Inhibition of TDP-43 aggregation by nucleic acid binding - PubMed (original) (raw)
Inhibition of TDP-43 aggregation by nucleic acid binding
Yi-Chen Huang et al. PLoS One. 2013.
Abstract
The aggregation of TAR DNA-binding protein (TDP-43) has been shown as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. While evidence has suggested that mutation or truncation in TDP-43 influences its aggregation process, nevertheless, the correlation between the TDP-43 aggregation propensity and its binding substrates has not been fully established in TDP-43 proteinopathy. To address this question, we have established a platform based on the in vitro protein expression system to evaluate the solubility change of TDP-43 in response to factors such as nucleotide binding and temperature. Our results suggest that the solubility of TDP-43 is largely influenced by its cognate single-strand DNA (ssDNA) or RNA (ssRNA) rather than hnRNP, which is known to associate with TDP-43 C-terminus. The direct interaction between the refolded TDP-43, purified from E.coli, and ssDNA were further characterized by Circular Dichroism (CD) as well as turbidity and filter binding assay. In addition, ssDNA or ssRNA failed to prevent the aggregation of the F147L/F149L double mutant or truncated TDP-43 (TDP208-414). Consistently, these two mutants form aggregates, in contrast with the wild-type TDP-43, when expressed in Neuro2a cells. Our results demonstrate an intimate relationship between the solubility of TDP-43 and its DNA or RNA binding affinity, which may shed light on the role of TDP-43 in ALS and FTLD.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. The solubility of human TDP-43 in rabbit reticulocyte cell-free system under different incubation conditions.
(A) A flow chart of solubility determination consisting of protein synthesis (step 1), time-course incubation (step 2), and the quantification of TDP-43 in different fractions (step 3). (B) TDP-43 in supernatant (S) and pellet (P) fractions are visualized by western blotting after SDS-PAGE under different incubation time (1–4 h) and temperature (30°C and 37°C) (step 2). (C) The solubility of TDP-43 was calculated by quantifying the gel bands of S and P in (B) with Image J and the formula, Solubility (%) = S/(S+P)*100.
Figure 2. The effects of ssDNA and ssRNA on TDP-43 solubility.
(A) The supernatant (S) and pellet (P) of TDP-43, incubated at 37°C for 3 h, in the absence (control) or presence of various ssDNA [i.e. (CA)6, (TG)3, (TG)6, (TG)9, and (TG)12] with different concentration (4–10 µM) were analyzed by western blotting. (B) The solubility of TDP-43 in the absence or presence of various ssDNA with different concentration was derived from the intensity of S and P in (A), and the P-values were calculated by Student’s t-test (**, P<0.01 and *, P<0.05) (C) The gel analysis of the TDP-43 supernatant (S) and pellet (P) incubated with 5 or 10 µM of ssRNA, (UG)12, for 3 h at 37°C with anti-Flag antibody. (D) The percentage of S and P was obtained from quantification of the intensity in (C) by using Image J .
Figure 3. The solubility of wild-type, mutant (F147L/F149L), and N-terminally truncated (TDP208–414) TDP-43 in the absence or presence of ssDNA or ssRNA.
(A) The schematics of wild-type and mutant (F147L/F149L) TDP-43. The DNA/RNA binding domains, RRM1 and RRM2, were highlighted in light and dark blue, respectively. The N-terminus FLAG tag (green) was designed for the recognition during western blotting. (B) The western blot analysis of the wild-type and mutant (F147L/F149L) TDP-43 under the incubation with 10 µM (TG)12 or (UG)12 for 3 h at 37°C with anti-Flag antibody. (C) The solubility of TDP-43 was derived from the band of S and P in (B). (D) The solubility of wild-type and mutation TDP-43 in the presence and absence of hnRNP A2.
Figure 4. Characterization of the refolded TDP-43 protein.
(A) The refolded wild-type and double-mutation (F147L/F149L) TDP-43 identified with coomassie blue in the SDS-PAGE, or by western blotting with TDP-43 antibody. (B) The secondary structure of refolding and native TDP-43 characterized by CD spectroscopy. The aggregation of the TDP-43 (C) and its mutant (F147L/F149L) (D) in the absence or presence of oligonucleotides incubated at 25°C with a continuous agitation from 0 to 4 h was monitored by the solution turbidity at 600 nm. The averaged results ± std are presented for protein alone (•), or incubated with (CA)6 (
□
), and (TG)6 (▵).
Figure 5. Confocal immunofluorescent images of Neuro2a cells of TDP-43 variants.
(A) Inclusions were observed only in the mutant (F147L/F149L, indicated by yellow arrow) and truncated (TDP208–414, indicated by red arrow) TDP-43 rather than the wild-type protein in the cell. Secondary anti-mouse antibody conjugated with Alexa 488 reacted with primary anti-FLAG antibodies hybridized with FLAG-tagged TDP-43 variants. DAPI was used for visualizing nuclei. (Scale bar = 10 µm). (B) The expression level for wild type, mutant (F147L/F149L), and truncated (TDP208–414) TDP-43 on the SDS-PAGE with western blotting. GAPDH is served as loading controls for each lane.
Figure 6. (A) The newly synthesized TDP-43 tends to aggregate when exposed to increased temperature (stress) in the rabbit reticulocyte cell free system.
This propensity is highly suppressed when it’s bound to its cognate DNA/RNA. (B) The proposed model of TDP-43 and its interacting partners within the Neuro2a cell. The cognate DNA/RNA may shift the equilibrium of TDP-43 toward its soluble form in the cytoplasm. (C) The mutated/truncated TDP-43 protein is failed to interact with DNA/RNA and result in the aggregation in the nucleus and the cytosol, respectively.
References
- Neumann M, Sampathu DM, Kwong LK, Truax AC, Micsenyi MC, et al. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314: 130–133. - PubMed
- Wang IF, Chang HY, Hou SC, Liou GG, Way TD, et al. (2012) The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies. Nat Commun 3: 766. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
This work was supported by the research grants from Academia Sinica and National Science Council, Taiwan (NSC 98-2113-M-001-015-MY2 and NSC 100-2113-M-001-013-MY2). Dr. K.-F.L. and Dr. R.-Y.H. were supported by the postdoctoral fellowship from Academia Sinica. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous