Effects of maturation on the conformational free-energy landscape of SOD1

Amyotrophic lateral sclerosis (ALS) is a devastating fatal syndrome characterized by very rapid degeneration of motor neurons. A leading hypothesis is that ALS is caused by toxic protein misfolding and aggregation, as also occurs in many other neurodegenerative disorders, such as prion, Alzheimer’s, Parkinson’s, and Huntington’s diseases. A prominent cause of familial ALS is mutations in the protein superoxide dismutase (SOD1), which promote the formation of misfolded SOD1 conformers that are prone to aberrant interactions both with each other and with other cellular components. We have shown previously that immature SOD1, lacking bound Cu and Zn metal ions and the intrasubunit disulfide bond (apoSOD12SH), has a rugged free-energy surface (FES) and exchanges with four other conformations (excited states) that have millisecond lifetimes and sparse populations on the order of a few percent. Here, we examine further states of SOD1 along its maturation pathway, as well as those off-pathway resulting from metal loss that have been observed in proteinaceous inclusions. Metallation and disulfide bond formation lead to structural transformations including local ordering of the electrostatic loop and native dimerization that are observed in rare conformers of apoSOD12SH; thus, SOD1 maturation may occur via a population-switch mechanism whereby posttranslational modifications select for preexisting structures on the FES. Metallation and oxidation of SOD1 stabilize the native, mature conformation and decrease the number of detected excited conformational states, suggesting that it is the immature forms of the protein that contribute to misfolded conformations in vivo rather than the highly stable enzymatically active dimer.

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Conformational free energy surface of the linear DPDPE peptide: cost of pre-organization for disulfide bond formation

Theoretical Chemistry Accounts ◽

10.1007/s002140050441 ◽

1999 ◽

Vol 101 (4) ◽

pp. 274-281 ◽

Cited By ~ 4

Author(s):

Yan Wang ◽

Krzysztof Kuczera

Keyword(s):

Free Energy ◽

Disulfide Bond ◽

Energy Surface ◽

Bond Formation ◽

Disulfide Bond Formation ◽

Free Energy Surface ◽

Conformational Free Energy

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The novel chaperone protein SRCP1 reduces insoluble SOD1 protein in vivo without extending ALS mouse lifespan

10.1101/2020.12.21.423691 ◽

2020 ◽

Author(s):

Ian W. Luecke ◽

Gloria Lin ◽

Stephanie Santarriaga ◽

K. Matthew Scaglione ◽

Allison D. Ebert

Keyword(s):

Protein Aggregation ◽

Motor Neurons ◽

Protein Misfolding ◽

Protein Quality ◽

Therapeutic Potential ◽

Dependent Manner ◽

Insoluble Protein ◽

Chaperone Protein

AbstractProtein misfolding and aggregation are shared features of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and protein quality control disruption contributes to neuronal toxicity. Therefore, reducing protein aggregation could hold therapeutic potential. We previously identified a novel chaperone protein, serine-rich chaperone protein 1 (SRCP1), that effectively prevents protein aggregation in cell culture and zebrafish models of Huntington’s disease. Here we tested whether this benefit extends to aggregated proteins found in ALS. We used viral-mediated expression of SRCP1 in in vitro and in vivo models of ALS. We found that SRCP1 reduced insoluble SOD1 protein levels in HEK293T cells overexpressing either the A4V or G93R mutant SOD1. However, the reduction of insoluble protein was not observed in either mutant C9orf72 or SOD1 ALS iPSC-derived motor neurons infected with a lentivirus expressing SRCP1. SOD1 G93A ALS mice injected with AAV-SRCP1 showed a small but significant reduction in insoluble and soluble SOD1 in both the brain and spinal cord, but SRCP1 expression did not improve mouse survival. These data indicate that SRCP1 likely reduces insoluble protein burden in a protein and/or context-dependent manner indicating a need for additional insight into SRCP1 function and therapeutic potential.

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A Synthetic SOD/Catalase Mimic Compound for the Treatment of ALS

Antioxidants ◽

10.3390/antiox10060827 ◽

2021 ◽

Vol 10 (6) ◽

pp. 827

Author(s):

Matan Soll ◽

Hagit Goldshtein ◽

Ron Rotkopf ◽

Niva Russek-Blum ◽

Zeev Gross

Keyword(s):

Locomotor Activity ◽

Motor Neurons ◽

Protein Misfolding ◽

Iron Homeostasis ◽

Treated Group ◽

Iron Complex ◽

Untreated Group ◽

Sporadic Als ◽

Familial Als

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. To date, the etiology of the disease is still unclear, with evidence of reactive oxygen species, mitochondrial dysfunction, iron homeostasis perturbation, protein misfolding and protein aggregation as key players in the pathology of the disease. Twenty percent of familial ALS and two percent of sporadic ALS instances are due to a mutation in Cu/Zn superoxide dismutase (SOD1). Sporadic and familial ALS affects the same neurons with similar pathology; therefore, the underlying hypothesis is that therapies effective in mutant SOD1 models could be translated to sporadic ALS. Corrole metal complexes have lately been identified as strong and potent catalytic antioxidants with beneficial effects in oxidative stress-related diseases such as Parkinson’s disease, Alzheimer’s disease, atherosclerosis, diabetes and its complications. One of the most promising candidates is the iron complex of an amphiphilic corrole, 1-Fe. In this study we used the SOD1 G93R mutant zebrafish ALS model to assess whether 1-Fe, as a potent catalytic antioxidant, displays any therapeutic merits in vivo. Our results show that 1-Fe caused a substantial increase in mutant zebrafish locomotor activity (up to 30%), bringing the locomotive abilities of the mutant treated group close to that of the wild type untreated group (50% more than the mutated untreated group). Furthermore, 1-Fe did not affect WT larvae locomotor activity, suggesting that 1-Fe enhances locomotor ability by targeting mechanisms underlying SOD1 ALS specifically. These results may pave the way for future development of 1-Fe as a viable treatment for ALS.

