: 2010-01-18 Skapad: 2010-01-18 Senast uppdaterad: 2019-11-08
    3. Native, amyloid fibrils and β-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly
    Öppna denna publikation i ny flik eller fönster >>Native, amyloid fibrils and β-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly
    2008 (Engelska)Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, nr 11, s. 3213-3221Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and β-oligomers) of recombinant human PrP (90–231 and 121–231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, β-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that β-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

    Nyckelord
    C1q, C4b-binding protein, Complement activation, Factor H, Human prion protein, Transmissible spongiform encephalopathies
    Nationell ämneskategori
    Teknik och teknologier
    Identifikatorer
    urn:nbn:se:liu:diva-45977 (URN)10.1016/j.molimm.2008.02.023 (DOI)
    Tillgänglig från: 2009-10-11 Skapad: 2009-10-11 Senast uppdaterad: 2019-11-08
    4. Properties of defined recombinant oligomeric forms of Aβ1‐42
    Öppna denna publikation i ny flik eller fönster >>Properties of defined recombinant oligomeric forms of Aβ1‐42
    Visa övriga...
    (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Oligomers of Aβ1-42 have been identified in human Alzheimer´s disease (AD) patients and in mouse models of AD. These species have attracted intense interest as possible neurological pathogens in AD. In our hands, expression of recombinant human Aβ1-42 in Escherichia coli followed by purification in the presence of cupric ions (CuCl2) afforded recovery of high quantities (>5 mg/L of culture) of well defined trimeric, hexameric, nonameric and dodecameric Aβ1-42. Strong denaturing conditions such as 6 M GuHCI, 8 M urea or boiling in 6.5 M urea supplemented with 2.5 % SDS all failed to separate the oligomers into smaller building blocks implicating that the oligomers are composed of covalently cross-linked Aβ1-42 monomers. Purification in the absence of cupric ions resulted in monomeric Aβ1-42. The Aβ1-42 oligomers were toxic and induced apoptosis when administered to neuroblastoma cells in culture. The described method producing oligomeric Aβ1-42 from a recombinant expression system paves the way for mechanistic studies, structural analysis, drug screening and opens up for vaccine development.

    Nationell ämneskategori
    Naturvetenskap
    Identifikatorer
    urn:nbn:se:liu:diva-53175 (URN)
    Tillgänglig från: 2010-01-18 Skapad: 2010-01-18 Senast uppdaterad: 2019-11-08
  • 82.
    Nyström, Sofie
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway2012Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, nr 31, s. 25975-25984Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met129 (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90–231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, Tm = 65–66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3–4 °C. The most destabilizing substitution was M129P, which lowered the Tm by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.

  • 83.
    Nyström, Sofie
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Propagating Artificial Amyloid Strains of Recombinant Human Prion Protein with Mutations in Position 1292010Ingår i: Prion, ISSN 1933-6896, E-ISSN 1933-690X, Vol. 4, nr 3, s. 124-124Artikel i tidskrift (Övrigt vetenskapligt)
    Abstract [en]

    The influence of the polymorphism M129V in the human PrPgene is well documented. Most cases of sporadic CJD afflicthomozygous individuals. Differences in codon 129 genotypegive rise to differences in phenotype regarding plaque and clinicalsymptoms. Despite this, little is known about the molecularbackground to this phenomenon.

    To study this phenomenon in greater detail we employedrecombinant human prion protein. Using several artificial mutationsallowed us to study the influence of different amino acidproperties on the formation of amyloid prion protein. The variantsused were 129A, 129V, 129L, 129M, 129W, 129P, 129E and129K. Three mutants were chosen to vary the hydrophobicity,the tryptophan mutant was chosen due to its bulkiness and theproline for its constraint of the polypeptide backbone. 129E and129K may give information regarding the effect of charge in this position.

    The protein was expressed in Escherichia coli, purified andsubjected to agitation at 37°C at physiological pH and salt concentration(Almstedt et al. Prion 2009). All mutants formedcongophilic and Thioflavine T positive aggregates within hours.Fibrillar morphology was also confirmed using transmission electronmicroscopy.

    Seeding the mutant proteins with preformed fibrils of themutant itself or of wild type protein revealed differences in seedingefficiency for the different mutants. By monitoring the fibrilsresulting from the seeded fibrillation reactions using luminescentconjugated polymers, a templating effect was seen. This strainlikebehavior was followed through several generations of fibrils.The fragility of the seeding fibrils was taken under considerationand was analyzed using urea denaturation.

