A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

Fay, James M.; Zhu, Cheng; Proctor, Elizabeth A.; Tao, Yazhong; Cui, Wenjun; Ke, Hengming; Dokholyan, Nikolay V.

doi:10.1016/j.str.2016.08.011

Cited by 41 publications

(30 citation statements)

References 64 publications

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“…The detailed procedures for Eris calculation were described previously (37). For mutations requiring significant backbone adjustment (e.g., small to large residues, Gly-to-Pro mutations), the flexible backbone mode was adopted in Eris, followed by molecular dynamic simulations as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

“…By perturbing (stabilizing or destabilizing) a series of sparsely populated conformational states along the SOD1 misfolding pathway, researchers have gained valuable insight into the nonnative SOD1 conformers and subsequent cytotoxicity. For instance, a phosphomimetic mutation results in increased thermodynamic stability of the SOD1 native dimer and positively impacts motor neuron survival in a model of ALS (8). In contrast, Proctor et al (9) identified trimer-stabilizing SOD1 mutants and demonstrated that the elevated population of SOD1 trimers promoted cell death.…”

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confidence: 99%

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Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

Zhu

Beck

Griffith

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Aberrant accumulation of misfolded Cu, Zn superoxide dismutase (SOD1) is a hallmark of SOD1-associated amyotrophic lateral sclerosis (ALS), an invariably fatal neurodegenerative disease. While recent discovery of nonnative trimeric SOD1-associated neurotoxicity has suggested a potential pathway for motor neuron impairment, it is yet unknown whether large, insoluble aggregates are cytotoxic. Here we designed SOD1 mutations that specifically stabilize either the fibrillar form or the trimeric state of SOD1. The designed mutants display elevated populations of fibrils or trimers correspondingly, as demonstrated by gel filtration chromatography and electron microscopy. The trimer-stabilizing mutant, G147P, promoted cell death, even more potently in comparison with the aggressive ALS-associated mutants A4V and G93A. In contrast, the fibril-stabilizing mutants, N53I and D101I, positively impacted the survival of motor neuron-like cells. Hence, we conclude the SOD1 oligomer and not the mature form of aggregated fibril is critical for the neurotoxic effects in the model of ALS. The formation of large aggregates is in competition with trimer formation, suggesting that aggregation may be a protective mechanism against formation of toxic oligomeric intermediates.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

Zhu

Beck

Griffith

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Ser59 and Ser98 are phosphorylated through a direct interaction with Dun1 in response to oxidative stress and this directs SOD1 to the nucleus where it acts as a transcription factor for genes involved in the oxidative stress response (Tsang et al, 2014). While production of phosphorylated SOD1 in amounts necessary for biophysical characterisation is challenging, Fay et al (2016) constructed and crystallised a SOD1 Thr2Asp phosphomimetic mutant. The wild-type SOD1 structure is maintained by the Thr2Asp substitution but strengthens the dimer interaction and slows dissociation rates even when the Ala4Val mutation was present along with glutathionylation at Cys111.…”

Section: Oxidationmentioning

confidence: 99%

The biophysics of superoxide dismutase-1 and amyotrophic lateral sclerosis

Wright

Antonyuk

Hasnain

2019

Quart. Rev. Biophys.

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Few proteins have come under such intense scrutiny as superoxide dismutase-1 (SOD1). For almost a century, scientists have dissected its form, function and then later its malfunction in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We now know SOD1 is a zinc and copper metalloenzyme that clears superoxide as part of our antioxidant defence and respiratory regulation systems. The possibility of reduced structural integrity was suggested by the first crystal structures of human SOD1 even before deleterious mutations in thesod1gene were linked to the ALS. This concept evolved in the intervening years as an impressive array of biophysical studies examined the characteristics of mutant SOD1 in great detail. We now recognise how ALS-related mutations perturb the SOD1 maturation processes, reduce its ability to fold and reduce its thermal stability and half-life. Mutant SOD1 is therefore predisposed to monomerisation, non-canonical self-interactions, the formation of small misfolded oligomers and ultimately accumulation in the tell-tale insoluble inclusions found within the neurons of ALS patients. We have also seen that several post-translational modifications could push wild-type SOD1 down this toxic pathway. Recently we have come to view ALS as a prion-like disease where both the symptoms, and indeed SOD1 misfolding itself, are transmitted to neighbouring cells. This raises the possibility of intervention after the initial disease presentation. Several small-molecule and biologic-based strategies have been devised which directly target the SOD1 molecule to change the behaviour thought to be responsible for ALS. Here we provide a comprehensive review of the many biophysical advances that sculpted our view of SOD1 biology and the recent work that aims to apply this knowledge for therapeutic outcomes in ALS.

