Wild-type Superoxide Dismutase 1 Research Articles

Design and investigation of celastrol‐peptide nanoassemblies and their binding interactions with superoxide dismutase 1 and its mutants

AbstractThe misfolding and aggregation of superoxide dismutase 1 (SOD1) and its mutants has been implicated in amyotrophic lateral sclerosis (ALS). In this study, we have created three peptide conjugates with the antioxidant pentacyclic terpene celastrol and examined their interactions with SOD1 and its mutants A4V and G93A. The peptides YYIVS, MPDAHL, and GSGGL are derived from natural sources and are known for their inherent antioxidant properties. Docking studies revealed that most conjugates showed strong binding with the metal binding and electrostatic loops as well as the β1, β5, and β6 hydrophobic core of SOD1. The conjugates were synthesized and self‐assembled into nanoassemblies. Surface plasmon resonance studies further confirmed the binding interactions of the nanoassemblies with the SOD1 proteins. The nanoassemblies were found to internalize into HEK293T cells. The HEK 293T cells were then transfected with GFP fused WT (Wild Type), A4V and G93A SOD1 mutants. Flow cytometry revealed that treatment with celastrol‐peptide nanoassemblies, affected the fluorescence of the SOD1 protein, implying their role in modulating SOD1, particularly for the mutants. N–Acetyl–Leu–Leu–Norleucinal (ALLN) induced SOD1 aggregation was also affected upon treatment with the nanoassemblies. These results suggest that the nanoassemblies may potentially modulate the activity and structure of SOD1.

Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

ALS-linked SOD1 mutations impair mitochondrial-derived vesicle formation and accelerate aging

Oxidative stress (OS) is regarded as the dominant theory for aging. While compelling correlative data have been generated to support the OS theory, a direct cause-and-effect relationship between the accumulation of oxidation-mediated damage and aging has not been firmly established. Superoxide dismutase 1 (SOD1) is a primary antioxidant in all cells. It is, however, susceptible to oxidation due to OS and gains toxic properties to cells. This study investigates the role of oxidized SOD1 derived from amyotrophic lateral sclerosis (ALS) linked SOD1 mutations in cell senescence and aging. Herein, we have shown that the cell line NSC34 expressing the G93A mutation of human SOD1 (hSOD1G93A) entered premature senescence as evidenced by a decreased number of the 5-ethynyl-2′-deoxyuridine (EdU)-positive cells. There was an upregulation of cellular senescence markers compared to cells expressing the wild-type human SOD1 (hSOD1WT). Transgenic mice carrying the hSOD1G93A gene showed aging phenotypes at an early age (135 days) with high levels of P53 and P16 but low levels of SIRT1 and SIRT6 compared with age-matched hSOD1WT transgenic mice. Notably, the levels of oxidized SOD1 were significantly elevated in both the senescent NSC34 cells and 135-day hSOD1G93A mice. Selective removal of oxidized SOD1 by our CT4-directed autophagy significantly decelerated aging, indicating that oxidized SOD1 is a causal factor of aging. Intriguingly, mitochondria malfunctioned in both senescent NSC34 cells and middle-aged hSODG93A transgenic mice. They exhibited increased production of mitochondrial-derived vesicles (MDVs) in response to mild OS in mutant humanSOD1 (hSOD1) transgenic mice at a younger age; however, the mitochondrial response gradually declined with aging. In conclusion, our data show that oxidized SOD1 derived from ALS-linked SOD1 mutants is a causal factor for cellular senescence and aging. Compromised mitochondrial responsiveness to OS may serve as an indicator of premature aging.

Protein kinetics of superoxide dismutase-1 in familial and sporadic amyotrophic lateral sclerosis.

