Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates.

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.

Proton-coupled electron transfer at a mis-metalated zinc site detected with protein charge ladders.

Distinguishing proton-coupled electron transfer (PCET) from uncoupled electron transfer (ET) in proteins can be challenging. A recent investigation [J. C. Koone, M. Simmang, D. L. Saenger, M. L. Hunsicker-Wang and B. F. Shaw, J. Am. Chem. Soc., 145, 16488-16497] reported that protein charge ladders and capillary electrophoresis can distinguish between single ET, PCET, and two-proton coupled ET (2PCET) by directly measuring the change in protein net charge upon reduction/oxidation (ΔZET). The current study used similar methods to assess PCET in zinc-free, "double copper" superoxide dismutase-1 (4Cu-SOD1), where one copper is bound at the copper site of each monomer and one copper is bound at the bridging zinc site, resulting in a quasi-type III Cu center. At pH 7.4, the net charge (Z) of the 4Cu-SOD1 dimer was unaffected by reduction of all four Cu2+ ions, i.e., ΔZ4ET = -0.09 ± 0.05 per dimer (-0.02 ± 0.01 per copper atom). These values suggest that PCET is taking place at all four Cu atoms of the homodimer. Molecular dynamics and Poisson-Boltzmann calculations suggest that a metal-coordinating histidine at the zinc site (His71) is the proton acceptor. These data show how ligands of a naturally occurring zinc site can help facilitate PCET when the right redox metal is bound.

Mass spectrometry imaging of SOD1 protein-metal complexes in SOD1G93A transgenic mice implicates demetalation with pathology.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the central nervous system (CNS). Mutations in the metalloenzyme SOD1 are associated with inherited forms of ALS and cause a toxic gain of function thought to be mediated by dimer destabilization and misfolding. SOD1 binds two Cu and two Zn ions in its homodimeric form. We have applied native ambient mass spectrometry imaging to visualize the spatial distributions of intact metal-bound SOD1G93A complexes in SOD1G93A transgenic mouse spinal cord and brain sections and evaluated them against disease pathology. The molecular specificity of our approach reveals that metal-deficient SOD1G93A species are abundant in CNS structures correlating with ALS pathology whereas fully metalated SOD1G93A species are homogenously distributed. Monomer abundance did not correlate with pathology. We also show that the dimer-destabilizing post-translational modification, glutathionylation, has limited influence on the spatial distribution of SOD1 dimers.

SoDCoD: a comprehensive database of Cu/Zn superoxide dismutase conformational diversity caused by ALS-linked gene mutations and other perturbations.

A structural alteration in copper/zinc superoxide dismutase (SOD1) is one of the common features caused by amyotrophic lateral sclerosis (ALS)-linked mutations. Although a large number of SOD1 variants have been reported in ALS patients, the detailed structural properties of each variant are not well summarized. We present SoDCoD, a database of superoxide dismutase conformational diversity, collecting our comprehensive biochemical analyses of the structural changes in SOD1 caused by ALS-linked gene mutations and other perturbations. SoDCoD version 1.0 contains information about the properties of 188 types of SOD1 mutants, including structural changes and their binding to Derlin-1, as well as a set of genes contributing to the proteostasis of mutant-like wild-type SOD1. This database provides valuable insights into the diagnosis and treatment of ALS, particularly by targeting conformational alterations in SOD1. Database URL: https://fujisawagroup.github.io/SoDCoDweb/.

Unveiling the double-edged sword: SOD1 trimers possess tissue-selective toxicity and bind septin-7 in motor neuron-like cells.

Misfolded species of superoxide dismutase 1 (SOD1) are associated with increased death in amyotrophic lateral sclerosis (ALS) models compared to insoluble protein aggregates. The mechanism by which structurally independent SOD1 trimers cause cellular toxicity is unknown but may drive disease pathology. Here, we uncovered the SOD1 trimer interactome-a map of potential tissue-selective protein-binding partners in the brain, spinal cord, and skeletal muscle. We identified binding partners and key pathways associated with SOD1 trimers and found that trimers may affect normal cellular functions such as dendritic spine morphogenesis and synaptic function in the central nervous system and cellular metabolism in skeletal muscle. We discovered SOD1 trimer-selective enrichment of genes. We performed detailed computational and biochemical characterization of SOD1 trimer protein binding for septin-7. Our investigation highlights key proteins and pathways within distinct tissues, revealing a plausible intersection of genetic and pathophysiological mechanisms in ALS through interactions involving SOD1 trimers.

Pathogenic R163W Variant of the Copper Chaperone for Sod1 (Ccs) Functions as an Anti-chaperone.

