An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Qualls, David; Crosby, Keith; Brown, Hilda; Borchelt, David R.

doi:10.1371/journal.pone.0083981

Cited by 7 publications

(21 citation statements)

References 15 publications

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“…To determine the ability for amino substitutions at tryptophan 32 (W32) to suppress aggregation of ALS mutant SOD1, we employed a cell model system that we have used in prior studies [71,[79][80][81]. We generated SOD1: YFP fusion constructs as described in Methods and transfected these into Chinese Hamster Ovary (CHO) cells.…”

Section: Effects Of the W32s-sod1 Mutation On Sod1 Aggregation In An mentioning

confidence: 99%

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

et al. 2020

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Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the "strain" of the conformer that is propagating.

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Section: Effects Of the W32s-sod1 Mutation On Sod1 Aggregation In An mentioning

confidence: 99%

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

et al. 2020

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“…; Qualls et al . ,b). Similarly, WT SOD1 tagged with green fluorescent protein does not readily form inclusions when expressed in cultured cells and is fully active (Stevens et al .…”

Section: Resultsmentioning

confidence: 99%

“…; Qualls et al . ,b). The fusion construct of WT‐SOD1:Dendra2 and A4V‐SOD1:Dendra2 was generated by amplifying the Dendra2 cDNA using oligonucleotides that modified the 5′ sequence of Dendra2 to remove the start codon and align the coding frame to be in‐frame with SOD1.…”

Section: Methodsmentioning

confidence: 99%

Relationship between mutant Cu/Zn superoxide dismutase 1 maturation and inclusion formation in cell models

Ayers

McMahon

Gill

et al. 2016

Journal of Neurochemistry

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A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis (ALS), is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with ALS mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In the present study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time-stamp that distinguishes pre-existing and newly made proteins. In cells that transiently over-express the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu co-factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly-made protein, are prone to misfolding and aggregation.

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“…WT-hSOD1mon fusions to RFP or YFP remain soluble and completely releasable by saponin [23]. Unexpectedly, fusions of WT-mSod1mon to either RFP or YFP produced inclusions that were saponin resistant (Figure 8).…”

Section: Resultsmentioning

confidence: 99%

Features of wild-type human SOD1 limit interactions with misfolded aggregates of mouse G86R Sod1

Qualls

Prudencio

Roberts

et al. 2013

Mol Neurodegeneration

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Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is well established that mutations in SOD1, associated with fALS, heighten the propensity of the protein to misfold and aggregate. Although aggregation appears to be a factor in the toxicity of mutant SOD1s, the precise nature of this toxicity has not been elucidated. A number of other studies have now firmly established that raising the levels of wild-type (WT) human SOD1 (hSOD1) proteins can in some manner augment the toxicity of mutant hSOD1 proteins. However, a recent study demonstrated that raising the levels of WT-hSOD1 did not affect disease in mice that harbor a mouse Sod1 gene (mSod1) encoding a well characterized fALS mutation (G86R). In the present study, we sought a potential explanation for the differing effects with WT-hSOD1 on the toxicity of mutant hSOD1 versus mutant mSod1. In the cell culture models used here, we observe poor interactions between WT-hSOD1 and misfolded G86R-mSod1, possibly explaining why over-expression of WT-hSOD1 does not synergize with mutant mSod1 to accelerate the course of the disease in mice.

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An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Cited by 7 publications

References 15 publications

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

Relationship between mutant Cu/Zn superoxide dismutase 1 maturation and inclusion formation in cell models

Features of wild-type human SOD1 limit interactions with misfolded aggregates of mouse G86R Sod1

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