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

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 id...

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Published in:The Journal of biological chemistry 2021-12, Vol.297 (6), p.101405-101405, Article 101405
Main Authors: Faruk, Mohammad Omar, Ichimura, Yoshinobu, Kageyama, Shun, Komatsu-Hirota, Satoko, El-Gowily, Afnan H., Sou, Yu-shin, Koike, Masato, Noda, Nobuo N., Komatsu, Masaaki
Format: Article
Language:English
Subjects:
amyotrophic lateral sclerosis
Amyotrophic Lateral Sclerosis - genetics
Amyotrophic Lateral Sclerosis - metabolism
Animals
autophagy
HEK293 Cells
Humans
Kelch-Like ECH-Associated Protein 1 - genetics
Kelch-Like ECH-Associated Protein 1 - metabolism
liquid droplet
Mice
Mutation, Missense
NF-E2-Related Factor 2 - genetics
NF-E2-Related Factor 2 - metabolism
NRF2
p62
Sequestosome-1 Protein - genetics
Sequestosome-1 Protein - metabolism
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/j.jbc.2021.101405