Amyloid fibrils are self-associating filamentous structures, the deposition of which is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease and various other disorders. Here we used single molecule manipulation methods to explore the mechanics and structural dynamics of amyloid fibrils. In mechanically manipulated amyloid fibrils, formed from either amyloid ␤ (A␤) peptides 1-40 or 25-35, ␤-sheets behave as elastic structures that can be "unzipped" from the fibril with constant forces. The unzipping forces were different for A␤1-40 and A␤25-35. Unzipping was fully reversible across a wide range of stretch rates provided that coupling, via the ␤-sheet, between bound and dissociated states was maintained. The rapid, cooperative zipping together of ␤-sheets could be an important mechanism behind the self-assembly of amyloid fibrils. The repetitive force patterns contribute to a mechanical fingerprint that could be utilized in the characterization of different amyloid fibrils.Amyloid fibrils are self-associating filamentous structures formed from the 39 -43-residue-long amyloid ␤-peptide (A␤) 1 or its subfragments (1). The deposition of amyloid oligomers (2) and fibrils is considered to be one of the most important factors in the pathogenesis of Alzheimer's disease (3) and other disorders (4). The structure of A␤-fibrils has for long been enigmatic because of insoluble aggregate formation that precludes the use of standard structural methods such as x-ray crystallography and solution NMR. Recent data from site-directed spin labeling (5), and particularly from solid-state NMR experiments (6, 7), have formed the basis of a high resolution model of the A␤1-40 fibril: ␤-hairpins lying perpendicular to the fibril axis are associated into ␤-sheets that line up to form protofilaments, which are then assembled parallel into fibrils. Protofilaments are thus thought to represent an ϳ2-3-nm-diameter structural unit within the amyloid fibril (1). During amyloidogenesis the formation of fibrils is preceded by the appearance of globular aggregates that are thought to fuse, by not fully understood mechanisms, into fibrillar structures (8). Recently, curved, beaded, ϳ200-nm-long and ϳ6 -8-nm-wide fibrillar precursors were described to appear in the amyloidogenetic pathway, which were called protofibrils (9 -12). The protofibrils are thought to go through a structural transition on their way to forming the amyloid fibril. The exact nature of structural dynamics within the amyloid fibril related to amyloidogenesis, however, remains to be resolved.Single molecule manipulation experiments have in the recent past provided unique and unprecedented insights not only into the structure and elasticity but also into mechanically driven transitions of molecular systems (13)(14)(15)(16)(17)(18)(19)(20)(21). In the present work we mechanically manipulated amyloid fibrils formed from either A␤1-40 or A␤25-35 peptides. We showed that filamentous entities most likely corresponding to ␤-sheets can be "unzipp...

Parts of the PEVK (Pro-Glu-Val-Lys) domain of the skeletal muscle isoform of the giant intrasarcomeric protein titin have been shown to bind F-actin. However, the mechanisms and physiological function of this are poorly understood. To test for actin binding along PEVK, we expressed contiguous N-terminal (PEVKI), middle (PEVKII), and C-terminal (PEVKIII) PEVK segments of the human soleus muscle isoform. We found a differential actin binding along PEVK in solid-state binding, cross-linking and in vitro motility assays. The order of apparent affinity is PEVKII>PEVKI>PEVKIII. To explore which sequence motifs convey the actin-binding property, we cloned and expressed PEVK fragments with different motif structure: PPAK, polyE-rich and pure polyE fragments. The polyE-containing fragments had a stronger apparent actin binding, suggesting that a local preponderance of polyE motifs conveys an enhanced local actin-binding property to PEVK. The actin binding of PEVK may serve as a viscous bumper mechanism that limits the velocity of unloaded muscle shortening towards short sarcomere lengths. Variations in the motif structure of PEVK might be a method of regulating the magnitude of the viscous drag.

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Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R α-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the γ-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37°C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R α-chain and the GPI-anchored PIBF receptor.

Amyloid fibrils are important components of tissue deposits in neurodegenerative and protein misfolding diseases. Because modified amyloid peptide subunits can be generated by synthetic methods and the nanometer-scale fibrils are stable under diverse conditions, amyloid fibrils have been suggested for use in nanotechnology applications. However, well-controlled and oriented growth of amyloid fibrils has not yet been accomplished. Here we show that amyloid β 25–35 (Aβ 25–35), a toxic fragment of Alzheimer’s beta peptide, forms trigonally oriented fibrils on mica. Oriented binding depends on an apparently cooperative interaction of a positively charged moiety on the Aβ 25–35 peptide with the K+-binding pocket of the mica lattice. Time-lapse in situ AFM revealed that the formation of oriented fibrils is the result of epitaxial polymerization rather than binding of already assembled fibrils from solution. By varying the K+ concentration the growth rate and the mesh size of the oriented amyloid fibril network may be tuned. The K+-controlled oriented assembly of Aβ 25–35 fibrils could be utilized in nanotechnology applications such as formation of oriented tracks for molecular devices and generation of nanoelectronic circuits.