    Almstedt, Nyström S, Nilsson P, Hammarström P. Prion2009; 3:224-35.

  • 84.
    Osterlund, Maria
    et al.
    Novo Nordisk AS, Prot Biotechnol, DK-2880 Bagsvaerd, Denmark Linkoping Univ, IFM, Dept Chem, Linkoping, Sweden Linkoping Univ, IFM, Dept Phys Chem, Linkoping, Sweden Novo Nordisk AS, Vasc Biochem, Malov, Denmark.
    Owenius, Rikard
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Karin
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Persson, Egon
    Vascular Biochemistry, Novo Nordisk A/S, Denmark.
    Lindgren, Mikael
    Department of Laser Systems, Division of Sensor Technology, Swedish Defence Research Agency, P.O. Box 1165, SE- 581 11 Linko¨ping, Sweden..
    Freskgård, Per-Ola
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Svensson, Magdalena
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa2001Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, nr 31, s. 9324-9328Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

  • 85.
    Osterlund, Marie
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Owenius, Rikard
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Persson, E.
    Tissue Factor/Factor VII Research, Novo Nordisk A/S, Måløv, Denmark.
    Lindgren, M.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Freskgard, P.-O.
    Freskgård, P.-O., Tissue Factor/Factor VII Research, Novo Nordisk A/S, Måløv, Denmark.
    Svensson, Magdalena
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Spectroscopic probing of the influence of calcium and the Gla domain on the interaction between the first EGF domain in factor VIla and tissue factor2000Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, nr 20, s. 6204-6211Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxy-glutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.

  • 86.
    Osterlund, Marie
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Owenius, Rikard
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Svensson, Magdalena
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Lindgren, M
    Persson, E
    Freskgård, Per-Ola
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Mapping local interactions and resolving association kinetics for a receptor-ligand system2000Konferensbidrag (Övrigt vetenskapligt)
  • 87.
    Owenius, Rikard
    et al.
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Jarl, Anngelica
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär genetik. Linköpings universitet, Tekniska högskolan.
    Carlsson, Uno
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Eaton, Sandra
    University of Denver, Department of Chemistry & Biochemistry Denver, USA.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    GroEL-induced stretching of a substrate protein: An EPR/SDSL study in BIOPHYSICAL JOURNAL, vol 88, issue 1, pp 562A-562A2005Ingår i: BIOPHYSICAL JOURNAL, Elsevier (Cell Press) / Biophysical Society , 2005, Vol. 88, nr 1, s. 562A-562AKonferensbidrag (Refereegranskat)
    Abstract [en]

    The Hsp60-type chaperonin GroEL assists in the folding of the enzyme Human Carbonic Anhydrase II (HCA II) and protects it from aggregation.It is still a controversy whether the action of GroEL is an active or passive process. Single- and double-cysteine mutants were specificallyspin labeled at a topological breakpoint in the β-core of HCA II. X-band electron paramagnetic resonance (EPR) was used at physiologicaltemperatures to monitor the GroEL-induced structural changes in this region of HCA II. Inter-residue distance calculations based on dipolarinteraction show that the proximity of the labeled positions F147 and K213 in the native state of HCA II is ~11±2 Å, and that it is virtuallyintact in the thermally-induced molten-globule state that binds to GroEL. However, upon interaction with GroEL a spin-spin distance increaseto ~22±3 Å indicates a conformational change in HCA II that is part of the GroEL-induced substrate stretch that enables structural rearrangementof a misfolded substrate protein.

  • 88.
    Owenius, Rikard
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemisk Fysik.
    Jarl, Anngelica
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Jonsson, Bengt-Harald
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    An unfolding machine in action: Streching by the chaperonin BroEL of the substrate protin core2006Ingår i: Cold Spring Harbor Molecular Chaperones and the heat shock response,2006, 2006Konferensbidrag (Övrigt vetenskapligt)
  • 89.
    Owenius, Rikard
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Osterlund, Marie
    Linköpings universitet, Institutionen för fysik, kemi och biologi. Linköpings universitet, Tekniska högskolan.
    Svensson, Magdalena
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Lindgren, M
    Persson, E
    Freskgård, Per-Ola
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites2001Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 81, nr 4, s. 2357-2369Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The specific complex between the extracellular part of tissue factor (sTF) and factor Vlla (FVlla) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVlla interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma -carboxyglutamic acid (Gla) domain of FVlla, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVlla. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVlla binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVlla, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

