The mutational landscape of a prion-like domain

Abstract

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

Nucleolin rescues TDP-43 toxicity in yeast and human cell models

“…2A, TDP-43 fusion protein localized in discrete foci in all TDP-43-expressing yeast strains soon after induction (6 h). Our observation is consistent with previous ndings reporting that yeast cells expressing TDP-43 (by one or more alleles) may contain a variable number of inclusion foci, which are also different in size and widely dispersed within the cells [58,59].…”

“…The above data demonstrated that expression of WT or ALS-associated mutant human TDP-43 markedly affects cell viability and growth in yeast strains carrying two or more copies of the transgene, while the expression of TDP-43 (either WT or mutated) from a single genomic copy was not su cient to cause cell death. This nding supports the contention that TDP-43 cytotoxicity in yeast is dose-dependent [30,55,59] and is suggestive of a saturating toxic effect of two copies of the TDP-43 transgene, either WT or mutated, that correlates with the accumulation of detergent-insoluble aggregates of the protein.…”

“…We also analysed the effects of two ALS-linked TDP-43 missense mutations (i.e., Q331K and M337V), that were previously reported known to increase TDP-43 toxicity in yeast cells [19,78]. However, according to a more recent study [59], we found that both disease-associated mutants behaved like the WT protein, as no cytotoxicity was observed when mutant proteins were expressed by a single genomic copy (Fig. S3).…”

The genetic landscape for amyloid beta fibril nucleation accurately discriminates familial Alzheimer’s disease mutations

“…These 'solubility scores' do not predict the effects of mutations on Aß nucleation (figure supplement 1C). Previously we identified a principal component of aa properties (PC1, related to changes in hydrophobicity) that predicts the aggregation and toxicity of the amyotrophic lateral sclerosis (ALS) protein TDP-43 (Bolognesi et al 2019). PC1 is also not a good predictor of Aß nucleation ( Figure 1D) but it does predict the previously reported changes in Aß solubility ( Figure 1E), suggesting that Aß is aggregating by a similar process to TDP-43 in this alternative selection assay (Gray et al 2019) but by a different mechanism in the nucleation selection.…”

An in vivo platform to select and evolve aggregation-resistant proteins

“…aggregation-prone or insoluble proteins). Secondly, using the TPBLA as a screen for deep mutational scanning 52 would allow areas of sequence frustration (i.e. destabilisation due to functional constraints or aggregation propensity) to be mapped and their underlying mechanisms of aggregation to be better understood, enabling their modification using rational engineering approaches.…”