![]() B, effects of D336N and D337E mutations on interaction with LC3. A, domain structure of p62 and disease-related mutations in the LIR and KIR. On the other hand, T350A mutation may indirectly weaken the interaction with KEAP1 by the following mechanism: the side chain of Thr350 forms an intramolecular hydrogen bond with the main chain of Glu352 ( Fig. 1 D) T350A mutation impairs this hydrogen bond, which may cause a conformational change in p62 KIR and thus lead to reduced affinity for KEAP1.įigure 1 Models for binding of disease-related p62 mutants with LC3 and KEAP1. P348L, S349T, and G351A mutations weaken the interaction with KEAP1 by causing a steric clash with Tyr525 (P348L and S349T) or Tyr572 (G351A) of KEAP1 ( Fig. 1 D). The side chain of Leu341 is bound to the L-site of LC3, and the binding affinity becomes lower by 元41V mutation due to poor fit ( Fig. 1 C). On the other hand, D337E mutation strengthens the interaction with LC3 by creating an electrostatic interaction between Glu337 and Lys49 ( Fig. 1 B) (Asp337 is located distally from Lys49 and thus cannot form an electrostatic interaction). Loss of a negative charge due to D336N mutation weakens this interaction and thereby reduces the affinity of p62 for LC3. As shown in Figure 1 B, p62 Asp336 forms an electrostatic interaction with LC3 Arg11. On the basis of these structural data, we constructed models of complexes formed between p62 mutants and either LC3 or KEAP1 ( Fig. 1, B– D). KeywordsĪbbreviations: ALS ( amyotrophic lateral sclerosis), BRET ( bioluminescence resonance energy transfer), FTD ( frontotemporal degeneration), GSTM1 ( Glutathione S-transferase Mu 1), KIR ( Kelch-like ECH-associated protein 1–interacting region), LIR ( LC3-interacting region), mTORC1 ( mechanistic target of rapamycin complex 1), NQO1 ( NAD(P)H dehydrogenase, quinone 1), NRF2 ( nuclear factor erythroid 2–related factor 2), PDB ( Paget’s disease of bone), RBP ( RNA-binding protein), TRAF6 ( tumor necrosis factor receptor–associated factor 6), UBA ( ubiquitin-associated), UGDH ( UDP-glucose 6-dehydrogenase) 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. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. 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. 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. 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. 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. 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. 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. Glycobiology and Extracellular Matrices. ![]()
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