Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function

Li, Wen; Reeb, Ashley N.; Lin, Binyan; Subramanian, Praveen; Fey, Erin E.; Knoverek, Catherine R.; French, Rachel L.; Bigio, Eileen H.; Ayala, Yuna M.

doi:10.1074/jbc.m116.753913

Cited by 46 publications

(70 citation statements)

References 55 publications

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“…Indeed, the Disheveled 2 DIX domain polymerization is inhibited post‐translationally by the addition of ubiquitin to multiple lysine residues (Madrzak et al , ), and both the axin 1 and Disheveled 2 DIX domains can be post‐translationally phosphorylated (Trinidad et al , ; Yi et al , ; Mertins et al , ). Furthermore, phosphorylation of other domains of TDP‐43 has been shown to tune function (Li et al , ). Therefore, future efforts examining the biophysical and cellular effects of TDP‐43 NTD phosphorylation will be useful to determine the pathways regulating TDP‐43 self‐assembly and function.…”

Section: Discussionmentioning

confidence: 99%

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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: Discussionmentioning

confidence: 99%

“…Splicing assays were carried out in HeLa cells transiently transfected with Lipofectamine 2000 and Oligofectamine in the case of siRNA (Life Technologies) according to manufacturer protocols. Splicing assays were performed using the CFTR reporter minigene as previously described (Li et al , ).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2018

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“…WT and mutant siRNA-resistant constructs were generated by site-directed mutagenesis in Nterminally FLAG-tagged pCMV2 64 . HeLa cells were grown in growth media-Dulbecco's modified Eagle's medium, 4,500 mg/l glucose, l-glutamine, and sodium bicarbonate and supplemented with filtered fetal bovine serum at 10%.…”

Section: Splicing Assaysmentioning

confidence: 99%

“…Cells were transiently transfected with Lipofectamine 2000 and Oligofectamine in the case of siRNA (Life Technologies) according to manufacturer protocols. RNAimediated downregulation of TDP-43 was carried out as previously described 64 and siCONTROL Nontargetting siRNA#1 (Dharmacon) was used as control. TDP-43 downregulation was confirmed by immunoblotting.…”

Section: Splicing Assaysmentioning

confidence: 99%

TDP-43 α-helical structure tunes liquid-liquid phase separation and function

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et al. 2019

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Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. Present in several MLOs, TDP-43 undergoes LLPS and is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some disease variants of TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes in a dimeric TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a mini-gene assay. Therefore, TDP-43 helical region serves as a short but uniquely tunable module that shows promise as for controlling assembly and function in cellular and synthetic biology applications of LLPS.

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“…A few studies have reported the purification of the N-terminal fragment of TDP-43 (33)(34)(35), on the 2 RRM domains (15)(16)(17), and on the CTD (21). Some investigators succeeded in purifying the full-length protein but to a yield of only a few micrograms that allowed a limited number of low-resolution studies and with a purification procedure that could not be reproduced (33,(36)(37)(38). The main problems reported when purifying the full-length protein were its aggregation and degradation (17,19,33,35,36,(38)(39)(40).…”

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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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Heat Shock-induced Phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK Kinase Regulates TDP-43 Function

Cited by 46 publications

References 55 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

TDP-43 α-helical structure tunes liquid-liquid phase separation and function

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

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