Accumulation of misfolded superoxide dismutase-1 (SOD1) is a pathological hallmark of SOD1-related amyotrophic lateral sclerosis (ALS) and is observed in sporadic ALS where its role in pathogenesis is controversial. Understanding in vivo protein kinetics may clarify how SOD1 influences neurodegeneration and inform optimal dosing for therapies that lower SOD1 transcripts. We employed stable isotope labeling paired with mass spectrometry to evaluate in vivo protein kinetics and concentration of soluble SOD1 in cerebrospinal fluid (CSF) of SOD1 mutation carriers, sporadic ALS participants and controls. A deaminated SOD1 peptide, SDGPVKV, that correlates with protein stability was also measured. In participants with heterozygous SOD1A5V mutations, known to cause rapidly progressive ALS, mutant SOD1 protein exhibited ~twofold faster turnover and ~ 16-fold lower concentration compared to wild-type SOD1 protein. SDGPVKV levels were increased in SOD1A5V carriers relative to controls. Thus, SOD1 mutations impact protein kinetics and stability. We applied this approach to sporadic ALS participants and found that SOD1 turnover, concentration, and SDGPVKV levels are not significantly different compared to controls. These results highlight the ability of stable isotope labeling approaches and peptide deamidation to discern the influence of disease mutations on protein kinetics and stability and support implementation of this method to optimize clinical trial design of gene and molecular therapies for neurological disorders. Clinicaltrials.gov: NCT03449212.

Molecular dynamics analysis of superoxide dismutase 1 mutations suggests decoupling between mechanisms underlying ALS onset and progression

Mutations in the superoxide dismutase 1 (SOD1) gene are the second most common known cause of ALS. SOD1 variants express high phenotypic variability and over 200 have been reported in people with ALS. It was previously proposed that variants can be broadly classified in two groups, ‘wild-type like’ (WTL) and ‘metal binding region’ (MBR) variants, based on their structural location and biophysical properties. MBR variants, but not WTL variants, were associated with a reduction of SOD1 enzymatic activity. In this study we used molecular dynamics and large clinical datasets to characterise the differences in the structural and dynamic behaviour of WTL and MBR variants with respect to the wild-type SOD1, and how such differences influence the ALS clinical phenotype. Our study identified marked structural differences, some of which are observed in both variant groups, while others are group specific. Moreover, collecting clinical data of approximately 500 SOD1 ALS patients carrying variants, we showed that the survival time of patients carrying an MBR variant is generally longer (∼6 years median difference, p < 0.001) with respect to patients with a WTL variant. In conclusion, our study highlighted key differences in the dynamic behaviour between WTL and MBR SOD1 variants, and between variants and wild-type SOD1 at an atomic and molecular level, that could be further investigated to explain the associated phenotypic variability. Our results support the hypothesis of a decoupling between mechanisms of onset and progression of SOD1 ALS, and an involvement of loss-of-function of SOD1 with the disease progression.

Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Modulate miRNA Biogenesis through Aberrant Interactions with Exportin 5.

Mutations in the SOD1 (superoxide dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 43 and 24 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of wild-type SOD1. Immunoprecipitation followed by western blot analysis indicated the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over the wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type protein. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to the diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, that is, through aberrant binding toward XPO5.

Screening of Natural Compounds Against SOD1 as a Therapeutic Target for Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is an uncommon and progressive neurological illness that predominantly includes the neurons liable for voluntary muscular activities. Starting from weakness or stiffness in muscles, this gradually exploits the strength and ability to speak, eat, move, and even breathe. Its exact mechanism is still not clear, but mutations in the SOD1 gene have been reported to cause ALS, and some studies also found involvement of SOD1 overexpression in the pathogenesis of ALS. As of now, there is no remedy available for its cure. Objective: The objective of this study is to identify the potential inhibitors for wild type 1HL5, l113T mutant, and A4V mutant of SOD1 (Superoxide Dismutase 1) protein. Methods: In this study, in silico approaches like virtual screening, molecular docking, pharmacokinetic parameters study, and molecular dynamics simulation were used to identify the best potential inhibitors against wild type and mutant SOD1 protein. Results: On the basis of binding affinity and binding energy, the top three compounds ZINC000095486263, ZINC000095485989, and ZINC000028462577 were observed as the best compounds. In the case of 1HL5, ZINC000095486263 had the highest binding affinity with docking score - 10.62 Kcal/mol, 1UXM with ZINC000095485989 had the highest docking score -12.03 Kcal/mol, and 4A7V with ZINC000028462577 was found -11.72 Kcal/mol. Further, Molecular Dynamic Simulations (MDS) results showed that the ZINC000095486263, ZINC000095485989, and ZINC000095485956 compounds were formed a stable complex with 1HL5, 1UXM, and 4A7V respectively. Conclusions: After analyzing the results, we hereby conclude that natural compounds such as ZINC000095486263, ZINC000095485989, and ZINC000095485956 could be used as a potential inhibitor of 1HL5, 1UXM, and 4A7V respectively for ALS treatment and could be used as a drug. Further, In vivo/vitro study of these compounds could be a future direction in the field of drug discovery.