  • 90.
    Palmqvist, Emma
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Mutagenesis of the sugar donor site of the Arabidopsis thaliana glycosyltransferase UGT72B12010Självständigt arbete på avancerad nivå (masterexamen), 20 poäng / 30 hpStudentuppsats (Examensarbete)
    Abstract [sv]

    Glykosyltransferaset UGT72B1 från Arabidopsis thaliana är ett av många enzymer som katalyserar reaktionen där en glukosenhet från UDP-glukos länkas till en acceptormolekyl, i det här fallet en kloranilin eller en klorfenol. Det är en del av ett detoxifieringssytem i växtcellen, som liknar det i människan, där ett glukuronosyltransferas möjliggör nedbrytning av bl.a. läkemedel. Det vore intressant att kunna undersöka de humana enzymernas aktivitet mot olika läkemedel och även fastställa effekten glukoslänkningen har på dessa substansers egenskaper. De humana enzymerna är dock membranprotein och är därför svåra att rena fram och att kristallisera. Här har istället ett försök gjorts för att ändra substratspecificiteten hos UGT72B1 från UDP-glukos till UDP-glukuronsyra. Kombinationer av de fyra punktmutationerna G18S, P139R, W367S och AG387ED introducerades i UGT72B1. Ingen aktivitet med UDP-glukuronsyra erhölls dock. Enkelmutanterna W367S och AG387ED bibehöll liknande aktivitet som vildtypen, medan P139R hade starkt reducerad aktivitet och G18S uttrycktes inte alls. Alla andra kombinationer av mutationer resulterade i ännu lägre aktivitet. Fyra chimeriska proteiner konstruerades också. De skapades genom kombination av UGT72B1 och det humana enzymet UGT2B4. Dessa var alla lösliga proteiner men ingen av dem uppvisade någon aktivitet.

  • 91. Persson, M
    et al.
    Zhou, A
    Mitri, R
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Eaton, GR
    Eaton, SS
    Distance determination between deeply buried position in human carbon anhydrase II2000Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 78, nr 1, s. 2255Pos-Konferensbidrag (Övrigt vetenskapligt)
  • 92.
    Persson, Malin
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Lindgren, M.
    Jonsson, Bengt-Harald
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Molekylär Bioteknik.
    Svensson, Magdalena
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    EPR Mapping of Interactions Between Spin-labeled Variants of Human Carbonic Anhydrase II and GroEL. Evidence for increased flexibility of the hydrophobic core by the interaction.1999Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, s. 432-441Artikel i tidskrift (Refereegranskat)
  • 93.
    Persson, Malin
    et al.
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi.
    Harbridge, JR
    Univ Denver, Dept Chem & Biochem, Denver, CO 80208 USA Linkoping Univ, Dept Chem, IFM, SE-58183 Linkoping, Sweden.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Mitri, R
    Univ Denver, Dept Chem & Biochem, Denver, CO 80208 USA Linkoping Univ, Dept Chem, IFM, SE-58183 Linkoping, Sweden.
    Mårtensson, Lars-Göran
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Carlsson, Uno
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Eaton, GR
    Univ Denver, Dept Chem & Biochem, Denver, CO 80208 USA Linkoping Univ, Dept Chem, IFM, SE-58183 Linkoping, Sweden.
    Eaton, SS
    Univ Denver, Dept Chem & Biochem, Denver, CO 80208 USA Linkoping Univ, Dept Chem, IFM, SE-58183 Linkoping, Sweden.
    Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II2001Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 80, nr 6, s. 2886-2897Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 W. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the noninteracting component was compared.

