S-acylation of SOD1, CCS, and a stable SOD1-CCS heterodimer in human spinal cords from ALS and non-ALS subjects

Sarah E Antinone,Raymond P Roos,William N Green,Ghanashyam D Ghadge,Lyle W Ostrow

doi:10.1038/srep41141

Sarah E Antinone, Raymond P Roos + Show 3 more

Open Access

https://doi.org/10.1038/srep41141

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Journal: Scientific Reports	Publication Date: Jan 25, 2017
Citations: 13	License type: open-access

Affiliation: University of Chicago, Johns Hopkins University

Abstract

Previously, we found that human Cu, Zn-superoxide dismutase (SOD1) is S-acylated (palmitoylated) in vitro and in amyotrophic lateral sclerosis (ALS) mouse models, and that S-acylation increased for ALS-causing SOD1 mutants relative to wild type. Here, we use the acyl resin-assisted capture (acyl-RAC) assay to demonstrate S-acylation of SOD1 in human post-mortem spinal cord homogenates from ALS and non-ALS subjects. Acyl-RAC further revealed that endogenous copper chaperone for SOD1 (CCS) is S-acylated in both human and mouse spinal cords, and in vitro in HEK293 cells. SOD1 and CCS formed a highly stable heterodimer in human spinal cord homogenates that was resistant to dissociation by boiling, denaturants, or reducing agents and was not observed in vitro unless both SOD1 and CCS were overexpressed. Cysteine mutations that attenuate SOD1 maturation prevented the SOD1-CCS heterodimer formation. The degree of S-acylation was highest for SOD1-CCS heterodimers, intermediate for CCS monomers, and lowest for SOD1 monomers. Given that S-acylation facilitates anchoring of soluble proteins to cell membranes, our findings suggest that S-acylation and membrane localization may play an important role in CCS-mediated SOD1 maturation. Furthermore, the highly stable S-acylated SOD1-CCS heterodimer may serve as a long-lived maturation intermediate in human spinal cord.

Highlights

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive paralysis leading to death from loss of motor neurons in the brain and spinal cord
We found that S-acylation predominantly occurs on the immature disulfide-reduced form of SOD1 and that S-acylation of the total SOD1 pool is increased under conditions where there is a higher proportion of the immature species[15]
In order to assay the S-acylation of the total pool of SOD1 protein, we used the acyl-RAC method, which is similar to acyl-biotin exchange method22 (ABE) but assesses proteins from the whole lysate as opposed to only immunoprecipitated proteins[21]

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive paralysis leading to death from loss of motor neurons in the brain and spinal cord. We previously reported that SOD1 undergoes S-acylation and that FALS-causing SOD1 mutations increase the proportion of S-acylated SOD1 when expressed in cell culture and in ALS mouse models. We find that endogenous SOD1 is S-acylated at relatively low levels in human patient samples, with a trend toward increased levels in both sporadic and familial ALS patients relative to non-ALS subjects. We report the presence of a stable and highly S-acylated SOD1-CCS heterodimer complex in human spinal cord tissue that was only observed in vitro when both SOD1 and CCS were overexpressed. The heterodimer was S-acylated in all samples with a higher proportion being S-acylated than observed for SOD1 and CCS monomers. The SOD1-CCS heterodimer may represent a long-lived maturation intermediate in human spinal cord

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