Co-deposition of SOD1, TDP-43 and p62 proteinopathies in ALS: evidence for multifaceted pathways underlying neurodegeneration

Multiple neurotoxic proteinopathies co-exist within vulnerable neuronal populations in all major neurodegenerative diseases. Interactions between these pathologies may modulate disease progression, suggesting they may constitute targets for disease-modifying treatments aiming to slow or halt neurodegeneration. Pairwise interactions between superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43) and ubiquitin-binding protein 62/sequestosome 1 (p62) proteinopathies have been reported in multiple transgenic cellular and animal models of amyotrophic lateral sclerosis (ALS), however corresponding examination of these relationships in patient tissues is lacking. Further, the coalescence of all three proteinopathies has not been studied in vitro or in vivo to date. These data are essential to guide therapeutic development and enhance the translation of relevant therapies into the clinic. Our group recently profiled SOD1 proteinopathy in post-mortem spinal cord tissues from familial and sporadic ALS cases, demonstrating an abundance of structurally-disordered (dis)SOD1 conformers which become mislocalized within these vulnerable neurons compared with those of aged controls. To explore any relationships between this, and other, ALS-linked proteinopathies, we profiled TDP-43 and p62 within spinal cord motor neurons of the same post-mortem tissue cohort using multiplexed immunofluorescence and immunohistochemistry. We identified distinct patterns of SOD1, TDP43 and p62 co-deposition and subcellular mislocalization between motor neurons of familial and sporadic ALS cases, which we primarily attribute to SOD1 gene status. Our data demonstrate co-deposition of p62 with mutant and wild-type disSOD1 and phosphorylated TDP-43 in familial and sporadic ALS spinal cord motor neurons, consistent with attempts by p62 to mitigate SOD1 and TDP-43 deposition. Wild-type SOD1 and TDP-43 co-deposition was also frequently observed in ALS cases lacking SOD1 mutations. Finally, alterations to the subcellular localization of the three proteins were tightly correlated, suggesting close relationships between the regulatory mechanisms governing the subcellular compartmentalization of these proteins. Our study is the first to profile spatial relationships between SOD1, TDP-43 and p62 pathologies in post-mortem spinal cord motor neurons of ALS patients, previously only studied in vitro. Our findings suggest interactions between these three key ALS-linked proteins are likely to modulate the formation of their respective proteinopathies, and perhaps the rate of motor neuron degeneration, in ALS patients.

Misfolding-Associated Exposure of Natively Buried Residues in Mutant SOD1 Facilitates Binding to TRAF6