The copper chaperone for Sod1 (Ccs) is a metallochaperone that plays a multifaceted role in the maturation of Cu,Zn superoxide dismutase (Sod1). The Ccs mutation R163W was identified in an infant with fatal neurological abnormalities. Based on a comprehensive structural and functional analysis, we developed the first data-driven model for R163W-related pathogenic phenotypes. The work here confirms previous findings that the substitution of arginine with tryptophan at this site, which is located adjacent to a conserved Zn binding site, creates an unstable Zn-deficient protein that loses its ability to efficiently activate Sod1. Intriguingly, R163W Ccs can reduce copper (i.e., Cu(II) → Cu(I)) bound in its Sod1-like domain (D2), and this novel redox event is accompanied by disulfide bond formation. The loss of Zn binding, along with the unusual ability to bind copper in D2, diverts R163W Ccs toward aggregation. The remarkably high affinity of D2 Cu(I) binding converts R163W from a Cu chaperone to a Cu scavenger that accelerates Sod1 deactivation (i.e., an Anti-chaperone). Overall, these findings present a first-of-its-kind molecular mechanism for Ccs dysfunction that leads to pathogenesis in humans.

Direct observation of prion-like propagation of protein misfolding templated by pathogenic mutants.

Many neurodegenerative diseases feature misfolded proteins that propagate via templated conversion of natively folded molecules. However, crucial questions about how such prion-like conversion occurs and what drives it remain unsolved, partly because technical challenges have prevented direct observation of conversion for any protein. We observed prion-like conversion in single molecules of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis. Tethering pathogenic misfolded SOD1 mutants to wild-type molecules held in optical tweezers, we found that the mutants vastly increased misfolding of the wild-type molecule, inducing multiple misfolded isoforms. Crucially, the pattern of misfolding was the same in the mutant and converted wild-type domains and varied when the misfolded mutant was changed, reflecting the templating effect expected for prion-like conversion. Ensemble measurements showed decreased enzymatic activity in tethered heterodimers as conversion progressed, mirroring the single-molecule results. Antibodies sensitive to disease-specific epitopes bound to the converted protein, implying that conversion produced disease-relevant misfolded conformers.

Terminal {Ni(II)-SH} complex promoted anaerobic catalytic sulfur atom transfer reaction: implication to the sulfide oxidase function of Cu/Zn-superoxide dismutase.

In mitochondria, the detoxification of molar excess H2S as polysulfide proceeded via an oxidation process promoted by Cu/Zn containing superoxide dismutase (SOD1) enzyme, which has been very recently reported as the alternative enzyme for cytosolic H2S oxidation. Herein, we present Ni(II) complexes bearing the terminal SH group as a synthetic functional analogue for the sulfide oxidase function of SOD1. Synthesis, crystal structure and complete spectroscopic characterization of two sets of complexes, [NiLOMe/tBu(PPh3)] (2OMe/tBu) and tetraethyl salt of [NiLOMe/tBu(SH)]-1 (3OMe/tBu), were described (LOMe = (E)-2-methoxy-6-(((2-sulfidophenyl)imino)methyl)phenolate and LtBu = (E)-2,4-di-tert-butyl-6-(((2-sulfidophenyl)imino)methyl)phenolate). Under anaerobic conditions, 3OMe/tBu responded to a catalytic sulfur atom transfer (SAT) reaction with PPh3 to produce SPPh3. The SAT reaction was analyzed using detailed studies of 1H and 31P NMR spectra. Finally, the SAT reactivity pattern was compared with the same in the native enzyme of SOD1.

Native State Stabilization of Amyloidogenic Proteins by Kinetic Stabilizers: Inhibition of Protein Aggregation and Clinical Relevance.

Proteinopathies or amyloidoses are a group of life-threatening disorders that result from misfolding of proteins and aggregation into toxic insoluble amyloid aggregates. Amyloid aggregates have low clearance from the body due to the insoluble nature, leading to their deposition in various organs and consequent organ dysfunction. While amyloid deposition in the central nervous system leads to neurodegenerative diseases that mostly cause dementia and difficulty in movement, several other organs, including heart, liver and kidney are also affected by systemic amyloidoses. Regardless of the site of amyloid deposition, misfolding and structural alteration of the precursor proteins play the central role in amyloid formation. Kinetic stabilizers are an emerging class of drugs, which act like pharmacological chaperones to stabilize the native state structure of amyloidogenic proteins and to increase the activation energy barrier that is required for adopting a misfolded structure or conformation, ultimately leading to the inhibition of protein aggregation. In this review, we discuss the kinetic stabilizers that stabilize the native quaternary structure of transthyretin, immunoglobulin light chain and superoxide dismutase 1 that cause transthyretin amyloidoses, light chain amyloidosis and familial amyotrophic lateral sclerosis, respectively.

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells.

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into ß-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in ß-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that ß-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

Purification and characterization of recombinant human superoxide dismutase integrated with resilin-like polypeptide.

