SwePub
Sök i SwePub databas

  Extended search

Träfflista för sökning "WFRF:(Linse Sara) "

Search: WFRF:(Linse Sara)

  • Result 1-50 of 280
Sort/group result
   
EnumerationReferenceCoverFind
1.
  • Cabaleiro-Lago, Celia, et al. (author)
  • Inhibition of IAPP and IAPP(20-29) fibrillation by polymeric nanoparticles
  • 2010
  • In: Langmuir. - : The American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 26:5, s. 3453-3461
  • Journal article (peer-reviewed)abstract
    • The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.
  •  
2.
  • Hellstrand, Erik, et al. (author)
  • Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion.
  • 2013
  • In: The FEBS Journal. - : Wiley. - 1742-464X. ; 280:11, s. 2675-2687
  • Journal article (peer-reviewed)abstract
    • This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.
  •  
3.
  • Linse, Björn, et al. (author)
  • Monte Carlo simulations of protein amyloid formation reveal origin of sigmoidal aggregation kinetics.
  • 2011
  • In: Molecular BioSystems. - : Royal Society of Chemistry (RSC). - 1742-2051 .- 1742-206X. ; 7, s. 2296-2303
  • Journal article (peer-reviewed)abstract
    • Severe conditions and lack of cure for many amyloid diseases make it highly desired to understand the underlying principles of formation of fibrillar aggregates (amyloid). Here, amyloid formation from peptides was studied using Monte Carlo simulations. Systems of 20, 50, 100, 200 or 500 hexapeptides were simulated. Association kinetics were modeled equal for fibrillar and other (inter- and intra-peptide) contacts and assumed to be faster the lower the effective contact order, which represents the distance in space. Attempts to form contacts were thus accepted with higher probability the lower the effective contact order, whereby formation of new contacts next to preexisting ones is favored by shorter physical separation. Kinetic discrimination was invoked by using two different life-times for formed contacts. Contacts within amyloid fibrils were assumed to have on average longer life-time than other contacts. We find that the model produces fibrillation kinetics with a distinct lag phase, and that the fibrillar contacts need to dissociate on average 5-20 times slower than all other contacts for the fibrillar structure to dominate at equilibrium. Analysis of the species distribution along the aggregation process shows that no other intermediate is ever more populated than the dimer. Instead of a single nucleation event there is a concomitant increase in average aggregate size over the whole system, and the occurrence of multiple parallel processes makes the process more reproducible the larger the simulated system. The sigmoidal shape of the aggregation curves arises from cooperativity among multiple interactions within each pair of peptides in a fibril. A governing factor is the increasing probability as the aggregation process proceeds of neighboring reinforcing contacts. The results explain the very strong bias towards cross β-sheet fibrils in which the possibilities for cooperativity among interactions involving neighboring residues and the repetitive use of optimal side-chain interactions are explored at maximum.
  •  
4.
  • Linse, Sara, et al. (author)
  • Protein folding through kinetic discrimination
  • 2007
  • In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:27, s. 8481-8486
  • Journal article (peer-reviewed)abstract
    • Proteins fold on a mu s-ms time scale. However, the number of possible conformations of the polypeptide backbone is so large that random sampling would not allow the protein to fold within the lifetime of the universe, the Levinthal paradox. We show here that a protein chain can fold efficiently with high fidelity if on average native contacts survive longer than non-native ones, that is, if the dissociation rate constant for breakage of a contact is lower for native than for non-native interactions. An important consequence of this finding is that no pathway needs to be specified for a protein to fold. Instead, kinetic discrimination among formed contacts is a sufficient criterion for folding to proceed to the native state. Successful protein folding requires that productive contacts survive long enough to obtain a certain level of probability that other native contacts form before the first interacting unit dissociates. If native contacts survive longer than non-native ones, this prevents misfolding and provides the folding process with directionality toward the native state. If on average all contacts survive equally long, the protein chain is deemed to fold through random search through all possible conformations (i.e., the Levinthal paradox). A modest degree of cooperativity among the native contacts, that is, decreased dissociation rate next to neighboring contacts, shifts the required ratio of dissociation rates into a realistic regime and makes folding a stochastic process with a nucleation step. No kinetic discrimination needs to be invoked in regards to the association process, which is modeled as dependent on the diffusion rate of chain segments.
  •  
5.
  • O'Connell, David, et al. (author)
  • Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins
  • 2010
  • In: Molecular & Cellular Proteomics. - 1535-9484. ; 9:6, s. 1118-1132
  • Journal article (peer-reviewed)abstract
    • Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca(2+) ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labelled calmodulin in the presence of Ca(2+). This protein array contains 37,200 redundant proteins, incorporating over 10,000 unique human neural proteins from a human brain cDNA library. We designed a screen to find high affinity (K(D) = 1 muM) binding partners of calmodulin and identified 76 human proteins from all intracellular compartments, of which 72 are novel. We measured the binding kinetics of 74 targets with calmodulin using a high throughput surface plasmon resonance assay. Most of the novel calmodulin-target complexes identified have low dissociation rates (koff = 10(3) s(-1)) and high affinity (K(D) = 1 muM), consistent with the design of the screen. Many of the identified proteins are known to assemble in neural tissue, forming assemblies such as the spectrin scaffold and the postsynaptic density. We have developed a microarray of the identified target proteins with which we can characterise the biochemistry of calmodulin for all targets in parallel. Four novel targets were verified in neural cells by co-immunoprecipitation, and four were selected for exploration of the calmodulin-binding regions. Using synthetic peptides and isothermal titration calorimetry, calmodulin binding motifs were identified in the potassium voltage gated channel Kv6.1, (residues 474-493), CaM kinase-like vesicle-associated protein (302-316), EF-hand domain family member A2 (202-216) and phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (400-415).