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily impacting motor neurons. Mutations in superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS. Several of these mutations lead to misfolding or toxic gain of function in the SOD1 protein. Recently, we reported that misfolded SOD1 interacts with TNF receptor-associated factor 6 (TRAF6) in the SOD1G93A rat model of ALS. Further, we showed in cultured cells that several mutant SOD1 proteins, but not wildtype SOD1 protein, interact with TRAF6 via the MATH domain. Here, we sought to uncover the structural details of this interaction through molecular dynamics (MD) simulations of a dimeric model system, coarse grained using the AWSEM force field. We used direct MD simulations to identify buried residues, and predict binding poses by clustering frames from the trajectories. Metadynamics simulations were also used to deduce preferred binding regions on the protein surfaces from the potential of the mean force in orientation space. Well-folded SOD1 was found to bind TRAF6 via co-option of its native homodimer interface. However, if loops IV and VII of SOD1 were disordered, as typically occurs in the absence of stabilizing Zn2+ ion binding, these disordered loops now participated in novel interactions with TRAF6. On TRAF6, multiple interaction hot-spots were distributed around the equatorial region of the MATH domain beta barrel. Expression of TRAF6 variants with mutations in this region in cultured cells demonstrated that TRAF6T475 facilitates interaction with different SOD1 mutants. These findings contribute to our understanding of the disease mechanism and uncover potential targets for the development of therapeutics.

Evaluation of Zn2+- and Cu2+-Binding Affinities of Native Cu,Zn-SOD1 and Its G93A Mutant by LC-ICP MS.

The tight binding of Cu and Zn ions to superoxide dismutase 1 (SOD1) maintains the protein stability, associated with amyotrophic lateral sclerosis (ALS). Yet, the quantitative studies remain to be explored for the metal-binding affinity of wild-type SOD1 and its mutants. We have investigated the demetallation of Cu,Zn-SOD1 and its ALS-related G93A mutant in the presence of different standard metal ion chelators at varying temperatures by using an LC-ICP MS-based approach and fast size-exclusion chromatography. Our results showed that from the slow first-order kinetics both metal ions Zn2+ and Cu2+ were released simultaneously from the protein at elevated temperatures. The rate of the release depends on the concentration of chelating ligands but is almost independent of their metal-binding affinities. Similar studies with the G93A mutant of Cu,Zn-SOD1 revealed slightly faster metal-release. The demetallation of Cu,Zn-SOD1 comes always to completion, which hindered the calculation of the KD values. From the Arrhenius plots of the demetallation in the absence of chelators ΔH‡ = 173 kJ/mol for wt and 191 kJ/mol for G93A mutant Cu,Zn-SOD1 was estimated. Obtained high ΔH values are indicative of the occurrence of protein conformational changes before demetallation and we concluded that Cu,Zn-SOD1 complex is in native conditions kinetically inert. The fibrillization of both forms of SOD1 was similar.

The E2 ubiquitin-conjugating enzyme HIP2 is a crucial regulator of quality control against mutant SOD1 proteotoxicity

Mutations in superoxide dismutase 1 (SOD1) leading to the formation of intracellular protein aggregates cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by a selective degeneration of motor neurons. The ALS-linked mutant SOD1 emerged as a possible target for ubiquitin–proteasome system (UPS)-mediated degradation. We aimed to elucidate the role of huntingtin interaction protein 2 (HIP2), an E2 ubiquitin-conjugating enzyme, in the proteotoxicity of mutant SOD1 aggregates. We found that HIP2 interacts with mutant SOD1, but not wild-type SOD1, and is upregulated in response to mutant SOD1 expression. Upregulation of HIP2 protein was observed in the spinal cord of 16-week-old SOD1-G93A transgenic mice. HIP2 further modified mutant SOD1 proteins via K48-linked polyubiquitination and degraded mutant SOD1 proteins through the UPS. Upregulation of HIP2 protected cells from mutant SOD1-induced toxicity. Taken together, our findings demonstrate that HIP2 is a crucial regulator of quality control against the proteotoxicity of mutant SOD1. Our results suggest that modulating HIP2 may represent a novel therapeutic strategy for the treatment of ALS.