  • 94.
    Philipson, O.
    et al.
    Uppsala University.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Portelius, E.
    Sahlgrenska University Hospital.
    Olofsson, T.
    Uppsala University.
    Ingelsson, M.
    Uppsala University.
    Hyman, B.T.
    Massachusetts General Hospital.
    Blennow, K.
    Sahlgrenska University Hospital.
    Lannfelt, L.
    Uppsala University.
    Kalimo, H.
    Uppsala University.
    Nilsson, L.N.G.
    Uppsala University.
    A highly insoluble state of Aβ similar to that of Alzheimers disease brain is found in Arctic APP transgenic mice2009Ingår i: Neurobiology of Aging, ISSN 0197-4580, Vol. 30, nr 9, s. 1393-1405Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Amyloid-β (Aβ) is a major drug target in Alzheimers disease. Here, we demonstrate that deposited Aβ is SDS insoluble in tgAPP-ArcSwe, a transgenic mouse model harboring the Arctic (E693G) and Swedish (KM670/671NL) APP mutations. Formic acid was needed to extract the majority of deposited Aβ in both tgAPP-ArcSwe and Alzheimers disease brain, but not in a commonly used type of mouse model with the Swedish mutation alone. Interestingly, the insoluble state of Arctic Aβ was determined early on and did not gradually evolve with time. In tgAPP-ArcSwe, Aβ plaques displayed a patchy morphology with bundles of Aβ fibrils, whereas amyloid cores in tgAPP-Swe were circular with radiating fibrils. Amyloid was more densely stacked in tgAPP-ArcSwe, as demonstrated with a conformation sensitive probe. A reduced increase in plasma Aβ was observed following acute administration of an Aβ antibody in tgAPP-ArcSwe, results that might imply reduced brain to plasma Aβ efflux. TgAPP-ArcSwe, with its insoluble state of deposited Aβ, could serve as a complementary model to better predict the outcome of clinical trials.

  • 95.
    Salagic, Belma
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Regulation of COX-2 signaling in the blood brain barrier2009Självständigt arbete på avancerad nivå (magisterexamen), 20 poäng / 30 hpStudentuppsats
    Abstract [en]

    Upon an inflammation the immune system signals the brain by secreted cytokines to elicit central nervous responses such as fever, loss of appetite and secretion of stress hormones. Since the blood brain barrier, (BBB) protects the brain from unwanted material, molecules like cytokines are not allowed to cross the barrier and enter the brain. However, it is clear that they in some way can signal the brain upon an inflammation. Many suggestions concerning this signaling has been made, one being that cytokines bind to receptors on the endothelial cells of the blood vessels of the brain and trigger the production of prostaglandins that can cross the BBB. This conversion is catalyzed by the enzyme cyclooxygenase-2, (COX-2), which is induced by transcription factors like NF-κB in response to cytokines. One of the central nervous responses to inflammatory stimuli is activation of the HPA-axis whose main purpose is glucocorticoid production. Glucocorticoids inhibit the inflammatory response by suppressing gene transcription of pro-inflammatory genes including those producing prostaglandins through direct interference with transcription factors such as NF-κB or initiation of transcription of anti-inflammatory genes like IκB or IL-10. It has however not been clear if glucocorticoids can target the endothelial cells of the brain in order to provide negative feed-back on the immune-to-brain signaling, and in that way inhibit central nervous inflammatory symptoms. An anatomical prerequisite for such a mechanism would be that the induced prostaglandin production occurs in cells expressing GR. This has however never been demonstrated. Here I show that a majority of the brain endothelial cells expressing the prostaglandin synthesizing enzyme COX-2 in response to immune challenge also express the glucocorticoid receptor, (GR). This indicates that immune-to-brain signaling is a target for negative regulation of inflammatory signaling executed by glucocorticoids and identifies brain endothelial GR as a possible future drug target for treatment of central nervous responses to inflammation such as fever and pain.

  • 96.
    Sandberg, Alexander
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Interaction studies of luminescent conjugated oligothiophenes with aggregated Amyloid β2013Självständigt arbete på avancerad nivå (masterexamen), 30 poäng / 45 hpStudentuppsats (Examensarbete)
    Abstract [en]

    Alzheimer’s disease is the most common cause of dementia and was responsible for over 2% of all deaths in Sweden 2012. One of the pathological hallmarks is amyloid plaques built by fibrillated Amyloid β. Luminescent conjugated oligothiophenes are known to stain and give characteristic fluorescence spectra when staining amyloid fibrils. Little is however known about the interactions between LCOs and fibrils. Studies have been performed on molecules more traditionally known to stain amyloid fibrils. Studies have also been performed on fibrils using limited proteolysis. So far no studies have been performed using LCOs combined with limited proteolysis in order to study the interaction pattern between LCOs and fibrils.

