Phe-Gly motifs drive fibrillization of TDP-43’s prion-like domain condensates

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.

Sequence Determinants of TDP-43 Ribonucleoprotein Condensate Formation and Axonal Transport in Neurons

“…Likewise, substituting threonine for tyrosine confers a polar side chain but eliminates the aromatic ring. Since GFG and FG motifs participate in TDP-43 LLPS and are critical for the formation of TDP-43 fibrils ( Pantoja-Uceda et al, 2021 ), we similarly replaced phenylalanine in GFG or FG/FS motifs with tyrosine or leucine (F276Y/L, F283Y/L, F289Y/L, F313L/Y, F313-316Y/L, F367Y/L, F397Y/L, and F401Y/L) ( Supplementary Figure S1 ). We hypothesized substitutions that abolish the aromatic ring would show more severe effects on TDP-43 RNP condensate formation and transport compared to mutations that preserve the aromatic structure.…”

“…We were interested to define the role of these phenylalanine residues in TDP-43 RNP granule transport. Prior studies have demonstrated that mutating or deleting GFG and FG LLPS motifs does not affect phase separation of purified TDP-43 but instead impacts TDP-43 fibrillization ( Li et al, 2018b ; Pantoja-Uceda et al, 2021 ). In addition, F to Y substitutions do not alter TDP-43 dynamics as measured by half bleach fluorescence recovery ( Schmidt et al, 2019 ).…”

“…In contrast, TDP-43 phase separation depends on a short stretch of conserved amino acids in LCD (320–340) that forms an α-helical domain and serves as a backbone to drive LLPS ( Conicella et al, 2016 ). The α-helical domain is flanked by two intrinsically disordered regions (IDR1: 274–316 and IDR2: 341–414) which contain LLPS (GFG/FG) motifs, tryptophan residues and a single tyrosine residue that also influence phase separation in vitro ( Conicella et al, 2016 ; Li et al, 2018b ; Schmidt et al, 2019 ; Pantoja-Uceda et al, 2021 ). In addition, IDR1 contains a low complexity, aromatic-rich, kinked segment (LARKS) at 312–317, which has been shown to mediate reversible adhesion between the LCDs of RNA binding proteins ( Guenther et al, 2018 ; Hughes et al, 2018 ).…”

“…Although recent work suggests assembly into biomolecular condensates may modulate the RNA-regulatory functions of TDP-43 and other constituents of RNP condensates, conflicting data exist, and many questions about the physiologic function of condensates in neurons and their pathophysiology in disease remain ( Schmidt et al, 2019 ; Conicella et al, 2020 ; Altman et al, 2021 ; Gao et al, 2021 ; Hallegger et al, 2021 ). Inspired by elegant work that uncovered molecular determinants of TDP-43 LLPS and fibrillization in vitro ( Conicella et al, 2016 ; Li et al, 2018b ; Schmidt et al, 2019 ; Pantoja-Uceda et al, 2021 ), in the present study we sought to determine whether 1) different ALS-linked TDP-43 mutations or 2) disruption of specific aromatic and charged residues in the LCD have distinct effects on the formation of TDP-43 positive RNP condensates and their functional impact on TDP-43 RNP transport in neurons.…”

Parkin beyond Parkinson’s Disease—A Functional Meaning of Parkin Downregulation in TDP-43 Proteinopathies

“…At the structural level, TDP-43 possesses a highly structured N-terminus domain (NTD) [ 66 , 67 ] that controls protein dimerization/oligomerization [ 66 , 68 ]. This NTD is followed by two RNA Recognition Motifs (RRMs) that are responsible for sequence-specific binding to RNA [ 69 , 70 ] and then by an unstructured C-terminus region that plays a fundamental role in phase separation and aggregation [ 71 , 72 , 73 , 74 ].…”