P53 Transactivation Domain Mediates Binding and Phase Separation with Poly-PR/GR

Sinem Usluer,Tobias Madl,Emil Spreitzer,Benjamin Bourgeois

doi:10.3390/ijms222111431

Abstract

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the presence of poly-PR/GR dipeptide repeats, which are encoded by the chromosome 9 open reading frame 72 (C9orf72) gene. Recently, it was shown that poly-PR/GR alters chromatin accessibility, which results in the stabilization and enhancement of transcriptional activity of the tumor suppressor p53 in several neurodegenerative disease models. A reduction in p53 protein levels protects against poly-PR and partially against poly-GR neurotoxicity in cells. Moreover, in model organisms, a reduction of p53 protein levels protects against neurotoxicity of poly-PR. Here, we aimed to study the detailed molecular mechanisms of how p53 contributes to poly-PR/GR-mediated neurodegeneration. Using a combination of biophysical techniques such as nuclear magnetic resonance (NMR) spectroscopy, fluorescence polarization, turbidity assays, and differential interference contrast (DIC) microscopy, we found that p53 physically interacts with poly-PR/GR and triggers liquid–liquid phase separation of p53. We identified the p53 transactivation domain 2 (TAD2) as the main binding site for PR25/GR25 and showed that binding of poly-PR/GR to p53 is mediated by a network of electrostatic and/or hydrophobic interactions. Our findings might help to understand the mechanistic role of p53 in poly-PR/GR-associated neurodegeneration.

Highlights

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with many common neuropathological, genetic, and clinical features [1,2]
Among the p53 transactivation domain (TAD), we showed that only the transactivation domain 2 (TAD2) subdomain is sufficient to mediate the interaction with dipeptide repeat proteins (DPRs), but the TAD1 contributes to the increase in the overall binding affinity
As previous studies have shown a link between DPRs, p53 activation, and stabilization [40], we wanted to assess whether the model DPRs poly(PR)25 and poly(GR)25 directly interact with p53 in vitro using the purified proteins and nuclear magnetic resonance (NMR) spectroscopy

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with many common neuropathological, genetic, and clinical features [1,2]. The most frequent genetic cause of ALS and FTD is a GGGGCC hexanucleotide repeat expansion in the first non-coding region of the chromosome 9 open reading frame 72 gene (C9orf72) [3,4] This mutation results in loss of function of the C9orf by inhibition of transcription [3,5,6]; generation of RNA foci [7], which sequester critical RNA-binding proteins [8,9] and/or interfere with essential cellular processes [10]; and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs) [11,12,13,14]. Toxicity of poly-PR/GR has been shown to be linked to multiple cellular mechanisms, such as mitochondrial stress [24], ER stress [25,26], impaired nucleocytoplasmic transport [20,27,28], oxidative stress [24], impaired splicing [29,30,31], impaired translation [32], interference with the formation of membrane-less organelles (MLOs) [33,34,35,36], and combinations thereof

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