  •  
6.
  • Szczepankiewicz, Olga, et al. (author)
  • N-Terminal Extensions Retard Aβ42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aβ42.
  • 2015
  • In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 137:46, s. 14673-14685
  • Journal article (peer-reviewed)abstract
    • Amyloid β-protein (Aβ) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid β-protein (Aβ) variants were believed to begin at or after the canonical β-secretase cleavage site within the amyloid β-protein precursor. However, N-terminally extended forms of Aβ (NTE-Aβ) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aβ42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aβ42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aβ42s form fibrils via the same mechanism as Aβ42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aβ42 and NTE-Aβ42 coaggregate to form mixed fibrils and fibrils of either Aβ42 or NTE-Aβ42 catalyze aggregation of all monomers. NTE-Aβ42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.
  •  
7.
  • Abou-Hachem, Maher, et al. (author)
  • Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
  • 2002
  • In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5720-5729
  • Journal article (peer-reviewed)abstract
    • Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
  •  
8.
  • Abou-Hachem, Maher, et al. (author)
  • The modular organisation and stability of a thermostable family 10 xylanase
  • 2003
  • In: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 21:5-6, s. 253-260
  • Journal article (peer-reviewed)abstract
    • The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.
  •  
9.
  •  
10.
  • Andersson, Alexandra, et al. (author)
  • Single-vesicle intensity and colocalization fluorescence microscopy to study lipid vesicle fusion, fission, and lipid exchange
  • 2022
  • In: Frontiers in Molecular Neuroscience. - : Frontiers Media SA. - 1662-5099. ; 15
  • Journal article (peer-reviewed)abstract
    • Interactions of lipid vesicles play important roles in a large variety of functions and dysfunctions in the human body. Vital for several biochemical functions is the interaction between monomeric proteins and lipid membranes, and the induced phenomena such as fusion between vesicles and cell membranes, lipid exchange between the membranes, or vesicle fission. Identification of single events and their frequency of occurrence would provide valuable information about protein-lipid interactions in both healthy and degenerative pathways. In this work, we present a single-vesicle intensity and colocalization fluorescence microscopy assay with a custom-written MATLAB analysis program. The assay can be used to study lipid exchange as well as vesicle fusion and fission between two vesicle populations labeled with different fluorescent dyes. Vesicles from the two populations are first mixed and docked to a glass surface. The sample is then simultaneously imaged using two separate wavelength channels monitoring intensity changes and colocalization of vesicles from the two populations. The monomeric pre-synaptic protein α-synuclein (α-syn) and small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DOPS), and monosialotetrahexosylganglioside (GM1) were used as a model system to evaluate the method. From our analysis, neither α-syn induced fusion nor lipid exchange was observed for vesicles consisting of DOPC:DOPS (7:3). However, including 10% GM1 in the vesicles resulted in a 91% increase of the number of vesicles within 10 min, combined with a 57% decrease in the average fluorescence intensity per vesicle, indicating that approximately half of the vesicles underwent fission. The method facilitates the study of lipid vesicle fusion, fission, and lipid exchange under controlled conditions. It also allows these events to be studied for systems with more complex composition including exosomes and lipid-based drug carriers, to enable a better understanding of their physicochemical properties.
  •  
11.
  • Andersson, Alexandra, et al. (author)
  • The density of anionic lipids modulates the adsorption of α-Synuclein onto lipid membranes
  • 2024
  • In: Biophysical Chemistry. - 0301-4622. ; 305
  • Journal article (peer-reviewed)abstract
    • α-Synuclein is an intrinsically disordered presynaptic protein associated with Parkinson's disease. The physiological role of α-Synuclein is not fully understood, but the protein is known to interact with lipid membranes. We here study how membrane charge affects the adsorption of α-Synuclein to (i) supported lipid bilayers and (ii) small unilamellar vesicles with varying amounts of anionic lipids. The results showed that α-Synuclein adsorbs onto membranes containing ≥5% anionic phosphatidylserine (DOPS) lipids, but not to membranes containing ≤1% DOPS. The density of adsorbed α-Synuclein increased steadily with the DOPS content up to 20% DOPS, after which it leveled off. The vesicles were saturated with α-Synuclein at a 3–5 times higher protein density compared to the supported bilayers, which suggests that a more deformable membrane binds more α-Synuclein. Altogether, the results show that both membrane charge density and flexibility influence the association of α-Synuclein to lipid membranes.
  •  
12.
  • André, Ingemar, et al. (author)
  • Measurement of Ca2+-binding constants of proteins and presentation of the CaLigator software.