    Amyloid β is expressed and purified using a simple few step purification protocol. The amyloid β peptide was then fibrillated in several generations in order to select for a homogenous fibril structure. This purification protocol also has the ability to purify different oligomers of Amyloid β that are interesting from a toxicity point of view. In this thesis optical characteristics and limited proteolysis with mass spectrometry are being used to studies the interactions between LCOs and fibrillated amyloid β. The proteolytic pattern was suggestive of an accessible N-terminal and a hidden C-terminal of Amyloid β M1-42 in the fibril. It was also shown that the proteolysis cleavage pattern of Chymotrypsin is not disrupted when the LCO pKTAA was used to stain fibrils. The emission spectra from the two LCOs pATAA and pKTAA changes differently when subjected to continuous excitation indicative of conformational changes or chemical modification.

  • 97. Sigurdson, C.J.
    et al.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Organisk Kemi. Linköpings universitet, Tekniska högskolan.
    Hornemann, S.
    Manco, G.
    Polymenidou, M.
    Schwarz, P.
    Leclerc, M.
    Hammarström, Per
    Linköpings universitet, Tekniska högskolan. Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi.
    Wütrich, K.
    Aguzzi, A.
    Prion strain discrimination using luminescent conjugated polymers2007Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 4, nr 12, s. 1023-1030Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The occurrence of multiple strains of prions may reflect conformational variability of PrPSc, a disease-associated, aggregated variant of the cellular prion protein, PrPC. Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and mouse-adapted Rocky Mountain Laboratory scrapie prions. Furthermore, using LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy, and identified noncongophilic deposits in prion-infected deer and sheep. We found that fibrils with distinct morphologies generated from chemically identical recombinant PrP yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct supramolecular PrP structures. LCPs may help to detect structural differences among discrete protein aggregates and to link protein conformational features with disease phenotypes.

  • 98.
    Sjöberg, Andreas P.
    et al.
    Lund University, Department of Laboratory Medicine, Division of Medical Protein Chemistry, Wallenberg Laboratory, Malmö, Sweden.
    Nyström, Sofie
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Blom, Anna M.
    Lund University, Department of Laboratory Medicine, Division of Medical Protein Chemistry, Wallenberg Laboratory, Malmö, Sweden.
    Native, amyloid fibrils and β-oligomers of the C-terminal domain of human prion protein display differential activation of complement and bind C1q, factor H and C4b-binding protein directly2008Ingår i: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 45, nr 11, s. 3213-3221Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Prion protein (PrP) is an endogenous protein involved in the pathogenesis of bovine spongiform encephalopathy and Creutzfeldt–Jakob disease. Murine PrP has been reported to bind C1q and activate the classical pathway of complement in a copper-dependent manner. Here we show that various conformational isoforms (native, amyloid fibrils, and β-oligomers) of recombinant human PrP (90–231 and 121–231) bind C1q and activate complement. PrP binds both the globular head and collagenous stalk domains of C1q. Native, β-oligomeric and amyloid fibrils of PrP all activate the classical and alternative pathways of complement to different extent. However, they do not trigger the lectin pathway. Of the tested PrP conformational isoforms we find that β-oligomers bind C1q and activate complement most strongly. Membrane attack complex formation initiated by PrP is subdued in comparison to deposition of early complement components. This is most likely attributed to the interaction between human PrP and complement inhibitors factor H and C4b-binding protein. Accordingly, PrP-triggered complement activation in the terminal pathway was increased in serum lacking C4b-binding protein. Taken together the present study indicates that complement activation may be an important factor in human prion diseases, suggesting that complement induced activities may prove relevant therapeutic targets.

