title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.
abstract: Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.
authors.affiliation: a School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Alice
authors.initials: A
authors.lastname: Goode
authors.affiliation: b School of Chemistry, University of Nottingham , Nottingham , UK.|c Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Butler
authors.affiliation: b School of Chemistry, University of Nottingham , Nottingham , UK.|c Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Jed
authors.initials: J
authors.lastname: Long
authors.affiliation: a School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: James
authors.initials: J
authors.lastname: Cavey
authors.affiliation: a School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Scott
authors.affiliation: a School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Barry
authors.initials: B
authors.lastname: Shaw
authors.affiliation: b School of Chemistry, University of Nottingham , Nottingham , UK.|c Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Jill
authors.initials: J
authors.lastname: Sollenberger
authors.affiliation: a School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gell
authors.affiliation: d Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø - The Arctic University of Norway , Tromsø , Norway.
authors.forename: Terje
authors.initials: T
authors.lastname: Johansen
authors.affiliation: b School of Chemistry, University of Nottingham , Nottingham , UK.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
authors.affiliation: b School of Chemistry, University of Nottingham , Nottingham , UK.|c Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.affiliation: a School of Life Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001343:Autophagy / Q000235:genetics* / Q000502:physiology; D057174:Frontotemporal Lobar Degeneration; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009046:Motor Neurons / Q000378:metabolism / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D000071456:Sequestosome-1 Protein / Q000235:genetics*
chemical_list: C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein
keywords: ALS; Atg8/LC3; FTLD; LIR; SQSTM1/p62; autophagy