TAR DNA-binding protein 43 (TDP-43) liquid–liquid phase separation is mediated by just a few aromatic residues

Abstract

Eukaryotic cells contain distinct organelles, but not all of these compartments are enclosed by membranes. Some intrinsically disordered proteins mediate membraneless organelle formation through liquid-liquid phase separation (LLPS). LLPS facilitates many biological functions such as regulating RNA stability and ribonucleoprotein assembly, and disruption of LLPS pathways has been implicated in several diseases. Proteins exhibiting LLPS typically have low sequence complexity and specific repeat motifs. These motifs promote multivalent connections with other molecules and the formation of higher-order oligomers, and their removal usually prevents LLPS. The intrinsically disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43), a protein involved in motor neuron disease and dementia lacks a dominant LLPS motif, however, and how this domain forms condensates is unclear. Using extensive mutagenesis of TDP-43, we demonstrate here that three tryptophan residues and, to a lesser extent, four other aromatic residues are most important for TDP-43 to undergo LLPS. Our results also suggested that only a few residues may be required for TDP-43 LLPS because the α-helical segment (spanning ∼20 residues) in the middle part of the C-terminal domain tends to self-assemble, reducing the number of motifs required for forming a multivalent connection. Our results indicating that a self-associating α-helical element with a few key residues regulates condensate formation highlight a different type of LLPS involving intrinsically disordered regions. The C-terminal domain of TDP-43 contains ∼50 disease-related mutations, with no clear physicochemical link between them. We propose that they may disrupt LLPS indirectly by interfering with the key residues identified here.

Shortened TDP43 isoforms upregulated by neuronal hyperactivity drive TDP43 pathology in ALS

“…within the TDP43 C-terminus promotes liquid-phase separation and aggregation (90)(91)(92)(93)(94). Even so, our observations and those of others (64,65) show that sTDP43 is relatively insoluble and prone to aggregation, despite lacking the LCD.…”

“…In keeping with previous studies 65 , overexpressed sTDP43 accumulates in the cytoplasm where it often forms large, insoluble inclusions. The low-complexity domain (LCD) within the TDP43 C-terminus is essential for liquid-phase separation [87][88][89][90] , and has been heavily implicated in TDP43 aggregation. Even so, our observations and those of others 64,65 show that sTDP43 is insoluble and prone to aggregation, despite lacking the LCD.…”

Phase transition and amyloid formation by a viral protein as an additional molecular mechanism of virus-induced cell toxicity

“…In addition, PNT3 contains a stretch of three contiguous tyrosines (aa 210-212 of P), and two non-contiguous tyrosines at positions 237 and 249 ( Figure 1A). The well-established effect of tyrosines [31] and, more generally, of π-orbital containing residues [32,33] in promoting phase separation provides an additional conceptual piece to rationalize the behavior of PNT3. Liquid-hydrogel transitions are thought to be preceded by a step of liquid-liquid phase separation (LLPS) [26,34] that can be tackled by a wide range of physico-chemical solution studies.…”

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4

“…Furthermore, alike the wildtype TDP-43 2C protein [71], concentration-dependent inhibition of the aggregation of TDP-43 2C -A315T was also observed in the presence of AIM4 and AIM1 ( Figure 6). like TDP-43 [5,95]. From our in silico studies, in the 6N3C-A315T mutant TDP-43 structure (aa: 288-319), AIM4 was predicted to interact with the major amino acids, glycine and phenylalanine, which are proposedly involved in its pathogenic LLPS [95].…”

“…like TDP-43 [5,95]. From our in silico studies, in the 6N3C-A315T mutant TDP-43 structure (aa: 288-319), AIM4 was predicted to interact with the major amino acids, glycine and phenylalanine, which are proposedly involved in its pathogenic LLPS [95]. Therefore, the effect of AIM4 on the in vitro LLPS of mutant TDP-43 2C -A315T, that also encompasses the aa: 288-319, was checked.…”