  • 99.
    Sole-Domenech, Santiago
    et al.
    Karolinska Institute, Sweden .
    Sjovall, Peter
    SP Technical Research Institute Sweden, Sweden .
    Vukojevic, Vladana
    Karolinska Institute, Sweden .
    Fernando, Ruani
    Hop St Eloi, France .
    Codita, Alina
    Karolinska Institute, Sweden .
    Salve, Sachin
    Karolinska Institute, Sweden .
    Bogdanovic, Nenad
    Karolinska Institute, Sweden .
    H Mohammed, Abdul
    Karolinska Institute, Sweden .
    Hammarström, Per
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Nilsson, Peter
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
    M LaFerla, Frank
    University of Calif Irvine, CA USA .
    Jacob, Stefan
    Karolinska Institute, Sweden .
    Berggren, Per-Olof
    Karolinska Institute, Sweden .
    Gimenez-Llort, Lydia
    University of Autonoma Barcelona, Spain .
    Schalling, Martin
    Karolinska Institute, Sweden .
    Terenius, Lars
    Karolinska Institute, Sweden .
    Johansson, Bjorn
    Karolinska Institute, Sweden .
    Localization of cholesterol, amyloid and glia in Alzheimers disease transgenic mouse brain tissue using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and immunofluorescence imaging2013Ingår i: Acta Neuropathologica, ISSN 0001-6322, E-ISSN 1432-0533, Vol. 125, nr 1, s. 145-157Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    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 Alzheimers 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 Alzheimers disease.

  • 100.
    Sörgjerd, Karin
    Linköpings universitet, Institutionen för fysik, kemi och biologi, Biokemi. Linköpings universitet, Tekniska högskolan.
    Molecular Aspects of Transthyretin Amyloid Disease2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [sv]

    Denna avhandling handlar om proteiner. Särskilt de som inte fungerar som de ska utan har blivit vad man kallar ”felveckade”. Anledningen till att proteiner veckas fel beror ofta (men inte alltid) på mutationer i arvsmassan. Felveckade proteiner kan leda till sjukdomar hos människor och djur (man brukar tala om amyloidsjukdomar), ofta av neurologisk karaktär. Exempel på amyloidsjukdomar är polyneuropati, där perifera nervsystemet är drabbat, vilket leder till begränsad rörelseförmåga och senare till förlamning; och Alzheimer´s sjukdom, där centrala nervsystemet är drabbat och leder till begränsad tankeförmåga och minnesförluster.

    Studierna som presenteras i denna avhandling har gått ut på att få en bättre förståelse för hur felveckade proteiner interagerar med det som vi har naturligt i cellerna och som fungerar som skyddande, hjälpande proteiner, så kallade chaperoner.

    Transtyretin (TTR) är ett protein som cirkulerar i blodet och transporterar tyroxin (som är ett hormon som bland annat har betydelse för ämnesomsättningen) samt retinol-bindande protein (vitamin A). I TTR genen har man funnit över 100 punktmutationer, vilka har kopplats samman med amyloidsjukdomar, bland annat ”Skellefteåsjukan”. Mutationer i TTR genen leder ofta till att proteinet blir instabilt vilket leder till upplösning av TTR tetrameren till monomerer. Dessa monomerer kan därefter sammanfogas på nytt men denna gång på ett sätt som är farligt för organismen. I denna avhandling har fokus legat på en mutation som kallas TTR D18G, vilken har identifierats i olika delar av världen och leder till en dödlig form av amyloidos i centrala nervsystemet.

    Det chaperon som vi har studerat benämns BiP och är beläget i en cellkomponent som kallas för det endoplasmatiska retiklet (ER). I ER finns cellens kontrollsystem i vilket det ses till att felveckade proteiner inte släpps ut utan istället bryts ned.

    Denna avhandling har visat att BiP kan fånga upp TTR D18G inuti celler och där samla mutanten i lösliga partiklar som i detta fall är ofarliga för cellen. Avhandligen har också visat att nedbrytningen av TTR D18G sker mycket långsammare när BiP finns i riklig mängd.