  • 2002
  • In: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 305:2, s. 195-205
  • Journal article (peer-reviewed)abstract
    • The complexity of Ca2+ cell signaling is dependent on a plethoria of Ca2+-binding proteins that respond to signals in different ranges of Ca2+ concentrations. Since the function of these proteins is directly coupled to their Ca2+-binding properties, there is a need for accurately determined equilibrium Ca2+-binding constants. In this work we outline the experimental techniques available to determine Ca2+-binding constants in proteins, derive the models used to describe the binding, and present CaLigator, software for least-square fitting directly to the measured quantity. The use of the software is illustrated for Ca2+-binding data obtained for two deamidated forms of calbindin D(9k), either an isospartate-56 (beta form) or a normal Asp-56 (alpha form). Here, the Ca2+-binding properties of the two isoforms have been studied using the chelator method. The alpha form shows similar Ca2+-binding properties to the wild type while the beta form has lost both cooperativety and affinity.
  •  
13.
  • André, Ingemar, et al. (author)
  • Residue-specific pK(a) determination of lysine and arginine side chains by indirect N-15 and C-13 NMR spectroscopy: Application to apo calmodulin
  • 2007
  • In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:51, s. 15805-15813
  • Journal article (peer-reviewed)abstract
    • Electrostatic interactions in proteins can be probed experimentally through determination of residue-specific acidity constants, We describe here triple-resonance NMR techniques for direct determination of lysine and arginine side-chain protonation states in proteins. The experiments are based on detection of nonexchangeable protons over the full range of pH and temperature and therefore are well suited for pK(a) determination of individual amino acid side chains. The experiments follow the side-chain N-15(zeta) (lysine) and N-15(epsilon) or C-13(zeta) (arginine) chemical shift, which changes due to sizable changes in the heteronuclear electron distribution upon (de)protonation. Since heteronuclear chemical shifts are overwhelmed by the charge state of the amino acid side chain itself, these methods supersede H-1-based NMR in terms of accuracy, sensitivity, and selectivity. Moreover, the N-15(zeta) and N-15(epsilon) nuclei may be used to probe changes in the local electrostatic environment. Applications to three proteins are described: apo calmodulin, calbindin D-9k, and FKBP12. For apo calmodulin, residue-specific pK(a) values of lysine side chains were determined to fall between 10.7 and 11.2 as a result of the high net negative charge on the protein surface. Ideal two-state titration behavior observed for all lysines indicates the absence of significant direct charge interactions between the basic residues. These results are compared with earlier studies based on chemical modification.
  •  
14.
  • André, Ingemar, et al. (author)
  • Salt enhances calmodulin-target interaction
  • 2006
  • In: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 90:8, s. 2903-2910
  • Journal article (peer-reviewed)abstract
    • Calmodulin (CaM) operates as a Ca2+ sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca2+-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.
  •  
15.
  • André, Ingemar, et al. (author)
  • Streptococcal M protein: Structural studies of the hypervariable region, free and bound to human C4BP
  • 2006
  • In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 45:14, s. 4559-4568
  • Journal article (peer-reviewed)abstract
    • Streptococcus pyogenes is a Gram-positive bacterium that causes several diseases, including acute tonsillitis and toxic shock syndrome. The surface-localized M protein, which is the most extensively studied virulence factor of S. pyogenes, has an similar to 50-residue N-terminal hypervariable region (HVR) that plays a key role in the escape of the host immunity. Despite the extensive sequence variability in this region, many HVRs specifically bind human C4b-binding protein (C4BP), a plasma protein that inhibits complement activation. Although the more conserved parts of M protein are known to have dimeric coiled-coil structure, it is unclear whether the HVR also is a coiled coil. Here, we use nuclear magnetic resonance (NMR) to study the conformational properties of HVRs from M4 and M22 proteins in isolation and in complex with the M protein binding portion of C4BP. We conclude that the HVRs of M4 and M22 are folded as coiled coils and that the folded nucleus of the M4 HVR has a length of similar to 27 residues. Moreover, we demonstrate that the C4BP binding surface of M4-N is found within a region of four heptad repeats. Using molecular modeling, we propose a model for the structure of the M4 HVR that is consistent with our experimental information from NMR spectroscopy.
  •  
16.
  • André, Ingemar, et al. (author)
  • The role of electrostatic interactions in calmodulin-peptide complex formation
  • 2004
  • In: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 87:3, s. 1929-1938
  • Journal article (peer-reviewed)abstract
    • The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from similar to-5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.
  •  
17.
  • Aprile, Francesco A., et al. (author)
  • Selective targeting of primary and secondary nucleation pathways in Ab42 aggregation using a rational antibody scanning method
  • 2017
  • In: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 3:6
  • Journal article (peer-reviewed)abstract
    • Antibodies targeting Ab42 are under intense scrutiny because of their therapeutic potential for Alzheimer’s disease. To enable systematic searches, we present an “antibody scanning” strategy for the generation of a panel of antibodies against Ab42. Each antibody in the panel is rationally designed to target a specific linear epitope, with the selected epitopes scanning the Ab42 sequence. By screening in vitro the panel to identify the specific microscopic steps in the Ab42 aggregation process influenced by each antibody, we identify two antibodies that target specifically the primary and the secondary nucleation steps, which are key for the production of Ab42 oligomers. These two antibodies act, respectively, to delay the onset of aggregation and to block the proliferation of aggregates, and correspondingly reduce the toxicity in a Caenorhabditis elegans model over-expressing Ab42. These results illustrate how the antibody scanning method described here can be used to readily obtain very small antibody libraries with extensive coverage of the sequences of target proteins.
