Templát-vezérelt amiloid fibrillogenézis biofizikai vizsgálata

The Aβ peptides form self-assembled fibrillar structures in Alzheimer's disease. The Aβ25-35 peptide used in our experiments is the biologically active, toxic fragment of the full-length Aβ peptide. We constructed a highly ordered, stable network from Aβ25-35 peptides showing trigonal orientation on mica. The same peptide can assemble itself into amyloid fibrils in solution as well. We mapped the morphological and nanomechanical features of the oriented network used later as an experimental model system. We were the first to compare the properties (morphology, nanomechanics and secondary structure) of epitaxially-grown and solution-grown amyloid fibrils by means of AFM, force spectroscopy and FTIR spectroscopy. Both types of fibrils displayed underlying fibrillar morphology. Nevertheless, in contrast to the homogeneity structure of epitaxially-grown fibrils, solution-grown fibrils showed higher structural hierarchy, polymorphism and topographical periodicity. Epitaxially grown fibrils formed linear, sheet-like structures. The fibrils differed in their growth kinetics: while fibril assembly in solution occurred on a time scale of hours to days, on mica surface fibrils appeared within a few minutes. The mica surface might act as a catalyzer accelerating the process of fibrillogenesis. Because of the accelerated fibrillogenesis, the effect of physical and chemical parameters on amyloid fibrillogenesis can be easily explored. Both types of fibrils displayed identical nanomechanical responses in form of force plateaus; the elementary force plateau was ∼30 pN. Both types of fibrils displayed an underlying β-sheet structure. The FTIR spectra also showed that fibrils assembled in solution have a more compact structure. We demonstrated that the epitaxially grown oriented network be considered as a good amyloid-fibril model. The stable, ordered network of Aβ25-35 fibrils on mica is a simple, reproducible system on the nanoscale. Studying on this model the effect of different factors interfering with fibrillogenesis, the changes of structural, nanomechanical properties of the Ab25-35 fibril network can be easily monitored by AFM. The effect of the BSB peptide LPFFD was investigated in an amyloid model system of oriented Aβ25-35 fibrils on mica. From force spectroscopy measurements we concluded that the LPFFD peptide may weaken the interaction between protofilaments. Our results indicate that the LPFFD peptide does not have a generalized beta-sheet-breaking effect in the amyloid assembly system utilized in the present experiment. By using mutant Aβ25-35 peptides containing amino acid residues with specific chemical reactivity the labeling of the oriented network with various biomolecules or conducting metals can be accomplished. For future applications the fine-tuning of the network's properties (e.g., average fibril length, number of branching points) was needed. We succeded in fine-tuning the properties of the mutant network by varying the cation-concentration. We demonstrated that the sulfhydryl group of Cys27 within the oriented fibril network is indeed exposed to the solution and is chemically accessible and reactive. We therefore demonstrated that the properties of the mutant Aβ25-35 network may provide important tools for future nanotechnological applications

Citoszkeletális fehérjék szerkezete, dinamikája, mechanikája és kölcsönhatásai: egyedi molekuláktól szupramolekuláris rendszerekig = Structure, dynamics, mechanics and interactions of cytoskeletal proteins: from single molecules to supramolecular systems

Pályázatunkban a citoszkeletális fehérjék szerkezetét, dinamikáját, mechanikáját és kölcsönhatásait vizsgáltuk elsősorban a harántcsíkolt izom különböző molekuláris rendszerein. Szintetikus miozin vastag filamentumok szerkezetét és nanomechanikáját atomerőmikroszkóppal (AFM) mértük. A titin Z-lemez horgonyzó komplex mechanikai stabilitását erőspektroszkópiával vizsgáltuk. Natív vázizom titin rugalmasságának és erővezérelt szerkezetváltozásainak vizsgálatára erővisszacsatolt lézercsipeszt fejlesztettünk. A titin PEVK domén konformációs dinamikáját FRET spektroszkópiával vizsgáltuk. A dezmin intermedier filamentumok és protofibrillumok szerkezetét és nanomechanikáját ugyancsak AFM-mel mértük meg. A szívizom típusú miozin-kötő C-fehérje molekuláris mechanikáját Monte-Carlo módszerrel szimuláltuk. Az aktomiozin motilitás pontosabb térbeli változásainak mérésére fluoreszcencia interferencia kontraszt (FLIC) mikroszkópia fejlesztését kezdtük meg. Az izommechanika organizmusban való mérésére speciális, AFM-alapú módszert dolgoztunk ki C. elegans rendszeren. A pályázat közvetlen támogatásával nyolc eredeti közlemény és egy könyvfejezet került publikálásra. | In our project we investigated the structure, dynamics, mechanics and interactions of cytoskeletal proteins mainly on muscle-derived molecular systems. The structure and nanomechanics of myosin thick filaments were explored by using atomic force microscopy (AFM). The mechanical stability of the Z-disc titin-anchoring complex was measured with single-molecule force spectroscopy. In order to measure the elasticity and force-driven structural changes in native titin molecules with high resolution, we developed a fast force-clamp optical tweezers apparatus. The structural dynamics of titin PEVK domain fragments was measured by using FRET spectroscopy. The structure and nanomechanical behavior of desmin intermediate filaments and protofibrils were also measured with AFM. For the simulation of the force versus extension of cardiac myosin-binding protein-C we used Monte-Carlo methods. To reveal greater spatial detail in the in vitro actomyosin motility we began developing a fluorescence interference contrast (FLIC) microscope system. In order to investigate the actomyosin mechanics within an organism, we developed a novel, AFM-based detection method based on C. elegans. With the direct support of the grant eight original papers and one book chapter were published

Epitaxial assembly dynamics of mutant amyloid beta25-35_N27C fibrils explored with time-resolved scanning force microscopy.

