Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Methods in Molecular Biophysics: Structure, Dynamics, Function for Biology and Medicine - 2nd Edition

International audienceCurrent techniques for studying biological macromolecules and their interactions are based on the application of physical methods, ranging from classical thermodynamics to more recently developed techniques for the detection and manipulation of single molecules. Reflecting the advances made in biophysics research over the past decade, and now including a new section on medical imaging, this new edition describes the physical methods used in modern biology. All key techniques are covered, including mass spectrometry, hydrodynamics, microscopy and imaging, diffraction and spectroscopy, electron microscopy, molecular dynamics simulations and nuclear magnetic resonance. Each method is explained in detail using examples of real-world applications. Short asides are provided throughout to ensure that explanations are accessible to life scientists, physicists and those with medical backgrounds. The book remains an unparalleled and comprehensive resource for graduate students of biophysics and medical physics in science and medical schools, as well as for research scientists looking for an introduction to techniques from across this interdisciplinary field

Overexpression of Wg leads to a dramatic loss of nipple arrays, correlating with the glossy eye phenotype.

<p>Three-dimensional AFM representation of nipple arrays of wild-type flies (A) and the <i>GMR-Gal4; UAS-Wg</i> flies overexpressing Wg in postmitotic eye cells (B). A catastrophic loss of nipples is observed upon Wg overexpression, with few remaining nipples randomly spaced with huge gaps between them. This loss of nipples correlates with the overall glossy appearance of the mutant eyes (B, insert), as opposed to the wild-type eyes (A, insert). The eye size in <i>GMR-Gal4; UAS-Wg</i> flies is also reduced due to photoreceptor loss. A light microscope was used to take images of the whole eyes shown in inserts.</p

Fine structure AFM images of <i>Drosophila</i> ommatidial surface reveal irregularities in the lens material deposition in <i>frizzled</i> mutants.

<p>Corneal surface of the wild-type (A, B) and <i>frizzled</i> mutant (C, D) eyes was analyzed at high resolution with AFM. Field of view is 10×10 µm. Arrows indicate intercalations of the lens material between ommatidial lens borders in the <i>frizzled</i> mutant (C, D). (A, C) represent top views, while (B, D) are their three-dimensional representations.</p

High-resolution analysis of the <i>Drosophila</i> nipple arrays.

<p>Corneal surface of the wild-type (A) and <i>frizzled</i> mutant (D) eyes was analyzed at high resolution with AFM. Field of view is 3×3 µm. Fourier transform spectra of the AFM images are shown as inserts in (A, D). (B, E) are cross-sectional profiles of representative scans of wild-type (B) and <i>frizzled</i> mutant (E) cornea of ca. 8 µm length. Blue lines in (B, E) are smoothing curves of the height recording curves depicted with the red lines. (C, F) are representative cross-sectional 4 µm-long profiles of flat areas of wild-type (C) and <i>frizzled</i> mutant (F) cornea such as those on (A, D).</p

Diffraction patterns of <i>Drosophila</i> cornea confirm lack of order in ommatidial arrangement in <i>frizzled</i> mutants.

<p>Corneal preparations from wild-type (A) and <i>frizzled</i> mutant (B) eyes were irradiated with a laser beam of 630 nm to collect diffraction patterns.</p

Different Oligomeric Properties and Stability of Highly Homologous A1 and Proto-Oncogenic A2 Variants of Mammalian Translation Elongation Factor eEF1

Translation elongation factor 1A (eEF1A) directs aminoacyl-tRNA to the A site of 80S ribosomes. In addition, more than 97% homologous variants of eEF1A, A1 and A2, whose expression in different tissues is mutually exclusive, may fulfill a number of independent moonlighting functions in the cell; for instance, the unusual appearance of A2 in an A1-expressing tissue was recently linked to the induction of carcinogenesis. The structural background explaining the different functional performance of the highly homologous proteins is unclear. Here, the main difference in the structural properties of these proteins was revealed to be the improved ability of A1 to self-associate, as demonstrated by synchrotron small-angle X-ray scattering (SAXS) and analytical ultracentrifugation. Besides, the SAXS measurements at different urea concentrations revealed the low resistance of the A1 protein to urea. Titration of the proteins by hydrophobic dye 8-anilino-1-naphthalenesulfonate showed that the A1 isoform is more hydrophobic than A2. As the different association properties, lipophilicity, and stability of the highly similar eEF1A variants did not influence considerably their translation functions, at least <i>in vitro</i>, we suggest this difference may indicate a structural background for isoform-specific moonlighting roles

Disassembly of YB-1 amyloid-like fibrils at physiological ionic strength.

<p>(A) and (B), YB-1 (56.8 µM) was incubated with 0.15 M or 2 M KCl for 24 h. An aliquot of YB-1 pre-incubated with 2 M KCl was diluted to 0.15 M KCl. Remaining samples were diluted to the same final protein concentration (4.26 µM) with appropriate KCl solutions to keep the salt concentration unchanged. The samples were incubated for 1 h at room temperature and analyzed by (A), ThT fluorescence (means and SD are shown (n = 3), and *** indicates <i>t</i>-test <i>p</i><0.001) and (B), EM imaging. (C), YB-1 (30 µM) was incubated with 0.15 M KCl or 2 M LiCl for 24 h. An aliquot of YB-1 pre-incubated with 2 M LiCl was dialyzed against 0.15 M KCl. Fibril formation was visualized by AFM imaging. Scale bars are 0.4 µm. Ionic strength conditions during the incubation are indicated.</p

Fibril formation by YB-1 and its fragments under physiological conditions.

<p>EM images of YB-1 (A), YB-1_1–219 (B), YB-1_1–129 (C), and YB-1_52–129 (D) (10 µM) incubated in the presence of 0.15 M KCl for 92 h. Scale bars are 0.4 µm.</p