  •  
18.
  • Areschoug, Thomas, et al. (author)
  • A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal beta Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface.
  • 2002
  • In: Journal of Bacteriology. - 0021-9193. ; 184:22, s. 6376-6383
  • Journal article (peer-reviewed)abstract
    • Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the beta protein of group B streptococci. The proline-rich region in beta, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a beta mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the beta protein. Comparison of the XPZ region in different beta proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.
  •  
19.
  • Arosio, Paolo, et al. (author)
  • Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation
  • 2016
  • In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697. ; 504, s. 7-13
  • Journal article (peer-reviewed)abstract
    • The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1-42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril-fibril assembly can play in the deposition of aggregation-prone peptides.
  •  
20.
  • Arosio, Paolo, et al. (author)
  • Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation
  • 2016
  • In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
  • Journal article (peer-reviewed)abstract
    • It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.
  •  
21.
  • Arosio, Paolo, et al. (author)
  • Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions.
  • 2016
  • In: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 10:1, s. 333-341
  • Journal article (peer-reviewed)abstract
    • Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.
  •  
22.
  • Arosio, Paolo, et al. (author)
  • On the lag phase in amyloid fibril formation
  • 2015
  • In: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 17:12, s. 7606-7618
  • Journal article (peer-reviewed)abstract
    • The formation of nanoscale amyloid fibrils from normally soluble peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: a lag phase, a growth phase and a final plateau regime. The question of which molecular events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. In this review, we discuss the nature and molecular origin of the lag-phase in amyloid formation by making use of tools and concepts from physical chemistry, in particular from chemical reaction kinetics. We discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in solution. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concentration that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least two -primary nucleation and elongation - and in many systems at least four - primary nucleation, elongation, secondary nucleation and fragmentation - microscopic processes occur during the lag phase. Moreover, these same processes occur during all three phases of the macroscopic aggregation process, albeit at different rates as governed by rate constants and by the concentration of reacting species at each point in time.
  •  
23.
  • Arosio, Paolo, et al. (author)
  • Quantification of the Concentration of A beta 42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay
  • 2014
  • In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:1, s. 219-225
  • Journal article (peer-reviewed)abstract
    • The aggregation of the amyloid beta peptide, A beta 42, implicated in Alzheimer's disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar A beta 42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.
  •  
24.
  • Aspuru-Guzik, A., et al. (author)
  • Charting a course for chemistry
  • 2019
  • In: Nature Chemistry. - : Springer Science and Business Media LLC. - 1755-4330 .- 1755-4349. ; 11:4, s. 286-294
  • Journal article (peer-reviewed)
  •  
25.
  •  
26.
  • Assarsson, Anna, et al. (author)
  • Charge dependent retardation of amyloid β aggregation by hydrophilic proteins
  • 2014
  • In: ACS Chemical Neuroscience. - 1948-7193 .- 1948-7193. ; 5:4, s. 266-74
  • Journal article (peer-reviewed)abstract
    • The aggregation of amyloid β peptides (Aβ) into amyloid fibrils is implicated in the pathology of Alzheimer's disease. In light of the increasing number of proteins reported to retard Aβ fibril formation, we investigated the influence of small hydrophilic model proteins of different charge on Aβ aggregation kinetics and their interaction with Aβ. We followed the amyloid fibril formation of Aβ40 and Aβ42 using thioflavin T fluorescence in the presence of six charge variants of calbindin D9k and single-chain monellin. The formation of fibrils was verified with transmission electron microscopy. We observe retardation of the aggregation process from proteins with net charge +8, +2, -2, and -4, whereas no effect is observed for proteins with net charge of -6 and -8. The single-chain monellin mutant with the highest net charge, scMN+8, has the largest retarding effect on the amyloid fibril formation process, which is noticeably delayed at as low as a 0.01:1 scMN+8 to Aβ40 molar ratio. scMN+8 is also the mutant with the fastest association to Aβ40 as detected by surface plasmon resonance, although all retarding variants of calbindin D9k and single-chain monellin bind to Aβ40.
  •  
27.
  • Assarsson, Anna, et al. (author)
  • Effects of polyamino acids and polyelectrolytes on amyloid β fibril formation
  • 2014
  • In: Langmuir. - : The American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 30:29, s. 8812-8
  • Journal article (peer-reviewed)abstract
    • The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly(acrylic acid), poly(ethylenimine), and poly(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide.
  •  
28.
  • Assarsson, Anna, et al. (author)
  • Effects of Polyamino Acids and Polyelectrolytes on Amyloid β Fibril Formation.
  • 2014
  • In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 30:29, s. 8812-8818
  • Journal article (peer-reviewed)abstract
    • The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly(acrylic acid), poly(ethylenimine), and poly(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide.
  •  
29.
