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      Cryo-EM structure of a neuronal functional amyloid implicated in memory persistence in Drosophila

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          Abstract

          How long-lived memories withstand molecular turnover is a fundamental question. Aggregates of a prion-like RNA-binding protein, cytoplasmic polyadenylation element–binding (CPEB) protein, is a putative substrate of long-lasting memories. We isolated aggregated Drosophila CPEB, Orb2, from adult heads and determined its activity and atomic structure, at 2.6-angstrom resolution, using cryo–electron microscopy. Orb2 formed ~75-nanometer-long threefold-symmetric amyloid filaments. Filament formation transformed Orb2 from a translation repressor to an activator and “seed” for further translationally active aggregation. The 31–amino acid protofilament core adopted a cross-β unit with a single hydrophilic hairpin stabilized through interdigitated glutamine packing. Unlike the hydrophobic core of pathogenic amyloids, the hydrophilic core of Orb2 filaments suggests how some neuronal amyloids could be a stable yet regulatable substrate of memory.

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          Most cited references23

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          Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.

          The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.
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            Is Open Access

            Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers

            Background Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils. Results We immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils. Conclusion Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.
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              Helical reconstruction in RELION

              We describe a new implementation for the reconstruction of helical assemblies in the empirical Bayesian framework of RELION. Our approach calculates optimal linear filters for the 3D reconstruction by embedding helical symmetry operators in Fourier-space, and deals with deviations from perfect helical symmetry through Gaussian-shaped priors on the orientations of individual segments. By incorporating our approach into the standard pipeline for single-particle analysis in RELION, our implementation aims to be easily accessible for non-experienced users. Although our implementation does not solve the problem that grossly incorrect structures can be obtained when the wrong helical symmetry is imposed, we show for four different test cases that it is capable of reconstructing structures to near-atomic resolution.
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                Author and article information

                Journal
                Science
                Science
                American Association for the Advancement of Science (AAAS)
                0036-8075
                1095-9203
                March 12 2020
                March 13 2020
                March 12 2020
                March 13 2020
                : 367
                : 6483
                : 1230-1234
                Affiliations
                [1 ]Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
                [2 ]Washington University Center for Cellular Imaging, Washington University School of Medicine, St. Louis, MO 63110, USA.
                [3 ]Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA.
                [4 ]MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
                [5 ]Departments of Neuroscience and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
                [6 ]Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA.
                Article
                10.1126/science.aba3526
                7182444
                32165583
                ead1caa3-8040-42a8-b4ee-7d9fef7dc680
                © 2020

                http://www.sciencemag.org/about/science-licenses-journal-article-reuse

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