Improving the catalytic efficiency and dimeric stability of Cu,Zn superoxide dismutase by combining structure-guided consensus approach with site-directed mutagenesis

Superoxide dismutase (SOD) leads the front line of defense against injuries mediated by the reactive oxygen species (ROS). The SOD from a high-altitude plant Potentilla atrosanguinea is a unique thermostable enzyme. In this study, we applied a structure-guided consensus approach on Cu,Zn SOD from Potentilla atrosanguinea plant, to improve its enzymatic properties. The polar uncharged amino acid (threonine) at position 97 of wild-type (WT) SOD was selected as a target residue for substitution by aspartate (T97D) through site-directed mutagenesis. The WT and T97D were examined by a combinative approach consisting of robust computational and experimental tools. The in-silico analysis indicated improved dimeric stability in T97D as compared to the WT. The strong interactions between the monomers were related to improved dimerization and enhanced catalytic efficiency of T97D. These results were validated by in-vitro assays showing improved dimer stability and catalytic efficiency in T97D than WT. Moreover, the mutation also improved the thermostability of the enzyme. The combined structural and functional data described the basis for improved specific activity and thermostability. This study could expand the scope of interface residue to be explored as targets for designing of SODs with improved kinetics.

In vitro evidence of propagation of superoxide dismutase-1 protein aggregation in canine degenerative myelopathy

Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disorder that has been linked to mutations in the superoxide dismutase 1 (SOD1) gene. The accumulation of misfolded protein aggregates in spinal neurons and astrocytes is implicated as an important pathological process in DM; however, the mechanism of protein aggregate formation is largely unknown. In human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), cell-to-cell propagation of disease-relevant proteins has been demonstrated. Therefore, in this study, propagation of aggregation-forming property of mutant SOD1 protein in DM in vitro was investigated.This study demonstrated that aggregates composed of canine wild type SOD1 protein were increased by co-transfection with canine mutant SOD1 (E40K SOD1), indicating intracellular propagation of SOD1 aggregates. Further, aggregated recombinant SOD1 proteins were released from the cells, taken up by other cells, and induced further aggregate formation of normally folded SOD1 proteins. These results suggest intercellular propagation of SOD1 aggregates. The hypothesis of cell-to-cell propagation of SOD1 aggregates proposed in this study may underly the progressive nature of DM pathology.

Misfolding-Associated Exposure of Buried Residues in Mutant Sod1 Facilitates Binding to TRAF6

Mutations of the protein Superoxide Dismutase 1 (SOD1) are implicated in a subset of Amyotrophic Lateral Sclerosis (ALS) cases. The C-terminal domain of TNF Receptor-Associated Factor 6 (TRAF6) is a newly discovered interactor of misfolded mutants of SOD1, but not wild-type SOD1. However no structural information is available yet to characterize how the proteins might directly interact. We sought to investigate possible mechanisms of this interaction through molecular dynamics (MD) and metadynamics simulations of a dimeric model system, coarse-grained using the AWSEM force field. We used direct MD simulations to predict possible binding poses by subjecting the trajectory to hierarchical clustering. Metadynamics simulations were used to deduce the preferred binding regions on the protein surfaces from the potential of the mean force in the spherical (θ, φ) orientation space around each protein. We found that the SOD1-interacting surface of TRAF6 is broadly distributed about the equatorial region of the beta barrel, while the TRAF6 interacting surface of SOD1 is relatively localized. When SOD1 is well-folded, the TRAF6 heterodimer interaction surface involves a co-option of the native SOD1 homodimer interface, while if SOD1 contains disordered loops IV and VII, as typically occurs in the absence of stabilizing Zn2+ ion binding, the disordered loops now partake in novel interactions with TRAF6. Using cDNA constructs, we expressed variants of TRAF6 with mutations in the predicted binding sites and found that TRAF6T475D no longer interacts with G93A and E100G mutants of SOD1 and has reduced interaction with SOD1A4V. These observations suggest a structural mechanism for the SOD1-TRAF6 interaction and may have implications for developing ALS therapeutics in the future.

Novel Selenium-based compounds with therapeutic potential for SOD1-linked amyotrophic lateral sclerosis.

BackgroundAmyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits.MethodsWe have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model.FindingWe have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds.InterpretationOur finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19).FundingProject funding was supported by the 10.13039/100000971ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the 10.13039/100009950Ministry of Education, Culture, Sports, Science and Technology (10.13039/501100001700MEXT), Japan.

Molecular and pharmacological chaperones for SOD1.