  • Axell, Emil, et al. (author)
  • The role of shear forces in primary and secondary nucleation of amyloid fibrils
  • 2024
  • In: Proceedings of the National Academy of Sciences of the United States of America. - 1091-6490. ; 121:25, s. 2322572121-2322572121
  • Journal article (peer-reviewed)abstract
    • Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for Aβ42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.
  •  
30.
  • Bahri, Salima, et al. (author)
  • 1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils
  • 2022
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 119:1
  • Journal article (peer-reviewed)abstract
    • Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aβ1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aβ1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.
  •  
31.
  • Bauer, Mikael, et al. (author)
  • Calmodulin Binding to the Polybasic C-Termini of STIM Proteins Involved in Store-Operated Calcium Entry.
  • 2008
  • In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:23, s. 6089-6091
  • Journal article (peer-reviewed)abstract
    • Translocation of STIM1 and STIM2 from the endoplasmic reticulum to the plasma membrane is a key step in store-operated calcium entry in the cell. We show by isothermal titration calorimetry that calmodulin binds in a calcium-dependent manner to the polybasic C-termini of STIM1 and STIM2, a region critical for their translocation to the plasma membrane ( K D
  •  
32.
  • Bauer, Mikael, et al. (author)
  • Identification of a high-affinity network of secretagogin-binding proteins involved in vesicle secretion.
  • 2011
  • In: Molecular BioSystems. - 1742-2051. ; 7, s. 2196-2204
  • Journal article (peer-reviewed)abstract
    • Secretagogin is a hexa EF-hand Ca(2+)-binding protein expressed in neuroendocrine, pancreatic endocrine and retinal cells. The protein has been noted for its expression in specific neuronal subtypes in the support of hierarchical organizing principles in the mammalian brain. Secretagogin has previously been found to interact with SNAP25 involved in Ca(2+)-induced exocytosis. Here, the cellular interaction network of secretagogin has been expanded with nine proteins: SNAP-23, DOC2alpha, ARFGAP2, rootletin, KIF5B, β-tubulin, DDAH-2, ATP-synthase and myeloid leukemia factor 2, based on screening of a high content protein array and validation and quantification of binding with surface plasmon resonance and GST pulldown assays. All targets have association rate constants in the range 10(4)-10(6) M(-1) s(-1), dissociation rate constants in the range 10(-3)-10(-5) s(-1) and equilibrium dissociation constants in the 100 pM to 10 nM range. The novel target SNAP23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion. Complementary roles in vesicle trafficking are known for ARFGAP2 and DOC2alpha in regulating fusion of vesicles to membranes, kinesin 5B and tubulin for transport of vesicles in the cell, while rootletin builds up the rootlet believed to function as a scaffold for vesicles. The identification of a discrete network of interacting proteins that mediate secretion and vesicle trafficking suggests a regulatory role for secretagogin in these processes.
  •  
33.
  • Bauer, Mikael, et al. (author)
  • Protein GB1 Folding and Assembly from Structural Elements.
  • 2009
  • In: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 10:4, s. 1552-1566
  • Journal article (peer-reviewed)abstract
    • Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in beta-hairpin structure for the C-terminal fragment (residues 41 - 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 - 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins.
  •  
34.
  • Bauer, Mikael, et al. (author)
  • Protein networks involved in vesicle fusion, transport, and storage revealed by array-based proteomics.
  • 2011
  • In: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 781, s. 47-58
  • Journal article (peer-reviewed)abstract
    • Secretagogin is a calcium-binding protein whose expression is characterised in neuroendocrine, pancreatic, and retinal cells. We have used an array-based proteomic approach with the prokaryotically expressed human protein array (hEx1) and the eukaryotically expressed human protein array (Protoarray) to identify novel calcium-regulated interaction networks of secretagogin. Screening of these arrays with fluorophore-labelled secretagogin in the presence of Ca(2+) ions led to the identification of 12 (hEx1) and 6 (Protoarray) putative targets. A number of targets were identified in both array screens. The putative targets from the hEx1 array were expressed, purified, and subjected to binding analysis using surface plasmon resonance. This identified binding affinities for nine novel secretagogin targets with equilibrium dissociation constants in the 100 pM to 10 nM range. Six of the novel target proteins have important roles in vesicle trafficking; SNAP-23, ARFGAP2, and DOC2alpha are involved in regulating fusion of vesicles to membranes, kinesin 5B and tubulin are essential for transport of vesicles in the cell, and rootletin builds up the rootlet, which is believed to function as scaffold for vesicles. Among the targets are two enzymes, DDAH-2 and ATP-synthase, and one oncoprotein, myeloid leukaemia factor 2. This screening method identifies a role for secretagogin in secretion and vesicle trafficking interacting with several proteins integral to these processes.
  •  
35.
  • Bauer, Mikael, et al. (author)
  • Zn2+ binding to human calbindin D(28k) and the role of histidine residues.
  • 2008
  • In: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 17:4, s. 760-767
  • Journal article (peer-reviewed)abstract
    • We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.
  •  
36.
  • Baumann, Kevin N., et al. (author)
  • A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Aβ42 Aggregation
  • 2023
  • In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 14:2, s. 323-329
  • Journal article (peer-reviewed)abstract
    • The aggregation of the amyloid β (Aβ) peptide is one of the molecular hallmarks of Alzheimer’s disease (AD). Although Aβ deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aβ assemblies. Because these intracellular Aβ aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aβ is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aβ (Aβ42). Intracellularly, Aβ42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aβ, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aβ42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aβ42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aβ aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aβ.