    Delarbeten
    1. Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy
    Öppna denna publikation i ny flik eller fönster >>Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy
    2005 (Engelska)Ingår i: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 88, nr 6, s. 4200-4212Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Transthyretin (TTR) is a protein linked to a number of different amyloid diseases including senile systemic amyloidosis and familial amyloidotic polyneuropathy. The transient nature of oligomeric intermediates of misfolded TTR that later mature into fibrillar aggregates makes them hard to study, and methods to study these species are sparse. In this work we explore a novel pathway for generation of prefibrillar aggregates of TTR, which provides important insight into TTR misfolding. Prefibrillar amyloidogenic oligomers and protofibrils of misfolded TTR were generated in vitro through induction of the molten globule type A-state from acid unfolded TTR through the addition of NaCl. The aggregation process produced fairly monodisperse oligomers (300–500 kD) within 2 h that matured after 20 h into larger spherical clusters (30–50 nm in diameter) and protofibrils as shown by transmission electron microscopy. Further maturation of the aggregates showed shrinkage of the spheres as the fibrils grew in length, suggesting a conformational change of the spheres into more rigid structures. The structural and physicochemical characteristics of the aggregates were investigated using fluorescence, circular dichroism, chemical cross-linking, and transmission electron microscopy. The fluorescent dyes 1-anilinonaphthalene-8-sulfonate (ANS), 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS), 4-(dicyanovinyl)-julolidine (DCVJ), and thioflavin T (ThT) were employed in both static and kinetic assays to characterize these oligomeric and protofibrillar states using both steady-state and time-resolved fluorescence techniques. DCVJ, a molecular rotor, was employed for the first time for studies of an amyloidogenic process and is shown useful for detection of the early steps of the oligomerization process. DCVJ bound to the early prefibrillar oligomers (300–500 kD) with an apparent dissociation constant of 1.6 mM, which was slightly better than for ThT (6.8 mM). Time-resolved fluorescence anisotropy decay of ANS was shown to be a useful tool for giving further structural and kinetic information of the oligomeric aggregates. ThT dramatically increases its fluorescence quantum yield when bound to amyloid fibrils; however, the mechanism behind this property is unknown. Data from this work suggest that unbound ThT is also intrinsically quenched and functions similarly to a molecular rotor, which in combination with its environmental dependence provides a blue shift to the characteristic 482nm wavelength when bound to amyloid fibrils.

    Nationell ämneskategori
    Biofysik
    Identifikatorer
    urn:nbn:se:liu:diva-12561 (URN)10.1529/biophysj.104.049700 (DOI)
    Tillgänglig från: 2008-09-15 Skapad: 2008-09-15 Senast uppdaterad: 2018-04-25Bibliografiskt granskad
    2. Prefibrillar Amyloid Aggregates and Cold Shocked Tetrameric Wild Type Transthyretin are Cytotoxic
    Öppna denna publikation i ny flik eller fönster >>Prefibrillar Amyloid Aggregates and Cold Shocked Tetrameric Wild Type Transthyretin are Cytotoxic
    Visa övriga...
    (Engelska)Manuskript (Övrigt vetenskapligt)
    Abstract [en]

    Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of TTR were produced by kinetic sampling from a TTR fibrillation reaction (A-state TTR, pH 2, 100 mM NaCl). The reaction was terminated at different time points, and different states in the aggregation process were captured and analyzed to elucidate the oligomer properties followed by sampling for cytotoxicity using exposure towards human SH-SYY5 neuroblastoma cells. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrenelabeled TTR, chemical cross-linking and electron microscopy we demonstrated that early formed oligomers from A-state TTR were soluble and comprised on the average 20-30 TTR monomers. Early oligomers were highly cytotoxic and induced apoptosis as indicated by the MTT assay and caspase-3 activation, whereas mature fibrils were non-toxic. We also indicate an activated unfolded protein response in cells exposed to oligomers as evidenced by an increased expression of the endoplasmic reticulum located molecular chaperone BiP. Following exposure, BiP appeared relocalized to the cytoplasm. Surprisingly, we also found that native tetrameric TTR purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The molecular basis for this pathogenicity is rather unclear but likely stems from previously reported increased sensitivity towards dissociation and denaturation of TTR at low temperatures and opens the possibility that rearranged tetrameric TTR is cytotoxic towards neuroblastoma cells.

    Nyckelord
    Amyloid, apoptosis, transthyretin, chaperone, misfolding, oligomer
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-12562 (URN)
    Tillgänglig från: 2008-09-15 Skapad: 2008-09-15 Senast uppdaterad: 2018-04-25
    3. Retention of Misfolded Mutant Transthyretin by the Chaperone BiP/GRP78 Mitigates Amyloidogenesi
    Öppna denna publikation i ny flik eller fönster >>Retention of Misfolded Mutant Transthyretin by the Chaperone BiP/GRP78 Mitigates Amyloidogenesi
    Visa övriga...
    2006 (Engelska)Ingår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 356, nr 2, s. 469-482 Artikel i tidskrift (Refereegranskat) Published
    Abstract [en]

