Abstract
Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.
Highlights
Transactive response DNA-binding Protein of 43 kDa (TDP-43) is an RNA-binding protein that forms aberrant aggregates associated with disease contexts, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [1], or the recently reported “LATE” dementia, which had been misdiagnosed as Alzheimer disease [2]
Because induced aggregation at higher pH values is reported to be driven by the central region containing a helical segment [9,10], we sought to confirm whether this general mechanism is operative under our conditions, using liquid-state NMR (LSNMR)
Considering that the TDP-43 prion-like domain (PrLD) is of low complexity, aggregation prone, and disordered, the LSNMR 13CO, 13Cα, 13Cβ, 15N, 1HN, 1Hα and 1Hβ correlations were obtained using a nonconventional strategy that affords robust and complete assignments with a minimal set of experiments
Summary
Transactive response DNA-binding Protein of 43 kDa (TDP-43) is an RNA-binding protein that forms aberrant aggregates associated with disease contexts, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) [1], or the recently reported “LATE” dementia, which had been misdiagnosed as Alzheimer disease [2]. TDP-43 aggregates into functional amyloids [3] and participates in the assembly of various biomolecular condensates [4,5], which highlight the need for structural studies to understand the interplay between these aggregate forms. TDP-43 contains a well-folded N-terminal [6] and 2 RNA-Recognition Motifs (RRM) domains [7] and a low-complexity, prion-like domain (PrLD) at its carboxyl terminus, which seems pivotal for the formation of the diverse amyloid and droplet forms. The TDP-43 PrLD is large (residues 267 to 414), comprising almost half the length of TDP-43, and is intrinsically disordered, with the exception of a short hydrophobic segment. TDP-43 fibrils at the condensate/solvent interface feature Phe residues (PathensTDP project) to E.B.; and Grant LCF/BQ/ PR19/11700003 from La Caixa Foundation (ID 100010434) to M.M. M.M. is a Ramon y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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