  •  
37.
  • Behnen, Petra, et al. (author)
  • Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes.
  • 2012
  • In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:34, s. 6718-6727
  • Journal article (peer-reviewed)abstract
    • Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.
  •  
38.
  • Berggård, Tord, et al. (author)
  • 140 mouse brain proteins identified by Ca2+-calmodulin affinity chromatography and tandem mass spectrometry.
  • 2006
  • In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 5:3, s. 669-687
  • Journal article (peer-reviewed)abstract
    • Calmodulin is an essential Ca2+-binding protein that binds to a variety of targets that carry out critical signaling functions. We describe the proteomic characterization of mouse brain Ca2+-calmodulin-binding proteins that were purified using calmodulin affinity chromatography. Proteins in the eluates from four different affinity chromatography experiments were identified by 1-DE and in-gel digestion followed by LC-MS/MS. Parallel experiments were performed using two related control-proteins belonging to the EF-hand family. After comparing the results from the different experiments, we were able to exclude a significant number of proteins suspected to bind in a nonspecific manner. A total of 140 putative Ca2+-calmodulin-binding proteins were identified of which 87 proteins contained calmodulin-binding motifs. Among the 87 proteins that contained calmodulin-binding motifs, 48 proteins have not previously been shown to interact with calmodulin and 39 proteins were known calmodulin-binding proteins. Many proteins with ill-defined functions were identified as well as a number of proteins that at the time of the analysis were described only as ORFs. This study provides a functional framework for studies on these previously uncharacterized proteins.
  •  
39.
  • Berggård, Tord, et al. (author)
  • Calbindin D28k exhibits properties characteristic of a Ca2+ sensor.
  • 2002
  • In: Journal of Biological Chemistry. - 1083-351X. ; 277:19, s. 16662-16672
  • Journal article (peer-reviewed)abstract
    • Calbindin D28k is a member of the calmodulin super-family of Ca2+ -binding proteins and contains six EF-hands. The protein is generally believed to function as a Ca2+ buffer, but the studies presented in this work indicate that it may also act as a Ca2+ sensor. The results show that Mg2+ binds to the same sites as Ca2+ with an association constant of approximately 1.4 x 10(3) M-1 in 0.15 M KCl. The four high-affinity sites in calbindin D28k bind Ca2+ in a non-sequential, parallel manner. In the presence of physiological concentrations of Mg2+, the Ca2+ -affinity is reduced by a factor of two and the cooperativity, which otherwise is modest, increases. Based on the binding constants determined in the presence of physiological salt concentrations, we estimate that at the Ca2+ concentration in a resting cell calbindin D28k is saturated to 40-75% with Mg2+, but to less than 9 % with Ca2+. In contrast, the protein is expected to be nearly fully saturated with Ca2+ at the Ca2+ level of an activated cell. A substantial conformational change is observed upon Ca2+ binding, but only minor structural changes take place upon Mg2+-binding. This suggests that calbindin D28k undergoes Ca2+ -induced structural changes upon Ca2+ activation of a cell. Thus, calbindin D28k displays several properties that would be expected for a protein involved in Ca2+ -induced signal transmission and hence may function not only as a Ca2+ buffer, but also as a Ca2+ sensor. Digestion patterns resulting from limited proteolysis of the protein suggest that the loop of EF-hand 2, a variant site that does not bind Ca2+, becomes exposed upon Ca2+ binding.
  •  
40.
  • Berggård, Tord, et al. (author)
  • Methods for the detection and analysis of protein-protein interactions
  • 2007
  • In: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 7:16, s. 2833-2842
  • Research review (peer-reviewed)abstract
    • A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.
  •  
41.
  • Berggård, Tord, et al. (author)
  • myo-Inositol monophosphatase is an activated target of calbindin D28k.
  • 2002
  • In: Journal of Biological Chemistry. - 1083-351X. ; 277:44, s. 41954-41959
  • Journal article (peer-reviewed)abstract
    • Calbindin D28k (calbindin) is a member of the calmodulin superfamily of Ca2+ -binding proteins. An intracellular target of calbindin was discovered using bacteriophage display. Human recombinant calbindin was immobilized on magnetic beads and used in affinity purification of phage-displayed peptides from a random 12-mer peptide library. One sequence, SYSSIAKYPSHS, was strongly selected both in the presence of Mg2+ and in the presence of Ca2+. Homology search against the protein sequence data base identified a closely similar sequence, ISSIKEKYPSHS, at residues 55-66 in myo-inositol-1(or 4)-monophosphatase (IMPase, EC 3.1.3.25), which constitute a strongly conserved, and exposed region in the 3D structure. IMPase is a key enzyme in the regulation of the activity of the phosphatidyl inositol signaling pathway. It catalyzes the hydrolysis of myo-inositol-1(or 4)-monophosphate to form free myo-inositol, maintaining a supply that represents the precursor for inositol phospholipid second messenger signaling systems. Fluorescence spectroscopy showed that isolated calbindin and IMPase interact with an apparent equilibrium dissociation constant, KD, of 0.9 mM. Both apo and Ca2+-bound calbindin was found to activate IMPase up to 250-fold, depending on the pH and substrate concentration. The activation is most pronounced at conditions which otherwise lead to a very low activity of IMPase, i.e. at reduced pH and at low substrate concentration.
