The N-terminal domain of ALS-linked TDP-43 assembles without misfolding

Tsoi, Phoebe S.; Choi, Kyoung‐Jae; Leonard, Paul G.; Sizovs, Antons; Moosa, Mahdi Muhammad; MacKenzie, Kevin R.; Ferreon, Josephine C.; Ferreon, Allan Chris M.

doi:10.1101/134072

Cited by 15 publications

(21 citation statements)

References 23 publications

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“…Several groups have reported that TDP‐43 NTD mediates assembly into dimers or other higher‐order structures (Shiina et al , ; Chang et al , ; Wang et al , ), though the details of the assembly have only recently begun to be visualized with atomistic resolution (Afroz et al , ; Jiang et al , ; Mompean et al , ; Tsoi et al , ). Recombinant TDP‐43 NTD (in our case: residues 1–80, with only three additional N‐terminal residues, GHM, resulting from the cloning strategy and cleavage with TEV protease) is highly soluble when expressed and purified from bacteria.…”

Section: Resultsmentioning

confidence: 99%

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

et al. 2018

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TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.

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Section: Resultsmentioning

confidence: 99%

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

et al. 2018

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“…We chose to use a construct that encompasses both the N-terminus and the RRM domains taking the start from our previous work on the RRM domains [23]. We decided to include also the N-terminus for various reasons: while the prion-like potential of the C-terminus has been associated to the pathological aggregation of TDP-43 [42], growing evidence indicates the importance of the N-terminally driven oligomerization for physiological self-assembly into stress granules [43], [44]. The N-terminal domain is also involved in the native functional dimerization of the protein, which is considered to increase the risk of aberrant self-interactions and aggregation of the protein [45], [46].…”

Section: Discussionmentioning

confidence: 99%

RNA as a key factor in driving or preventing self-assembly of the TAR DNA-binding protein 43

Zacco

Graña-Montes

Martin

et al. 2019

Journal of Molecular Biology

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Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are incurable motor neuron diseases associated with muscle weakness, paralysis and respiratory failure. Accumulation of TAR DNA-binding protein 43 (TDP-43) as toxic cytoplasmic inclusions is one of the hallmarks of these pathologies. TDP-43 is an RNA-binding protein responsible for regulating RNA transcription, splicing, transport and translation. Aggregated TDP-43 does not retain its physiological function. Here, we exploit the ability of TDP-43 to bind specific RNA sequences to validate our hypothesis that the native partners of a protein can be used to interfere with its ability to self-assemble into aggregates. We propose that binding of TDP-43 to specific RNA can compete with protein aggregation. This study provides a solid proof of concept to the hypothesis that natural interactions can be exploited to increase protein solubility and could be adopted as a more general rational therapeutic strategy.

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“…In the various structural studies so far reported, the isolated NTD domain was found to adopt either a dimeric (34,39,55,56) or oligomeric (18,19,57,58) state. Moreover, it was suggested that the native (nonmisfolded) full-length protein might adopt a complex oligomeric state because of the ability of the CTD to increase the oligomerization state of the protein (18,19).…”

Section: Refolded Pelb-tdp-43-his Is a Dimermentioning

confidence: 99%

Isolation and characterization of soluble human full‐length TDP‐43 associated with neurodegeneration

et al. 2019

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The involvement of transactivation response (TAR) DNA‐binding protein 43 (TDP‐43) in neurodegenerative diseases was revealed in 2006, when it was first reported to be the main component of the intracellular inclusions in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. After 12 yr it is not yet possible to purify to a reasonable yield and in a reproducible manner a stable full‐length protein, which has limited so far the characterization of its structure, function, molecular interactors, and pathobiology. Using a novel protocol we have achieved the purification of the full‐length TDP‐43, with both a short pectate lyase B tag and a glutathione S‐transferase tag, which consisted in its expression in bacteria, solubilization from inclusion bodies, purification under denaturing conditions, refolding, and a final size exclusion chromatography (SEC) step. Differential scanning fluorimetry was used to find the best buffers and combination of additives to increase both its solubility and its stability. The protein is pure, as determined with electrophoresis, Western blotting, and mass spectrometry; properly refolded, as revealed by circular dichroism and fluorescence spectroscopies; functional, because it binds to DNA and protein partners; and stable to degradation and aggregation in a physiologic solution. Analyses with dynamic light scattering and SEC revealed that the protein is a dimer.—Vivoli Vega, M., Nigro, A., Luti, S., Capitini, C., Fani, G., Gonnelli, L., Boscaro, F., Chiti, F. Isolation and characterization of soluble human full‐length TDP‐43 associated with neurodegeneration. FASEB J. 33, 10780–10793 (2019). http://www.fasebj.org

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The N-terminal domain of ALS-linked TDP-43 assembles without misfolding

Cited by 15 publications

References 23 publications

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing

RNA as a key factor in driving or preventing self-assembly of the TAR DNA-binding protein 43

Isolation and characterization of soluble human full‐length TDP‐43 associated with neurodegeneration

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