In conclusion, the M337V variant of TDP-43, but not the A90V variant, impacted the expression of known targets of TDP-43 in a manner consistent with the effects of a knockdown of TDP-43 function
In conclusion, the M337V variant of TDP-43, but not the A90V variant, impacted the expression of known targets of TDP-43 in a manner consistent with the effects of a knockdown of TDP-43 function. Open in a separate window FIGURE 6 The M337V, but not the A90V variant, leads to a downregulation of G3BP and HDAC6. and Song, 2015; Mompean et al., 2015; Lim et al., 2016; Mompean et al., 2016). DPC-423 Note that these structures C as well as the available X-ray structures 4IUF, 4Y00, and 4Y0F (Kuo et al., 2014; Chiang et al., 2016) C do not span the entire sequence, and large portions are either missing or poorly resolved, for example S90 in the largely unstructured terminus of 2CQG at the A90V site. (C) Composite TDP-43 schematic of available NMR structures. While iterative homology modeling and loop building techniques were used, a suitable template structure spanning across multiple domains was not found, and this composite structure should be considered as a schematic rather than a confident prediction of folding. Note that in this orientation the S409/S410 phosphorylation site is at the back of the structure near the N-terminus. The vast majority of mutations are missense mutations, with all but three located in the C-terminal glycine-rich domain (Buratti, 2015). Two mutations, P112H and D169G, are located in the RRM1 domain (Kabashi et al., 2008; Buratti, 2015; Moreno et al., 2015). The third, an alanine to valine substitution at residue 90 (A90V), is found between the bipartite NLS (Winton et al., 2008b; Chiang et al., 2012). Winton et al. (2008b) showed that the A90V mutation leads to aberrant cytoplasmic localization and decreased solubility of TDP-43, two pathological hallmarks of TDP-43 proteinopathies, and 4C (Beckman Optima TLX ultracentrifuge with TLA100.3 rotor and Delrin adaptors). The supernatant was collected as the RIPA-soluble fraction. The pellet was washed in RIPA buffer and centrifuged DPC-423 for an additional 30 min at 100,000 and 4C. The supernatant was discarded and the pellet was re-extracted in 100 L urea buffer [7 M urea, 2 M thiourea, 30 DPC-423 mM Tris pH 8.5, 4% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS, Sigma)]. The samples were sonicated 2x 15 s and centrifuged at room temperature for 30 min at 100,000 0.05. Experiments were replicated a minimum of three times. Results Analysis of TDP-43 Protein and Determining Whether It Can Predict the Impact of Mutations on Protein Structure Nuclear magnetic resonance and X-ray structural analysis of individual domains of WT and mutant TDP-43 continue to reveal insights into the relationships between disease-related mutations and the biophysical stability of the protein. We reasoned that assembling a three-dimensional model of TDP-43 may help us predict the structural impact of the A90V variant and support our efforts in elucidating its effects. Thus, we attempted to collate all structural information obtained from NMR and X-ray crystallography available in the public domain and obtain a complete 3D model of TDP-43 (Figure ?Figure11). However, structures including all domains which might enable a comprehensive examination of the effects of Fgfr1 specific mutations on overall structure and stability are challenging and not yet realized. The human TDP-43 sequence (Figure ?Figure1A1A) and NMR structures (Figure ?Figure1B1B) in Figure ?Figure11 highlight the NLS, NES, RRM1, RRM2, glycine-rich domain, as well as the A90V and M337V mutation sites and the S409/S410 phosphorylation site (He et al., 2004; Suzuki et al., 2005; Kuo et al., 2014; Lim and Song, 2015; Mompean et al., 2015, 2016; Chiang et al., 2016; Lim et al., 2016). The piecewise solutions of NMR and X-ray structures provide valuable domain-level information, but key portions of the protein remain either unresolved or missing. Iterative homology modeling and loop building techniques can be used to infer composite structures from their individual domains. However, in the case of TDP-43 a suitable template structure spanning across multiple domains does not exist, prohibiting the ability to confidently assess the overall structure (only an approximate schematic of the overall structure is shown, Figure ?Figure1C1C) and DPC-423 thus the impact of the A90V and M337V amino acid substitutions. Without assumptions of the impact of the A90V variant based on a complete model, we then proceeded to investigate the consequences of.