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“…Previous novel SOD1 mutants have been constructed by point mutation, including W32S/W32F (34,35), C111S(36), T2D (37), and E133Q (38), however, these studies did not use predictions of thermodynamic stability as a condition for their selection. Other studies have analyzed the force--induced mechanical unfolding of a de novo designed SOD1 construct .…”

Section: Discussionmentioning

confidence: 99%

In Silico Determined Properties of Designed Superoxide Dismutase-1 Mutants Predict ALS-like Phenotypes In Vitro and In Vivo

DuVal

McAlary

Habibi

et al. 2018

Preprint

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The underlying physical causes of SOD1---related ALS are still not well---understood. We address this problem here by computationally designing two de novo mutants, A89R and K128N, which were predicted theoretically to be either significantly destabilizing or stabilizing respectively. We subjected these in silico designed mutants to a series of experimental tests, including in vitro measures of thermodynamic stability, cell---based aggregation and toxicity assays, and an in vivo developmental model of zebrafish motor neuron axonopathy. The experimental tests validated the theoretical predictions: A89R is an unstable, highly---deleterious mutant, and K128N is a stable, non--toxic mutant. Moreover, K128N is predicted computationally to form an unusually stable heterodimer with the familial ALS mutant A4V. Consistent with this prediction, co---injection of K128N and A4V into zebrafish shows profound rescue of motor neuron pathology. The demonstrated success of these first principles calculations to predict the physical properties of SOD1 mutants holds promise for rationally designed therapies to counter the progression of ALS. SignificanceMutations in the protein superoxide dismutase cause ALS, and many of these mutants have decreased folding stability. We sought to pursue this thread using a synthetic biology approach, where we designed two de novo mutations, one stabilizing and one destabilizing, as predicted using computational molecular dynamics simulations. We then tested these mutants using in vitro, cell--based, and in vivo zebrafish models. We found that the unstable mutant was toxic, and induced a severe ALS phenotype in zebrafish; the predicted stable mutant, on the other hand, behaved even better than WT. In fact, it was able to rescue the ALS phenotype caused by mutant SOD1. We propose a mechanism for this rescue, which may provide an avenue for therapeutic intervention.\bodyMain Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons in the motor cortex, brainstem and spinal cord, with a lifetime risk of about 1:400(1). Most cases of ALS cases are sporadic with no consistently identified genetic mutation, however, a significant fraction of familial ALS (fALS) cases are due mutations in Cu,Zn superoxide dismutase (SOD1)(2---4). The ubiquity of disease---associated mutations (currently over 160 (5)) throughout the primary sequence of SOD1 suggests a physico---chemical origin for SOD1--related fALS.When natively folded, SOD1 is a 32 kDa homodimer that catalyzes the dismutation of oxygen radicals to less harmful molecular oxygen and hydrogen peroxide(4). A properly folded monomer binds one zinc and one copper atom, and contains an intramolecular disulfide bond, all of which are important for the stability of the protein(4). Loss of any of these factors increases the likelihood of SOD1 misfolding into a toxic disease state due to a decrease in stability and invariable formation of toxic aggregates(6). Indeed, SOD1---related fALS mutations ar...

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A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

Cited by 41 publications

References 64 publications

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

The biophysics of superoxide dismutase-1 and amyotrophic lateral sclerosis

In Silico Determined Properties of Designed Superoxide Dismutase-1 Mutants Predict ALS-like Phenotypes In Vitro and In Vivo

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