Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity

Mohammad Omar Faruk,Yoshinobu Ichimura,Shun Kageyama,Satoko Komatsu-Hirota,Afnan H El-Gowily,Yu-Shin Sou,Masato Koike,Nobuo N Noda,Masaaki Komatsu

doi:10.1016/j.jbc.2021.101405

Mohammad Omar Faruk, Yoshinobu Ichimura + Show 7 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101405

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Abstract

Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid–liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget’s disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein–protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.

Highlights

SQSTM1, the human gene encoding p62/SQSTM1, is localized on chromosome 5 and comprises eight exons up to 16 kb in length
P62 can be degraded by endosomal microautophagy [12], but like ubiquitinated cargos, it is primarily degraded during selective autophagy through interaction with FIP200/RB1-inducible coiled-coil protein 1, an upstream factor for autophagosome formation, and the subsequent mutually exclusive interaction with LC3, an autophagosome-localizing protein [13,14,15]. p62 is known as a multifunctional signaling hub, as it participates in its aforementioned adaptor role in selective autophagy, and in the activation of mechanistic target of rapamycin complex 1 during nutrient sensing and NF-κB activation during inflammation, apoptosis, and activation of the Keap1-Nrf2 pathway for antioxidant response [16]
T350A mutation may indirectly weaken the interaction with Kelch-like ECHassociated protein 1 (KEAP1) by the following mechanism: the side chain of Thr350 forms an intramolecular hydrogen bond with the main chain of Glu352 (Fig. 1D); T350A mutation impairs this hydrogen bond, which may cause a conformational change in p62 KEAP1-interacting region (KIR) and lead to reduced affinity for KEAP1

Summary

Introduction

SQSTM1, the human gene encoding p62/SQSTM1 (hereafter referred to as p62), is localized on chromosome 5 and comprises eight exons up to 16 kb in length. But especially K63, are able to promote clustering, whereas free monoubiquitin or unanchored ubiquitin chains, the K48 linkage, inhibit p62 clustering [18] These p62 structures arise from existing p62 filaments that are cross-linked by polyubiquitinated substrates [25, 26]. Inside these clustered droplets, p62 appears to retain little mobility, whereas ubiquitin, LC3, and KEAP1 may be able to diffuse more within the cluster and the surrounding cytosol [17, 18, 21]. We aimed to identify the p62 mutation (related diseases)

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