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

Abstract

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

New lessons on TDP-43 from the killifishN. furzeri

“…We used in vitro studies to compare the individual behaviour of different regions of the proteins from human and N. furzeri using recombinant fragments spanning the C-termini and the tandem RRM domains. We observed a strong tendency to aggregate of the C-termini in agreement with what has been observed for the human protein (Vega et al, 2019;Capitini et al, 2020). This region is so aggregation prone that the recombinant human C-terminus could be obtained in a soluble form only by cleaving off the glycine-rich region in agreement with previous studies (Capitini et al, 2020).…”

RNA Is a Double-Edged Sword in ALS Pathogenesis

“…Until recently, the lack of a high-yield protocol to produce pure, full-length TDP-43 has plagued the field. However, a major breakthrough by the Chiti group has made this possible ( Vivoli Vega et al, 2019 ), opening the door for future investigations into the full structure and function of TDP-43 in its monomeric and homodimerized forms. Additionally, shortened isoforms of TDP-43 (sTDP-43) have been discovered in neurons that exhibit hyperactivity, further increasing the need for structural studies of full length and sTDP-43 ( Weskamp et al, 2020 ).…”

Purification and Structural Characterization of Aggregation-Prone Human TDP-43 Involved in Neurodegenerative Diseases

“…Although the structures of individual domains of TDP-43 have been elucidated using several techniques that have revealed some aspects of the molecular functions of this protein (Afroz et al, 2017;Chiang et al, 2016;Guenther et al, 2018;Kuo et al, 2014;Mompeá n et al, 2016), the structure of full-length TDP-43 has been refractory to characterization due to the difficulty of purifying soluble and stable protein in sufficient amounts for analysis (Johnson et al, 2009;Kitamura et al, 2018;Li et al, 2017). A recent report overcame this challenge using denaturing conditions, but this could change the native TDP-43 structure (Vivoli Vega et al, 2019). In this work, we describe full-length wild-type TDP-43 and TDP-43 WtoA , both successfully purified by non-denaturing methods.…”