High-Throughput Microplate-Based Fluorescence Assays for Studying Stochastic Aggregation of Superoxide Dismutase-1

Abdolvahabi, Alireza; Rasouli, Sanaz; Croom, Corbin M.; Plewman, Devon L.

doi:10.1007/978-1-4939-8820-4_6

Cited by 1 publication

(1 citation statement)

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“…Protein purity was verified using TGX Stain-Free gels (Bio-Rad) on ChemiDoc Touch Imaging System (Bio-Rad) and each protein was determined to be >90% pure. After elution from a nickel-charged column, aggregationprone SOD1 was generated by demetallization with EDTA under reducing conditions (Abdolvahabi et al, 2019). Purification was performed in the presence of guanidine HCl to induce dimer subunit disassociation, followed by three stage dialysis buffer exchange in the presence of EDTA at 5 mM to remove metal ions.…”

Section: His-tag Recombinant Protein Productionmentioning

confidence: 99%

FAIM Opposes Aggregation of Mutant SOD1 That Typifies Some Forms of Familial Amyotrophic Lateral Sclerosis

et al. 2020

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative illness that is unremittingly fatal and for which no effective treatment exists. All forms of ALS are characterized by protein aggregation. In familial forms of ALS, specific and heritable aggregation-prone proteins have been identified, such as mutant superoxide dismutase (SOD1). It has been suggested that a factor capable of preventing mutant SOD1 protein aggregation and/or disassembling mutant SOD1 protein aggregates would ameliorate SOD1-associated forms of familial ALS. Here we identify Fas Apoptosis Inhibitory Molecule (FAIM), a highly evolutionarily conserved 20 kDa protein, as an agent with this activity. We show FAIM counteracts intracellular accumulation of mutant SOD1 protein aggregates, which is increased in the absence of FAIM, as determined by pulseshape analysis and filter trap assays. In a cell-free system, FAIM inhibits aggregation of mutant SOD1, and further disassembles and solubilizes established mutant SOD1 protein aggregates, as determined by thioflavin T (ThT), filter trap, and sedimentation assays. In sum, we report here a previously unknown activity of FAIM that opposes ALS disease-related protein aggregation and promotes proteostasis of an aggregation-prone ALS protein.

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Section: His-tag Recombinant Protein Productionmentioning

confidence: 99%