    Carriers of the D18G transthyretin (TTR) mutation display an unusual central nervous system (CNS) phenotype with late onset of disease. D18G TTR is monomeric and highly prone to misfold and aggregate even at physiological conditions. Extremely low levels of mutant protein circulate both in human serum and cerebrospinal fluid, indicating impaired secretion of D18G TTR. Recent data show efficient selective ER-associated degradation (ERAD) of D18G TTR. One essential component of the ER-assisted folding machinery is the molecular chaperone BiP. Co-expression of BiP and D18G TTR, or BiP and wild-type (wt) TTR, or mutants A25T TTR and L55P TTR in Escherichia coli showed that only D18G TTR was significantly captured by BiP. Negligible capture of wt TTR and L55P TTR was seen and a sixfold smaller amount of A25T TTR bound to BiP compared to D18G TTR. These data correlate very well with thermodynamic and kinetic stability of the TTR variants, indicating that folding efficiency is inversely correlated to BiP capture. The complexes between BiP and D18G TTR were stable and could be isolated through affinity chromatography. Analytical ultracentrifugation and size-exclusion chromatography revealed that D18G TTR and BiP formed a mixture of 1:1 complexes and large soluble oligomers. The stoichiometry of captured D18G TTR versus BiP increased with increasing size of the oligomers. This indicates that BiP either worked as a molecular shepherd collecting the aggregation-prone mutant into stable oligomers or that BiP could bind to oligomers formed from misfolded mutant protein. Sequence analysis of bound TTR peptides to BiP revealed a bound sequence corresponding to residues 88–103 of TTR, comprising β-strand F in the folded TTR monomer constituting part of the hydrogen bonding tetramer interface in native TTR. The F-strand has also been suggested as a possible elongation region of amyloid fibrils, implicating how substoichiomeric amounts of BiP could sequester prefibrillar amyloidogenic oligomers through binding to this part of TTR. BiP binding to D18G TTR was abolished by addition of ATP. The released D18G TTR completely misfolded into amyloid aggregates as shown by ThT fluorescence and Congo red binding.

    Nyckelord
    BiP; misfolding; intermediate; amyloid; oligomer
    Nationell ämneskategori
    Biokemi och molekylärbiologi
    Identifikatorer
    urn:nbn:se:liu:diva-12563 (URN)10.1016/j.jmb.2005.11.051 (DOI)
    Tillgänglig från: 2008-09-15 Skapad: 2008-09-15 Senast uppdaterad: 2018-04-25
    4. BiP can function as a molecular shepherd that alleviates oligomer toxicity and amass amyloid
    Öppna denna publikation i ny flik eller fönster >>BiP can function as a molecular shepherd that alleviates oligomer toxicity and amass amyloid
    Visa övriga...
    (Engelska)Manuskript (Övrigt vetenskapligt)
    Abstract [en]

    A wide range of diseases are linked to protein misfolding and aggregation inside and outside the cell. It is of utmost interest to understand how the molecular chaperone machinery of the endoplasmic reticulum (ER) handles the expression of highly amyloidogenic proteins. We explored the hypothesis that the ER located Hsp70 molecular chaperone BiP plays a crucial role in amyloid diseases and influence the misfolding process and disease progression. We used the transthyretin mutant TTR D18G associated with an unusual central nervous system amyloid disease as the model substrate because it represents the most destabilized and degraded TTR variant known. Over-expression of TTR D18G in concert with BiP showed that BiP selectively recognize the amyloidogenic mutant protein as compared to wild type in human cells and collects the mutant in stable intermediate size oligomers within the ER. Furthermore, whereas TTR D18G was found to be highly cytotoxic to neuroblastoma cells, TTR D18G preincubated with BiP was non-toxic indicating that BiP protects the cell from cytotoxicity. BiP was also found present in cerebellar amyloid deposits and co-localized with TTR in a TTR D18G patient suggesting that the complex can be found in the extracellular space. We promote a fundamental role of BiP in misfolding diseases and describe a molecular shepharding function of BiP in sequestrating amyloidogenic protein molecules in benign oligomeric states.

    Nyckelord
    Amyloid, transthyretin, chaperone, misfolding, oligomer, cytotoxicity
    Nationell ämneskategori
    Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
    Identifikatorer
    urn:nbn:se:liu:diva-12564 (URN)
    Tillgänglig från: 2008-09-15 Skapad: 2008-09-15 Senast uppdaterad: 2018-04-25
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