  •  
42.
  • Boza-Serrano, Antonio, et al. (author)
  • Galectin-3, a novel endogenous TREM2 ligand, detrimentally regulates inflammatory response in Alzheimer’s disease
  • 2019
  • In: Acta Neuropathologica. - : Springer Science and Business Media LLC. - 0001-6322 .- 1432-0533. ; 138:2, s. 251-273
  • Journal article (peer-reviewed)abstract
    • Alzheimer’s disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (Aβ) peptide, fibrillary tangles of intraneuronal tau and microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimer’s disease) mice and found specifically expressed in microglia associated with Aβ plaques. Single-nucleotide polymorphisms in the LGALS3 gene, which encodes gal3, were associated with an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data revealed that gal3 was required to fully activate microglia in response to fibrillar Aβ. Gal3 deletion decreased the Aβ burden in 5xFAD mice and improved cognitive behavior. Interestingly, a single intrahippocampal injection of gal3 along with Aβ monomers in WT mice was sufficient to induce the formation of long-lasting (2 months) insoluble Aβ aggregates, which were absent when gal3 was lacking. High-resolution microscopy (stochastic optical reconstruction microscopy) demonstrated close colocalization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 carbohydrate recognition domain. Furthermore, gal3 was shown to stimulate TREM2–DAP12 signaling in a reporter cell line. Overall, our data support the view that gal3 inhibition may be a potential pharmacological approach to counteract AD.
  •  
43.
  • Braun, Gabriel A., et al. (author)
  • Amyloid-beta peptide 37, 38 and 40 individually and cooperatively inhibit amyloid-beta 42 aggregation
  • 2022
  • In: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 13:8, s. 2423-2439
  • Journal article (peer-reviewed)abstract
    • The pathology of Alzheimer's disease is connected to the aggregation of beta-amyloid (A beta) peptide, which in vivo exists as a number of length-variants. Truncations and extensions are found at both the N- and C-termini, relative to the most commonly studied 40- and 42-residue alloforms. Here, we investigate the aggregation of two physiologically abundant alloforms, A beta(37) and A beta(38), as pure peptides and in mixtures with A beta(40) and A beta(42). A variety of molar ratios were applied in quaternary mixtures to investigate whether a certain ratio is maximally inhibiting of the more toxic alloform A beta(42). Through kinetic analysis, we show that both A beta(37) and A beta(38) self-assemble through an autocatalytic secondary nucleation reaction to form fibrillar beta-sheet-rich aggregates, albeit on a longer timescale than A beta(40) or A beta(42). Additionally, we show that the shorter alloforms co-aggregate with A beta(40), affecting both the kinetics of aggregation and the resulting fibrillar ultrastructure. In contrast, neither A beta(37) nor A beta(38) forms co-aggregates with A beta(42); however, both short alloforms reduce the rate of A beta(42) aggregation in a concentration-dependent manner. Finally, we show that the aggregation of A beta(42) is more significantly impeded by a combination of A beta(37), A beta(38), and A beta(40) than by any of these alloforms independently. These results demonstrate that the aggregation of any given A beta alloform is significantly perturbed by the presence of other alloforms, particularly in heterogeneous mixtures, such as is found in the extracellular fluid of the brain.
  •  
44.
  • Braun, Gabriel A., et al. (author)
  • On the Mechanism of Self-Assembly by a Hydrogel-Forming Peptide
  • 2020
  • In: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 21:12, s. 4781-4794
  • Journal article (peer-reviewed)abstract
    • Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogel-forming peptide, SgI37-49. Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.
  •  
45.
  • Buell, Alexander K., et al. (author)
  • Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation
  • 2014
  • In: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:21, s. 7671-7676
  • Journal article (peer-reviewed)abstract
    • The formation of amyloid fibrils by the intrinsically disordered protein alpha-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that alpha-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of alpha-synuclein aggregation and aggregate spreading in the context of Parkinson disease.
  •  
46.
  • Cabaleiro-Lago, Celia, et al. (author)
  • Dual Effect of Amino Modified Polystyrene Nanoparticles on Amyloid beta Protein Fibrillation
  • 2010
  • In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 1:4, s. 279-287
  • Journal article (peer-reviewed)abstract
    • The fibrillation kinetics of the amyloid beta peptide is analyzed in presence of cationic polystyrene nanoparticles of different size. The results highlight the importance of the ratio between the peptide and particle concentration. Depending on the specific ratio, the kinetic effects vary from acceleration of the fibrillation process by reducing the lag phase at low particle surface area in solution to inhibition of the fibrillation process at high particle surface area. The kinetic behavior can be explained if we assume a balance between two different pathways: first fibrillation of free monomer in solution and second nucleation and fibrillation promoted at the particle surface. The overall rate of fibrillation will depend on the interplay between these two pathways, and the predominance of one mechanism over the other will be determined by the relative equilibrium and rate constants.
  •  
47.
