TAR去氧核糖核酸結合酶-43 (TAR DNA-binding protein 43)在正常細胞是一個多功能的蛋白質，扮演轉錄因子以及調控信使核糖核酸剪接 (mRNA splicing) 的角色。TDP-43結構包含兩個核糖核酸結合區 (RNA binding motif; RRM)，RRM1以及RRM2，和一個富含甘氨酸的尾端區域 (C-termainal glycin rich region)。在病理細胞內，TDP-43形成包含體 (inclusion)，導致某些神經退化性疾病。在包含體內的TDP-43被證實其前端 (N-terminus) 已被去除，而形成一個大小約為25 kDa的片段，此片段包含部分的RRM2區域和glycin rich區域組成。TDP-43如何由正常的功能性蛋白，轉變成纖維狀的異常包含體，其致病機制目前尚未得知。為了了解TDP-43的結構區排列方式，我們使用小角度X光散射法取得了低解析度的 TDP-43s (包含RRM1 及RRM2 功能區)結構。再加上GST pull-down 檢驗法的得到的實驗數據，研究結果顯示 TDP-43 是以二聚體形式存在，以頭對頭的方式使兩個RRM1功能區做結合，另兩個RRM2功能區朝外延展。此外我們發現RRM2結構區裡的□3和□5有能力形成纖維。這些纖維並不能被thioflavin T澱粉樣蛋白纖維染劑 (amyloid-binding dye)，以及抗澱粉樣蛋白纖維抗體(anti-amyloid antibody) 所辨識，此結果和病理上的TDP-43纖維是一致的。因此，綜合上述結果我們推測在移除RRM1功能區和RRM2beta1後，不正常摺疊的RRM2 使beta3和beta5暴露出來，因而造成TDP-43蛋白質不正常堆疊和病理上纖維的形成。

REFERENCES
Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., et al. (2006). TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., et al. (2006). TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochemical and biophysical research communications 351, 602-611.
Benson, M.D., and Kincaid, J.C. (2007). The molecular biology and clinical features of amyloid neuropathy. Muscle Nerve 36, 411-423.
Blake, C., and Serpell, L. (1996). Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta-sheet helix. Structure (London, England : 1993) 4, 989-998.
Buratti, E., Brindisi, A., Giombi, M., Tisminetzky, S., Ayala, Y.M., and Baralle, F.E. (2005). TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail: an important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing. The Journal of biological chemistry 280, 37572-37584.
Buratti, E., Dörk, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F.E. (2001). Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping. The EMBO journal 20, 1774-1784.
Chen, A.K.-H., Lin, R.Y.-Y., Hsieh, E.Z.-J., Tu, P.-H., Chen, R.P.-Y., Liao, T.-Y., Chen, W., Wang, C.-H., and Huang, J.J.-T. (2010). Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis. Journal of the American Chemical Society 132, 1186-1187.
Chen, Y.R., and Glabe, C.G. (2006). Distinct early folding and aggregation properties of Alzheimer amyloid-beta peptides Abeta40 and Abeta42: stable trimer or tetramer formation by Abeta42. J Biol Chem 281, 24414-24422.
Chiti, F., and Dobson, C.M. (2006). Protein Misfolding, Functional Amyloid, and Human Disease. Annual Review of Biochemistry 75, 333-366.
Chiti, F., and Dobson, C.M. (2009). Amyloid formation by globular proteins under native conditions. Nature chemical biology 5, 15-22.
Cléry, A., Blatter, M., and Allain, F.H.-T. (2008). RNA recognition motifs: boring? Not quite. Current opinion in structural biology 18, 290-298.
Crichlow, G.V., Zhou, H., Hsiao, H.-h., Frederick, K.B., Debrosse, M., Yang, Y., Folta-Stogniew, E.J., Chung, H.-J., Fan, C., De La Cruz, E.M., et al. (2008). Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition. The EMBO journal 27, 277-289.
Dormann, D., Capell, A., Carlson, A.M., Shankaran, S.S., Rodde, R., Neumann, M., Kremmer, E., Matsuwaki, T., Yamanouchi, K., Nishihara, M., et al. (2009). Proteolytic processing of TAR DNA binding protein-43 by caspases produces C-terminal fragments with disease defining properties independent of progranulin. Journal of neurochemistry 110, 1082-1094.
