Abstract
The transcription factor Nrf2 and its repressor protein Keap1 play key roles in the regulation of antioxidant stress responses and both Keap1-Nrf2 signalling and oxidative stress have been implicated in the pathogenesis of the ALS-FTLD spectrum of neurodegenerative disorders. The Keap1-binding partner and autophagy receptor SQSTM1/p62 has also recently been linked genetically to ALS-FTLD, with some missense mutations identified in patients mapping within or close to its Keap1-interacting region (KIR, residues 347–352). Here we report the effects on protein function of four different disease associated mutations of SQSTM1/p62 which affect the KIR region. Only mutations mapping precisely to the KIR (P348L and G351A) were associated with a loss of Keap1 binding in co-immunoprecipitations comparable to wild-type SQSTM1/p62. These selective effects on Keap1 recognition were entirely rational based on protein structural models. Consistent with impaired Keap1 binding, the P348L and G351A KIR mutants showed reduced ability to activate Nrf2 signalling compared to wild-type SQSTM1/p62 in antioxidant response element (ARE)-luciferase reporter assays. The results suggest that SQSTM1 mutations within the KIR of SQSTM1/p62 contribute to aetiology of some cases of ALS-FTLD through a mechanism involving aberrant expression or regulation of oxidative response genes.
Highlights
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are devastating neurological diseases that lie within the same clinicopathological spectrum, with approximately 5-10% of cases being familial
Consistent with previous reports using MBP pull-down assays (Jain et al 2010), the G351A Keap1-interacting region (KIR) mutant was unable to capture endogenous Keap1; further, we found that the P348L KIR mutant was defective in Keap1 binding
A densitometric analysis of the blot presented in Fig. 2A indicates that the normalised Keap1 band intensities in the P348L and G351A IP lanes are in the order of only ~10% of the intensities for wild-type
Summary
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are devastating neurological diseases that lie within the same clinicopathological spectrum, with approximately 5-10% of cases being familial. Coexistence of PDB and ALS-FTLD is apparently rare and precisely how different SQSTM1 mutations (some of which are common to both disorders) can lead to either neurodegeneration or PDB is currently unknown, in some cases co-occurrence of an additional mutation such as a pathogenic C9orf expansion may account for the neurodegenerative phenotype (Almeida et al 2015). SQSTM1 encodes the SQSTM1/p62 protein and the overwhelming majority of mutations associated with PDB cluster within and around the C-terminal ubiquitin-associated (UBA) domain, whereas mutations associated with ALS-FTLD occur more widely throughout the protein sequence (Rea et al 2014, Majcher et al 2015). In addition to the UBA domain, the multi-domain SQSTM1/p62 protein consists of an N-terminal Phox and Bem1p (PB1) domain, zinc finger domain (ZZ), TRAF6-binding domain, LC3interacting region (LIR), Keap1-interacting region (KIR) and two PEST sequences.
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