Amyloid beta25-35 (Abeta25-35) is a toxic fragment of Alzheimer's beta peptide. We have previously shown that Abeta25-35 fibrils form a trigonally oriented network on mica by epitaxial growth mechanisms. Chemical reactivity can be furnished to the fibril by introducing a cysteine residue (Abeta25-35_N27C) while maintaining oriented assembly properties. Previously we have shown that fibril binding to mica is strongly influenced by KCl concentration. In the present work we explored the kinetics of epitaxial assembly of the mutant fibrils at different peptide and KCl concentrations by using in situ time-resolved AFM. We measured the length of Abeta25-35_N27C fibrils as a function of time. Increasing free peptide concentration enhanced fibril growth rate, and the critical peptide concentration of fibril assembly was 3.92muM. Increasing KCl concentration decreased the number of fibrils bound to the mica surface, and above 20mM KCl fibril formation was completely abolished even at high peptide concentrations. By modulating peptide and KCl concentrations in the optimal ranges established here the complexity of the Abeta25-35_N27C network can be finely tuned

Structural and nanomechanical comparison of epitaxially and solution-grown amyloid β25-35 fibrils

Aβ25-35, the fibril-forming, biologically active toxic fragment of the full-length amyloid β-peptide also forms fibrils on mica by an epitaxial assembly mechanism. Here we investigated, by using atomic force microscopy, nanomechanical manipulation and FTIR spectroscopy, whether the epitaxially-grown fibrils display structural and mechanical features similar to the ones evolving under equilibrium conditions in bulk solution. Unlike epitaxially-grown fibrils, solution-grown fibrils displayed a heterogeneous morphology and an apparently helical structure. While fibril assembly in solution occurred on a time scale of hours, on mica surface fibrils appeared within a few minutes. Both types of fibrils showed a similar plateau-like nanomechanical response characterized by the appearance of force staircases. The IR spectra of both fibril types contained an intense peak between 1620 and 1640 cm-1 indicating that β- sheets dominate their structure. A shift in the amide I band towards greater wavenumbers in epitaxially assembled fibrils suggests that their structure is less compact than that of solution grown fibrils. Thus, equilibrium conditions are required for a full structural compaction. Epitaxial Aβ25-35 fibril assembly, while significantly accelerated, may trap the fibrils in less compact configurations. Considering that under in vivo conditions the assembly of amyloid fibrils is influenced by the presence of extracellular matrix components, the ultimate fibril structure is likely to be influenced by the features of underlying matrix elements

Effects of Estrogen on Beta-Amyloid-Induced Cholinergic Cell Death in the Nucleus Basalis Magnocellularis

Alzheimer disease is characterized by accumulation of beta-amyloid (Abeta) and cognitive dysfunctions linked to early loss of cholinergic neurons. As estrogen-based hormone replacement therapy has beneficial effects on cognition of demented patients, and it may prevent memory impairments, we investigated the effect of estrogen-pretreatment on Abeta-induced cholinergic neurodegeneration in the nucleus basalis magnocellularis (NBM). We tested which Abeta species induces the more pronounced cholinotoxic effect in vivo. We injected different Abeta assemblies in the NBM of mice, and measured cholinergic cell and cortical fiber loss. Spherical Abeta oligomers had the most toxic effect. Pretreatment of ovariectomized mice with estrogen before Abeta injection decreased cholinergic neuron loss and partly prevented fiber degeneration. By using proteomics, we searched for proteins involved in estrogen-mediated protection and in Abeta toxicity 24 h following injection. The change in expression of, e.g., DJ-1, NADH ubiquinone oxidoreductase, ATP synthase, phosphatidylethanolamine-binding protein 1, protein phosphatase 2A and dimethylarginine dimethylaminohydrolase 1 support our hypothesis that Abeta induces mitochondrial dysfunction, decreases MAPK signaling, and increases NOS activation in NBM. On the other hand, altered expression of, e.g., MAP kinase kinase 1 and 2, protein phosphatase 1 and 2A by Abeta might increase MAPK suppression and NOS signaling in the cortical target area. Estrogen pretreatment reversed most of the changes in the proteome in both areas. Our experiments suggest that regulation of the MAPK pathway, mitochondrial pH and NO production may all contribute to Abeta toxicity, and their regulation can be prevented partly by estrogen pretreatment