Fernandez-Escamilla, A.-M., Rousseau, F., Schymkowitz, J., and Serrano, L. (2004). Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. Nature biotechnology 22, 1302-1306.
Fiesel, F.C., Voigt, A., Weber, S.S., Van Den Haute, C., Waldenmaier, A., Görner, K., Walter, M., Anderson, M.L., Kern, J.V., Rasse, T.M., et al. (2010). Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6. The EMBO journal 29, 209-221.
Gendron, T.F., Josephs, K.A., and Petrucelli, L. (2010). Review: transactive response DNA-binding protein 43 (TDP-43): mechanisms of neurodegeneration. Neuropathology and applied neurobiology 36, 97-112.
Handa, N., Nureki, O., Kurimoto, K., Kim, I., Sakamoto, H., Shimura, Y., Muto, Y., and Yokoyama, S. (1999). Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature 398, 579-585.
Igaz, L.M., Kwong, L.K., Chen-Plotkin, A., Winton, M.J., Unger, T.L., Xu, Y., Neumann, M., Trojanowski, J.Q., and Lee, V.M.-Y. (2009). Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies. The Journal of biological chemistry 284, 8516-8524.
Igaz, L.M., Kwong, L.K., Xu, Y., Truax, A.C., Uryu, K., Neumann, M., Clark, C.M., Elman, L.B., Miller, B.L., Grossman, M., et al. (2008). Enrichment of C-terminal fragments in TAR DNA-binding protein-43 cytoplasmic inclusions in brain but not in spinal cord of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. The American journal of pathology 173, 182-194.
Jones, D.T. (1999). Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292, 195-202.
Kabashi, E., Valdmanis, P.N., Dion, P., Spiegelman, D., McConkey, B.J., Vande Velde, C., Bouchard, J.-P., Lacomblez, L., Pochigaeva, K., Salachas, F., et al. (2008). TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nature genetics 40, 572-574.
Kim, S.H., Shanware, N., Bowler, M.J., and Tibbetts, R.S. (2010). ALS-associated proteins TDP-43 and FUS/TLS function in a common biochemical complex to coregulate HDAC6 mRNA. The Journal of biological chemistry 285, 34097-34105.
Konarev, P.V., Volkov, V.V., Sokolova, A.V., Koch, M.H.J., and Svergun, D.I. (2003). PRIMUS : a Windows PC-based system for small-angle scattering data analysis. Journal of Applied Crystallography 36, 1277-1282.
Krecic, A.M., and Swanson, M.S. (1999). hnRNP complexes: composition, structure, and function. Current opinion in cell biology 11, 363-371.
Kuo, P.-H., Doudeva, L.G., Wang, Y.-T., Shen, C.-K.J., and Yuan, H.S. (2009). Structural insights into TDP-43 in nucleic-acid binding and domain interactions. Nucleic acids research 37, 1799-1808.
Lagier-Tourenne, C., Polymenidou, M., and Cleveland, D.W. (2010). TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Human molecular genetics 2009, 1-19.
Letunic, I., Doerks, T., and Bork, P. (2009). SMART 6: recent updates and new developments. Nucleic Acids Res 37, D229-232.
Maris, C., Dominguez, C., and Allain, F.H.-T. (2005). The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. The FEBS journal 272, 2118-2131.
Maurer-Stroh, S., Debulpaep, M., Kuemmerer, N., de La Paz, M.L., Martins, I.C., Reumers, J., Morris, K.L., Copland, A., Serpell, L., Serrano, L., et al. (2010). Exploring the sequence determinants of amyloid structure using position-specific scoring matrices. Nature methods 7, 237-242.
Naiki, H., Higuchi, K., Hosokawa, M., and Takeda, T. (1989). Fluorometric determination of amyloid fibrils using the fluorescent dye, thioflavine T. Analytical Biochemistry 177, 244-249.
Neumann, M. (2009). Molecular Neuropathology of TDP-43 Proteinopathies. International journal of molecular sciences 10, 232-246.
Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi, M.C., Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C.M., et al. (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science (New York, NY) 314, 130-133.
Nishimoto, Y., Ito, D., Yagi, T., Nihei, Y., Tsunoda, Y., and Suzuki, N. (2010). Characterization of alternative isoforms and inclusion body of the TAR DNA-binding protein-43. The Journal of biological chemistry 285, 608-619.