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The Timing and Extent of Motor Neuron Vulnerability in ALS Correlates with Accumulation of Misfolded SOD1 Protein in the Cortex and in the Spinal Cord

Cells ◽

10.3390/cells9020502 ◽

2020 ◽

Vol 9 (2) ◽

pp. 502

Author(s):

Baris Genc ◽

Oge Gozutok ◽

Nuran Kocak ◽

P. Hande Ozdinler

Keyword(s):

Spinal Cord ◽

Drug Discovery ◽

Motor Neuron ◽

Motor Neurons ◽

Selective Vulnerability ◽

Motor Neuron Diseases ◽

Neuron Populations ◽

Misfolded Sod1 ◽

Over Time

Understanding the cellular and molecular basis of selective vulnerability has been challenging, especially for motor neuron diseases. Developing drugs that improve the health of neurons that display selective vulnerability relies on in vivo cell-based models and quantitative readout measures that translate to patient outcome. We initially developed and characterized UCHL1-eGFP mice, in which motor neurons are labeled with eGFP that is stable and long-lasting. By crossing UCHL1-eGFP to amyotrophic lateral sclerosis (ALS) disease models, we generated ALS mouse models with fluorescently labeled motor neurons. Their examination over time began to reveal the cellular basis of selective vulnerability even within the related motor neuron pools. Accumulation of misfolded SOD1 protein both in the corticospinal and spinal motor neurons over time correlated with the timing and extent of degeneration. This further proved simultaneous degeneration of both upper and lower motor neurons, and the requirement to consider both upper and lower motor neuron populations in drug discovery efforts. Demonstration of the direct correlation between misfolded SOD1 accumulation and motor neuron degeneration in both cortex and spinal cord is important for building cell-based assays in vivo. Our report sets the stage for shifting focus from mice to diseased neurons for drug discovery efforts, especially for motor neuron diseases.

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AAV2/9-mediated overexpression of MIF inhibits SOD1 misfolding, delays disease onset, and extends survival in mouse models of ALS

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904665116 ◽

2019 ◽

Vol 116 (29) ◽

pp. 14755-14760 ◽

Cited By ~ 8

Author(s):

Marcel F. Leyton-Jaimes ◽

Joy Kahn ◽

Adrian Israelson

Keyword(s):

Spinal Cord ◽

Motor Neurons ◽

Therapeutic Potential ◽

Disease Onset ◽

Mutant Sod1 ◽

Protein Levels ◽

Specific Accumulation ◽

Intracellular Organelles ◽

Misfolded Sod1

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by the loss of upper and lower motor neurons. Transgenic mice that overexpress mutant SOD1 develop paralysis and accumulate misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and endoplasmic reticulum (ER). Recently, macrophage migration inhibitory factor (MIF) was shown to directly inhibit mutant SOD1 misfolding and binding to intracellular membranes. In addition, complete elimination of endogenous MIF accelerated disease onset and late disease progression, as well as shortened the lifespan of mutant SOD1 mice with higher amounts of misfolded SOD1 detected within the spinal cord. Based on these findings, we used adeno-associated viral (AAV) vectors to overexpress MIF in the spinal cord of mutant SOD1G93A and loxSOD1G37R mice. Our data show that MIF mRNA and protein levels were increased in the spinal cords of AAV2/9-MIF–injected mice. Furthermore, mutant SOD1G93A and loxSOD1G37R mice injected with AAV2/9-MIF demonstrated a significant delay in disease onset and prolonged survival compared with their AAV2/9-GFP–injected or noninjected littermates. Moreover, these mice accumulated reduced amounts of misfolded SOD1 in their spinal cords, with no observed effect on glial overactivation as a result of MIF up-regulation. Our findings indicate that MIF plays a significant role in SOD1 folding and misfolding mechanisms and strengthen the hypothesis that MIF acts as a chaperone for misfolded SOD1 in vivo and may have further implications regarding the therapeutic potential role of up-regulation of MIF in modulating the specific accumulation of misfolded SOD1.

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TNF receptor–associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011215 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3808-3825 ◽

Cited By ~ 1

Author(s):

Sabrina Semmler ◽

Myriam Gagné ◽

Pranav Garg ◽

Sarah R. Pickles ◽

Charlotte Baudouin ◽

...

Keyword(s):

Superoxide Dismutase ◽

Motor Neurons ◽

Ubiquitin Ligase ◽

Protein Misfolding ◽

E3 Ubiquitin Ligase ◽

Tnf Receptor ◽

Superoxide Dismutase 1 ◽

Associated Factor ◽

Independent Events ◽

Misfolded Sod1

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor–associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

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TNF receptor associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation

10.1101/780460 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sabrina Semmler ◽

Myriam Gagné ◽

Pranav Garg ◽

Sarah Pickles ◽

Charlotte Baudouin ◽

...

Keyword(s):

Superoxide Dismutase ◽

Motor Neurons ◽

Ubiquitin Ligase ◽

Protein Misfolding ◽

E3 Ubiquitin Ligase ◽

Tnf Receptor ◽

Gene Encoding ◽

Associated Factor ◽

Independent Events ◽

Misfolded Sod1

ABSTRACTAmyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as with a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor and determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6-interacting from TRAF6 non-interacting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former, but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

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