  • Cabaleiro-Lago, Celia, et al. (author)
  • Dual effect of amino modified polystyrene nanoparticles on amyloid β protein fibrillation
  • 2010
  • In: ACS Chemical Neuroscience. - 1948-7193 .- 1948-7193. ; 1:4, s. 279-87
  • Journal article (peer-reviewed)abstract
    • The fibrillation kinetics of the amyloid β peptide is analyzed in presence of cationic polystyrene nanoparticles of different size. The results highlight the importance of the ratio between the peptide and particle concentration. Depending on the specific ratio, the kinetic effects vary from acceleration of the fibrillation process by reducing the lag phase at low particle surface area in solution to inhibition of the fibrillation process at high particle surface area. The kinetic behavior can be explained if we assume a balance between two different pathways: first fibrillation of free monomer in solution and second nucleation and fibrillation promoted at the particle surface. The overall rate of fibrillation will depend on the interplay between these two pathways, and the predominance of one mechanism over the other will be determined by the relative equilibrium and rate constants.
  •  
48.
  • Cabaleiro-Lago, Celia, et al. (author)
  • Inhibition of Amyloid beta Protein Fibrillation by Polymeric Nanoparticles
  • 2008
  • In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 130:46, s. 15437-15443
  • Journal article (peer-reviewed)abstract
    • Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer's disease-associated amyloid beta protein (A beta). We found that these nanoparticles affect mainly the nucleation step of A beta fibrillation. The elongation step is largely unaffected by the particles, and once the M is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of A beta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that A beta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric A beta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of A beta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.
  •  
49.
  • Cabaleiro-Lago, C., et al. (author)
  • Inhibition of IAPP and IAPP((20-29)) Fibrillation by Polymeric Nanoparticles
  • 2010
  • In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 26:5, s. 3453-3461
  • Journal article (peer-reviewed)abstract
    • The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP((20-29))) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation its a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied front 50:50 to 100:0, The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP((20-29)) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed its it delayed onset of ThT fluorescence increase, plus it reduction of the amount of fibrils formed its indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP((20-29))) the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.
  •  
50.
  • Cabaleiro-Lago, Celia, et al. (author)
  • The Effect of Nanoparticles on Amyloid Aggregation Depends on the Protein Stability and Intrinsic Aggregation Rate
  • 2012
  • In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 28:3, s. 1852-1857
  • Journal article (peer-reviewed)abstract
    • Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst 2010 7 (2), 521-532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolyrneric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For find the opposite-a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.
  •  
Skapa referenser, mejla, bekava och länka
  • Result 1-50 of 280
Type of publication
journal article (256)
conference paper (8)
research review (8)
doctoral thesis (3)
book chapter (3)
other publication (2)
show more...
show less...
Type of content
peer-reviewed (271)
other academic/artistic (8)
pop. science, debate, etc. (1)
Author/Editor
Linse, Sara (275)
Knowles, Tuomas P.J. (67)
Sparr, Emma (34)
Dobson, Christopher ... (33)
Cedervall, Tommy (32)
Vendruscolo, Michele (32)
show more...
Thulin, Eva (25)
Frohm, Birgitta (22)
Michaels, Thomas C T (19)
Arosio, Paolo (18)
Cabaleiro-Lago, Celi ... (18)
Cohen, Samuel I A (16)
Hellstrand, Erik (15)
Lundqvist, Martin (15)
Bauer, Mikael (15)
Chia, Sean (13)
Bernfur, Katja (12)
Habchi, Johnny (12)
Lynch, Iseult (11)
Dawson, Kenneth A. (11)
Lindman, Stina (11)
Berggård, Tord (10)
Cukalevski, Risto (10)
Nilsson, Hanna (10)
Buell, Alexander K. (10)
Andre, Ingemar (9)
Dear, Alexander J. (9)
Thacker, Dev (9)
Szczepankiewicz, Olg ... (9)
Emanuelsson, Cecilia (8)
Akke, Mikael (8)
Makasewicz, Katarzyn ... (8)
Olsson, Ulf (8)
Griffin, Robert G. (8)
Dahlbäck, Björn (7)
Malmendal, Anders (7)
Xue, Wei-Feng (7)
Walsh, Dominic M. (7)
Deppert, Knut (6)
Rissler, Jenny (6)
Svanborg, Catharina (6)
Bohgard, Mats (6)
Hansson, Lars-Anders (6)
Pagels, Joakim (6)
Mossberg, Anki (6)
Messing, Maria (6)
Silvers, Robert (6)
O'Connell, David J (6)
Sanagavarapu, Kalyan ... (6)
Gaspar, Ricardo (6)
show less...
University
Lund University (272)
Kristianstad University College (8)
Royal Institute of Technology (5)
Uppsala University (5)
Stockholm University (5)
Karolinska Institutet (5)
show more...
University of Gothenburg (4)
Swedish University of Agricultural Sciences (4)
Malmö University (2)
Linköping University (1)
show less...
Language
English (279)
Swedish (1)
Research subject (UKÄ/SCB)
Natural sciences (218)
Medical and Health Sciences (72)
Engineering and Technology (17)

Year

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.

 
pil uppåt Close

Copy and save the link in order to return to this view