Ou, S.H., Wu, F., Harrich, D., García-Martínez, L.F., and Gaynor, R.B. (1995). Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. Journal of virology 69, 3584-3596.
Pérez-Cañadillas, J.M. (2006). Grabbing the message: structural basis of mRNA 3'UTR recognition by Hrp1. The EMBO journal 25, 3167-3178.
Petoukhov, M.V., and Svergun, D.I. (2005). Global rigid body modeling of macromolecular complexes against small-angle scattering data. Biophysical journal 89, 1237-1250.
Putnam, C.D., Hammel, M., Hura, G.L., and Tainer, J.A. (2007). X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Quarterly reviews of biophysics 40, 191-285.
Quintas, A., Vaz, D.C., Cardoso, I., Saraiva, M.J., and Brito, R.M. (2001). Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants. J Biol Chem 276, 27207-27213.
Ratnaswamy, G., Koepf, E., Bekele, H., Yin, H., and Kelly, J.W. (1999). The amyloidogenicity of gelsolin is controlled by proteolysis and pH. Chem Biol 6, 293-304.
Rochet, J.C., and Lansbury, P.T. (2000). Amyloid fibrillogenesis: themes and variations. Current opinion in structural biology 10, 60-68.
Serpell, L., and Fraser, P. (1999). X-ray fiber diffraction of amyloid fibrils. Methods in enzymology 309, 526-536.
Sigrist, C.J., Cerutti, L., de Castro, E., Langendijk-Genevaux, P.S., Bulliard, V., Bairoch, A., and Hulo, N. (2010). PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res 38, D161-166.
Stromer, T., and Serpell, L.C. (2005). Structure and morphology of the Alzheimer's amyloid fibril. Microscopy research and technique 67, 210-217.
Svergun, D. (1999). Restoring Low Resolution Structure of Biological Macromolecules from Solution Scattering Using Simulated Annealing. Biophysical Journal 76, 2879-2886.
Svergun, D., Barberato, C., and Koch, M.H.J. (1995). CRYSOL – a Program to Evaluate X-ray Solution Scattering of Biological Macromolecules from Atomic Coordinates. Journal of Applied Crystallography 28, 768-773.
Svergun, D.I. (1992). Determination of the regularization parameter in indirect-transform methods using perceptual criteria. Journal of Applied Crystallography 25, 495-503.
Teplow, D.B. (1998). Structural and kinetic features of amyloid beta-protein fibrillogenesis. Amyloid 5, 121-142.
Thorpe, J.R., Tang, H., Atherton, J., and Cairns, N.J. (2008). Fine structural analysis of the neuronal inclusions of frontotemporal lobar degeneration with TDP-43 proteinopathy. Journal of neural transmission (Vienna, Austria : 1996) 115, 1661-1671.
Volkov, V.V., and Svergun, D.I. (2003). Uniqueness of ab initio shape determination in small-angle scattering. Journal of Applied Crystallography 36, 860-864.
Wang, H.-Y., Wang, I.-F., Bose, J., and Shen, C.-K.J. (2004). Structural diversity and functional implications of the eukaryotic TDP gene family. Genomics 83, 130-139.
Wang, X., and Hall, T.M.T. (2001). Structural basis for recognition of AU-rich element RNA by the HuD protein. Nature Structural & Molecular Biology 8, 141-145.
Wils, H., Kleinberger, G., Janssens, J., Pereson, S., Joris, G., Cuijt, I., Smits, V., Ceuterick-de Groote, C., Van Broeckhoven, C., and Kumar-Singh, S. (2010). TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. Proceedings of the National Academy of Sciences of the United States of America 107, 3858-3863.
Xu, R.M., Jokhan, L., Cheng, X., Mayeda, A., and Krainer, A.R. (1997). Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs. Structure (London, England : 1993) 5, 559-570.
Zhang, Y.-J., Xu, Y.-F., Cook, C., Gendron, T.F., Roettges, P., Link, C.D., Lin, W.-L., Tong, J., Castanedes-Casey, M., Ash, P., et al. (2009). Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proceedings of the National Academy of Sciences of the United States of America 106, 7607-7612.
Zhang, Y.-J., Xu, Y.-f., Dickey, C.A., Buratti, E., Baralle, F., Bailey, R., Pickering-Brown, S., Dickson, D., and Petrucelli, L. (2007). Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43. The Journal of neuroscience : the official journal of the Society for Neuroscience 27, 10530-10534.