Abstract
BackgroundMutations in the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of familial forms of amyotrophic lateral sclerosis (ALS), and mutant SOD1 has been shown to have increased surface hydrophobicity in vitro. Mutant SOD1 may adopt a complex array of conformations with varying toxicity in vivo. We have used a novel florescence-based proteomic assay using 4,4’-bis-1-anilinonaphthalene-8-sulfonate (bisANS) to assess the surface hydrophobicity, and thereby distinguish between different conformations, of SOD1and other proteins in situ.ResultsCovalent bisANS labeling of spinal cord extracts revealed that alterations in surface hydrophobicity of H46R/H48Q mutations in SOD1 provoke formation of high molecular weight SOD1 species with lowered solubility, likely due to increased exposure of hydrophobic surfaces. BisANS was docked on the H46R/H48Q SOD1 structure at the disordered copper binding and electrostatic loops of mutant SOD1, but not non-mutant WT SOD1. 16 non-SOD1 proteins were also identified that exhibited altered surface hydrophobicity in the H46R/H48Q mutant mouse model of ALS, including proteins involved in energy metabolism, cytoskeleton, signaling, and protein quality control. Heat shock proteins (HSPs) were also enriched in the detergent-insoluble fractions with SOD1. Given that chaperones recognize proteins with exposed hydrophobic surfaces as substrates and the importance of protein homeostasis in ALS, we crossed SOD1 H46R/H48Q mutant mice with mice over-expressing the heat shock factor 1 (HSF1) transcription factor. Here we showed that HSF1 over-expression in H46R/H48Q ALS mice enhanced proteostasis as evidenced by increased expression of HSPs in motor neurons and astrocytes and increased solubility of mutant SOD1. HSF1 over-expression significantly reduced body weight loss, delayed ALS disease onset, decreases cases of early disease, and increased survival for the 25th percentile in an H46R/H48Q SOD1 background. HSF1 overexpression did not affect macroautophagy in the ALS background, but was associated with maintenance of carboxyl terminus of Hsp70 interacting protein (CHIP) expression which declined in H46R/H48Q mice.ConclusionOur results uncover the potential importance of changes in protein surface hydrophobicity of SOD1 and other non-SOD1 proteins in ALS, and how strategies that activate HSF1 are valid therapies for ALS and other age-associated proteinopathies.
Highlights
Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of familial forms of amyotrophic lateral sclerosis (ALS), and mutant Cu/Zn superoxide dismutase 1 (SOD1) has been shown to have increased surface hydrophobicity in vitro
Soluble Mutant SOD1 in spinal cord extracts has increased surface hydrophobicity In order to assess the global distribution of proteins with altered exposure of surface hydrophobicity in the spinal cords of symptomatic ALS mice, the soluble S1 fraction was labeled with bisANS and separated by 2D gel electrophoresis (Figure 1A)
We show that changes in surface hydrophobicity as detected by bisANS relate to important gains or losses of function in soluble SOD1 and the non-SOD1 proteins we identified
Summary
Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of familial forms of amyotrophic lateral sclerosis (ALS), and mutant SOD1 has been shown to have increased surface hydrophobicity in vitro. A number of different mutations in genes encoding Cu/Zn superoxide dismutase (SOD1) [1], TAR DNA binding protein 43 (TDP-43) [2], and 17 others are associated with familial forms of ALS (fALS), which make up 10% of total ALS cases. Despite being responsible for the disproportionation of superoxide radicals to oxygen and hydrogen peroxide, mutations in SOD1 are known to be toxic due to a gain of function rather than a loss of function [6]. Exposed surface hydrophobicity is a significant recognition signal for HSP binding and subsequent re-folding or degradation by chaperones and co-chaperones via the ubiquitin proteasome system (UPS) [15] or autophagy [16]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
