The mutational landscape of a prion-like domain

Benedetta Bolognesi,Ben Lehner,Mireia Seuma,Andre J Faure,Gian Gaetano Tartaglia,Jörn M Schmiedel

doi:10.1038/s41467-019-12101-z

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.

Highlights

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases
We introduced the library into Saccharomyces cerevisiae cells, induced expression and used deep sequencing before and after induction to quantify the relative effects of each variant on growth in three biological replicates (Fig. 1a)
We find that mutations that decrease TAR DNA-binding protein 43 (TDP-43) hydrophobicity and increase TDP-43 toxicity increase the number of the small foci at the nuclear periphery and reduce the number of large distal foci (Fig. 3b, c, f, Fig. 3 Mutations leading to formation of solid-like aggregates rescue toxicity. a Representative fluorescence microscopy images of yeast cells expressing indicated YFP-tagged TDP-43 variants (W334K TDP-43 = toxic, A328V TDP-43 = non-toxic)

Summary

Introduction

Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in most cases of amyotrophic lateral sclerosis (ALS). 1234567890():,; The conversion of specific proteins into insoluble aggregates is a hallmark of many neurodegenerative disorders, including Alzheimer’s, Parkinson’s, Huntington’s, and Amyotrophic Lateral Sclerosis (ALS) with dominantly inherited mutations in aggregate-forming proteins causing rare familial forms of these diseases[1,2,3,4,5,6]. Aggregation depends critically on intrinsically disordered regions with a low sequence complexity resembling that of infectious yeast prions These prion-like domains (PRDs) are enriched in proteins that can form liquid-like cellular condensates[20,21,22] through liquid-demixing. Despite extensive investigation, the molecular form of the protein that causes cellular toxicity is still unknown[7,47]

Methods

Results

Conclusion