The efficacy of superoxide dismutase-1 (SOD1) folding impacts neuronal loss in motor system neurodegenerative diseases. Mutations can prevent SOD1 post-translational processing leading to misfolding and cytoplasmic aggregation in familial amyotrophic lateral sclerosis (ALS). Evidence of immature, wild-type SOD1 misfolding has also been observed in sporadic ALS, non-SOD1 familial ALS and Parkinson's disease. The copper chaperone for SOD1 (hCCS) is a dedicated and specific chaperone that assists SOD1 folding and maturation to produce the active enzyme. Misfolded or misfolding prone SOD1 also interacts with heat shock proteins and macrophage migration inhibitory factor to aid folding, refolding or degradation. Recognition of specific SOD1 structures by the molecular chaperone network and timely dissociation of SOD1-chaperone complexes are, therefore, important steps in SOD1 processing. Harnessing these interactions for therapeutic benefit is actively pursued as is the modulation of SOD1 behaviour with pharmacological and peptide chaperones. This review highlights the structural and mechanistic aspects of a selection of SOD1-chaperone interactions together with their impact on disease models.

Quercetin and Baicalein Act as Potent Antiamyloidogenic and Fibril Destabilizing Agents for SOD1 Fibrils.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that has been associated with the deposition of aggregates of superoxide dismutase 1 (SOD1). Effective therapeutics against SOD1 fibrillation is still an area of active research. Herein, we demonstrate the potential of two naturally occurring flavonoids (quercetin and baicalein) to inhibit fibrillation of wild-type SOD1 with the aid of a series of biophysical techniques. Our seeding experiments reveal that both of these flavonoids significantly affect the fibril elongation. Interestingly, our ThT binding assay, TEM, and SDS-PAGE experiments suggest that these flavonoids also disintegrate the fibrils into shorter fragments but do not completely depolymerize them into monomers. Binding parameters obtained from the analysis of UV-vis spectra suggest that these flavonoids bind moderately to native SOD1 dimer and have different binding sites. Docking of these flavonoids with a non-native monomer, non-native trimer, and oligomer derived from the 11-residue segment of SOD1 indicates that both quercetin and baicalein can bind to these species and thus can arrest the elongation of fibrils by blocking the fibrillar core regions on the intermediate species formed during aggregation of SOD1. MTT assay data revealed that both the flavonoids reduced the cytotoxicity of SOD1 fibrils. Experimental data also show the antiamyloidogenic potential of both flavonoids against A4V SOD1 mutant fibrillation. Thus, our findings may provide a direction for designing effective therapeutic agents against ALS which can act as promising antiamyloidogenic and fibril destabilizing agents.

Reduced thermodynamic stability as prerequisite for aggregation of SOD1 mutants: a path through the reduction in intramolecular disulfide bonds

Amyotrophic lateral sclerosis is a fatal, devastating, rapidly progressive, adult onset neurodegenerative disease, which is involved in the formation of proteinaceous virulent aggregates from superoxide dismutase 1 (SOD1) as a Cu/Zn metalloenzyme in motor neurons. However, the particular mechanism of mutant SOD1 toxicity has remained elusive. Here, we use complementary biochemical and biophysical methods to investigate thermodynamic parameters of wild-type (WT), E100K and D125H mutants in both metal-free (apo) and metalized (holo) forms of SOD1, to find out if there is any relationship between the structural/thermodynamic stability with aggregation propensity in apo and holo forms of WT/mutant SOD1 variants. Also, in this study, we compared the aggregation propensity of WT SOD1 and the mutants (E100K, D125H) under reducing conditions; the aggregation process and protein surface hydrophobicity were monitored by Thioflavin T (ThT) and ANS fluorescence, respectively. To determine molecular sizes, and intramolecular secondary structures, dynamic light scattering (DLS) and X-ray diffraction analyses were also applied, respectively. In addition to thermodynamic parameters measurements of both apo- and holo-proteins, melting temperature (Tm) of apo proteins was assessed. Furthermore, for comparative evaluation of toxicity of SOD1 aggregates, LDH cytotoxicity as well as hemolysis assays were performed. The hydrodynamic diameters of all SOD1 variants were the same. ThT/ANS results, however, confirmed that DTT-mediated reduction in intramolecular S–S bonds induces gross conformational changes followed by the protein assembly. Thermodynamic studies showed that neither metal deficiency/mutation nor intramolecular S–S reduction, alone, can significantly reduce stability of SOD1 proteins, and demetallation (reduced stability) and intramolecular S–S reduction, together, are required for initiation of SOD1 misfolding/aggregation. CR binding, CD and XRD results, also together, confirmed the non-amyloid character of relatively toxic SOD1 aggregates. It appears that reduced thermodynamic stability as prerequisite for aggregation of SOD1 mutants must be accompanied by intramolecular disulfides reduction.

