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      The Truncated C-terminal RNA Recognition Motif of TDP-43 Protein Plays a Key Role in Forming Proteinaceous Aggregates*

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          Abstract

          Background: TDP-43 forms aggregates in various neurodegenerative disorders.

          Results: The C-terminal-truncated RRM2 of TDP-43 forms non-amyloid fibrils in vitro and plays a dominant role in forming inclusions in vivo.

          Conclusion: The proteolytic cleavage of TDP-43 that removes the N-terminal dimerization domain may produce unassembled truncated RRM2 fragments for aggregation.

          Significance: This result provides a new direction for the prevention and treatment of TDP-43-associated diseases.

          Abstract

          TDP-43 is the major pathological protein identified in the cellular inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenic forms of TDP-43 are processed C-terminal fragments containing a truncated RNA-recognition motif (RRM2) and a glycine-rich region. Although extensive studies have focused on this protein, it remains unclear how the dimeric full-length TDP-43 is folded and assembled and how the processed C-terminal fragments are misfolded and aggregated. Here, using size-exclusion chromatography, pulldown assays, and small angle x-ray scattering, we show that the C-terminal-deleted TDP-43 without the glycine-rich tail is sufficient to form a head-to-head homodimer primarily via its N-terminal domain. The truncated RRM2, as well as two β-strands within the RRM2, form fibrils in vitro with a similar amyloid-negative staining property to those of TDP-43 pathogenic fibrils in diseases. In addition to the glycine-rich region, the truncated RRM2, but not the intact RRM2, plays a key role in forming cytoplasmic inclusions in neuronal cells. Our data thus suggest that the process that disrupts the dimeric structure, such as the proteolytic cleavage of TDP-43 within the RRM2 that removes the N-terminal dimerization domain, may produce unassembled truncated RRM2 fragments with abnormally exposed β-strands, which can oligomerize into high-order inclusions.

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          Most cited references33

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          Determination of domain structure of proteins from X-ray solution scattering.

          An ab initio method for building structural models of proteins from x-ray solution scattering data is presented. Simulated annealing is employed to find a chain-compatible spatial distribution of dummy residues which fits the experimental scattering pattern up to a resolution of 0.5 nm. The efficiency of the method is illustrated by the ab initio reconstruction of models of several proteins, with known and unknown crystal structure, from experimental scattering data. The new method substantially improves the resolution and reliability of models derived from scattering data and makes solution scattering a useful technique in large-scale structural characterization of proteins.
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            TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity.

            Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS and FTLD-U patients [corrected] . The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations.
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              RNA recognition motifs: boring? Not quite.

              The RNA recognition motif (RRM) is one of the most abundant protein domains in eukaryotes. While the structure of this domain is well characterized by the packing of two alpha-helices on a four-stranded beta-sheet, the mode of protein and RNA recognition by RRMs is not clear owing to the high variability of these interactions. Here we report recent structural data on RRM-RNA and RRM-protein interactions showing the ability of this domain to modulate its binding affinity and specificity using each of its constitutive elements (beta-strands, loops, alpha-helices). The extreme structural versatility of the RRM interactions explains why RRM-containing proteins have so diverse biological functions.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                29 March 2013
                31 January 2013
                31 January 2013
                : 288
                : 13
                : 9049-9057
                Affiliations
                From the []Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan,
                the [§ ]Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsin Chu 30013, Taiwan,
                the []Department of Life Sciences, Institute of Genome Sciences, National Yang-Ming University, Taipei 11221, Taiwan,
                the []Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan,
                the [** ]Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 70457, Taiwan,
                the [‡‡ ]Center of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan 70457, Taiwan, and
                the [§§ ]Graduate Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei 10048, Taiwan
                Author notes
                [2 ] To whom correspondence should be addressed: Inst. of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan. Tel.: 886-2-27884151; Fax: 886-2-27826085; E-mail: hanna@ 123456sinica.edu.tw .
                [1]

                Both authors contributed equally to this work.

                Article
                M112.438564
                10.1074/jbc.M112.438564
                3610977
                23372158
                95bbda49-6283-4cc2-981b-bf14703a0bfb
                © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 20 November 2012
                : 23 January 2013
                Categories
                Protein Structure and Folding

                Biochemistry
                aggregation,neurodegenerative diseases,protein folding,protein self-assembly,protein structure

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