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Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity.
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2021-11-12 , DOI: 10.1016/j.jbc.2021.101405 Mohammad Omar Faruk 1 , Yoshinobu Ichimura 2 , Shun Kageyama 2 , Satoko Komatsu-Hirota 2 , Afnan H El-Gowily 3 , Yu-Shin Sou 4 , Masato Koike 4 , Nobuo N Noda 5 , Masaaki Komatsu 2
Journal of Biological Chemistry ( IF 5.5 ) Pub Date : 2021-11-12 , DOI: 10.1016/j.jbc.2021.101405 Mohammad Omar Faruk 1 , Yoshinobu Ichimura 2 , Shun Kageyama 2 , Satoko Komatsu-Hirota 2 , Afnan H El-Gowily 3 , Yu-Shin Sou 4 , Masato Koike 4 , Nobuo N Noda 5 , Masaaki Komatsu 2
Affiliation
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.
中文翻译:
肌萎缩侧索硬化相关 p62/SQSTM1 突变体的相分离蛋白液滴显示出内部流动性降低。
几种肌萎缩侧索硬化 (ALS) 相关蛋白,如 FUS、TDP-43 和 hnRNPA1 表现出液-液相分离,并且它们的疾病相关突变与它们的液滴形式转变为聚集体相关。SQSTM1/p62 中的错义突变已在整个基因中发现,与 ALS、额颞叶变性 (FTD) 和佩吉特骨病有关。SQSTM1/p62 蛋白通过与泛素化蛋白相互作用形成液滴,这些液滴分别通过 p62 的 LC3 相互作用区 (LIR) 和 KEAP1 相互作用区 (KIR) 作为自噬体形成和抗氧化应激反应的平台。然而,目前尚不清楚 LIR 和 KIR 中与 ALS/FTD 相关的 p62 突变是否会破坏液滴形成,从而导致自噬缺陷、应激反应或两者兼而有之。为了评估 LIR 和 KIR 中与 ALS/FTD 相关的 p62 突变对主要氧化应激系统 Keap1-Nrf2 途径以及自噬转换的影响,我们开发了系统以高灵敏度监测其中的每一个。这些方法如细胞内蛋白质-蛋白质相互作用测定、多西环素诱导基因表达系统和用重组腺病毒将基因表达到原代培养细胞中表明,一些突变体(但不是全部)导致 NRF2 活化减少和自噬货物周转延迟。相反,虽然所有 p62 突变体都表现出足够的形成液滴的能力,所有这些液滴也表现出降低的内部流动性。这些结果表明,与其他 ALS 相关突变蛋白一样,p62 错义突变通过 p62 液滴流动性的质变导致 ALS/FTD 的主要缺陷。
更新日期:2021-11-11
中文翻译:
肌萎缩侧索硬化相关 p62/SQSTM1 突变体的相分离蛋白液滴显示出内部流动性降低。
几种肌萎缩侧索硬化 (ALS) 相关蛋白,如 FUS、TDP-43 和 hnRNPA1 表现出液-液相分离,并且它们的疾病相关突变与它们的液滴形式转变为聚集体相关。SQSTM1/p62 中的错义突变已在整个基因中发现,与 ALS、额颞叶变性 (FTD) 和佩吉特骨病有关。SQSTM1/p62 蛋白通过与泛素化蛋白相互作用形成液滴,这些液滴分别通过 p62 的 LC3 相互作用区 (LIR) 和 KEAP1 相互作用区 (KIR) 作为自噬体形成和抗氧化应激反应的平台。然而,目前尚不清楚 LIR 和 KIR 中与 ALS/FTD 相关的 p62 突变是否会破坏液滴形成,从而导致自噬缺陷、应激反应或两者兼而有之。为了评估 LIR 和 KIR 中与 ALS/FTD 相关的 p62 突变对主要氧化应激系统 Keap1-Nrf2 途径以及自噬转换的影响,我们开发了系统以高灵敏度监测其中的每一个。这些方法如细胞内蛋白质-蛋白质相互作用测定、多西环素诱导基因表达系统和用重组腺病毒将基因表达到原代培养细胞中表明,一些突变体(但不是全部)导致 NRF2 活化减少和自噬货物周转延迟。相反,虽然所有 p62 突变体都表现出足够的形成液滴的能力,所有这些液滴也表现出降低的内部流动性。这些结果表明,与其他 ALS 相关突变蛋白一样,p62 错义突变通过 p62 液滴流动性的质变导致 ALS/FTD 的主要缺陷。