Plasma microRNA miR-26b as a potential diagnostic biomarker of degenerative myelopathy in Pembroke welsh corgis

BackgroundDegenerative myelopathy (DM) is a progressive neurodegenerative disease frequently found in Pembroke Welsh Corgis (PWCs). Most DM-affected PWCs are homozygous for the mutant superoxide dismutase 1 (SOD1) allele; however, the genetic examination for the SOD1 mutation does not exclusively detect symptomatic dogs. In order to identify novel biomarkers, the plasma microRNA (miRNA) profiles of PWCs with DM were investigated.ResultsQuantification of the plasma levels of 277 miRNAs by an RT-qPCR array identified 11 up-regulated miRNAs and 7 down-regulated miRNAs in DM-affected PWCs from those in wild-type SOD1 PWCs. A pathway analysis identified 3 miRNAs: miR-26b, miR-181a, and miR-196a, which potentially regulate several genes associated with SOD1. In order to validate the diagnostic accuracy of the candidate miRNAs in the aged PWC population, candidate miRNAs in plasma were measured by RT-qPCR and a receiver operating characteristic (ROC) curve analysis was performed. miR-26b had the largest area under the ROC curve for distinguishing DM PWCs from healthy PWCs (sensitivity, 66.7%; specificity, 87.0%). The plasma level of miR-26b was significantly higher in the DM group than in the healthy control group. A positive correlation was observed between increases in the plasma level of miR-26b and disease progression.ConclusionsThese results suggest that plasma miR-26b is a potential novel diagnostic biomarker of DM.

A hyperthermophilic protein G variant engineered via directed evolution prevents the formation of toxic SOD1 oligomers.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective death of motor neurons in the brainstem, motor cortex, and spinal cord, leading to muscle atrophy and eventually to death. It is currently held that various oligomerization-inducing mutations in superoxide dismutase 1 (SOD1), an amyloid-forming protein, may be implicated in the familial form of this fast-progressing highly lethal neurodegenerative disease. A possible therapeutic approach could therefore lie in developing inhibitors to SOD1 mutants. By screening a focused mutagenesis library, mutated randomly in specific "stability patch" positions of the B1 domain of protein G (HTB1), we previously identified low affinity inhibitors of aggregation of SOD1G93A and SOD1G85R mutants. Herein, with the aim to generate a more potent inhibitor with higher affinity to SOD1 mutants, we employed an unbiased, random mutagenesis approach covering the entire sequence space of HTB1 to optimize as yet undefined positions for improved interactions with SOD1. Using affinity maturation screens in yeast, we identified a variant, which we designated HTB1M3 , that bound strongly to SOD1 misfolded mutants but not to wild-type SOD1. In-vitro aggregation assays indicated that in the presence of HTB1M3 misfolded SOD1 assembled into oligomeric species that were not toxic to NSC-34 neuronal cells. In addition, when NSC-34 cells were exposed to misfolded SOD1 mutants, either soluble or preaggregated, in the presence of HTB1M3 , this inhibitor prevented the prion-like propagation of SOD1 from one neuronal cell to another by blocking the penetration of SOD1 into the neuronal cells.