Human superoxide dismutase (hSOD1) plays an important role in the aerobic metabolism and free radical eliminating process in the body. However, the production of existing SOD faces problems such as complex purification methods, high costs, and poor product stability. This experiment achieved low-cost, rapid, and simple purification of hSOD1 through ammonium sulfate precipitation method and heat resistance of recombinant protein. We constructed a recombinant protein hSOD1-LR containing a resilin-like polypeptide tag and expressed it. The interest protein was purified by ammonium sulfate precipitation method, and the results showed that the purification effect of 1.5 M (NH4)2SO4 was the best, with an enzyme activity recovery rate of 80 % after purification. Then, based on its thermal stability, further purification of the interest protein at 60 °C revealed a purification fold of up to 24 folds, and the purification effect was similar to that of hSOD1-6xHis purified by nickel column affinity chromatography. The stability of hSOD1-LR showed that the recombinant protein hSOD1-LR has better stability than hSOD-6xHis. hSOD1-LR can maintain 76.57 % activity even after 150 min of reaction at 70 °C. At same time, hSOD1-LR had activity close to 80 % at pH < 5, indicating good acid resistance. In addition, after 28 days of storage at 4 °C and 40 °C, hSOD1-LR retained 92 % and 87 % activity, respectively. Therefore, the method of purifying hSOD1-LR through salt precipitation may have positive implications for the study of SOD purification.

MS785-MS27 Reactive Misfolded/Non-Native Zn-Deficient SOD1 Species Exhibit Cytotoxicity and Adopt Heterozygous Conformations in Motor Neurons.

Misfolding of superoxide dismutase-1 (SOD1) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) with SOD1 mutations. The development of antibodies specific for misfolded SOD1 deepens our understanding of how the protein participates in ALS pathogenesis. Since the term "misfolding" refers to various disordered conformers other than the natively folded one, which misfolded species are recognized by specific antibodies should be determined. Here, we molecularly characterized the recognition by MS785-MS27, an antibody cocktail experimentally confirmed to recognize over 100 ALS-linked SOD1 mutants. Indirect ELISA revealed that the antibody cocktail recognized Zn-deficient wild-type and mutated SOD1 species. It also recognized conformation-disordered wild-type and mutated SOD1 species, such as unfolded and oligomeric forms, but had less affinity for the aggregated form. Antibody-reactive SOD1 exhibited cytotoxicity to a motor neuron cell model, which was blocked by Zn treatment with Zn-deficient SOD1. Immunohistochemistry revealed antibody-reactive SOD1 mainly in spinal motor neurons of SOD1G93A mice throughout the disease course, and the distribution after symptomatic stages differed from that of other misfolded SOD1 species. This suggests that misfolded/non-native SOD1 species exist as heterogeneous populations. In conclusion, MS785-MS27 recognizes various conformation-disordered SOD1 species lacking the Zn ion.

Amyotrophic Lateral Sclerosis-Associated Mutants of SOD1 Perturb mRNA Splicing through Aberrant Interactions with SRSF2.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the degeneration of neurons in the brain and spinal cord. Although a substantial number of studies have been conducted, much remains to be learned about the cellular mechanisms underlying ALS. In this study, we employed an engineered ascorbate peroxidase (APEX)-based proximity biotinylation, together with affinity pull-down of the ensuing biotinylated peptides, to investigate the proximity proteomes of human SOD1 and its two ALS-linked mutants, G85R and G93A. We were able to identify 25 common biotinylated peptides with preferential enrichment in the proximity proteomes of SOD1G85R and SOD1G93A over wild-type SOD1. Our coimmunoprecipitation followed by Western blot analyses revealed that one of these proteins, SRSF2, binds more strongly with the two SOD1 mutants than its wild-type counterpart. We also observed aberrant splicing of mRNAs in cells with ectopic expression of the two SOD1 mutants relative to cells expressing the wild-type protein. In addition, the aberrations in splicing elicited by the SOD1 variants were markedly attenuated upon knockdown of SRSF2. Collectively, we uncovered that ALS-liked SOD1G85R and SOD1G93A mutants interact more strongly with SRSF2, where the aberrant interactions perturbed mRNA splicing. Thus, our work offered novel mechanistic insights into the contributions of the ALS-linked SOD1 mutants to disease etiology.

Early Aggregation Mechanism of SOD128-38 Based on Force Field Parameter of 5-Cyano-Tryptophan.

The aggregation of superoxide dismutase 1 (SOD1) results in amyloid deposition and is involved in familial amyotrophic lateral sclerosis, a fatal motor neuron disease. There have been extensive studies of its aggregation mechanism. Noncanonical amino acid 5-cyano-tryptophan (5-CN-Trp), which has been incorporated into the amyloid segments of SOD1 as infrared probes to increase the structural sensitivity of IR spectroscopy, is found to accelerate the overall aggregation rate and potentially modulate the aggregation process. Despite these observations, the underlying mechanism remains elusive. Here, we optimized the force field parameters of 5-CN-Trp and then used molecular dynamics simulation along with the Markov state model on the SOD128-38 dimer to explore the kinetics of key intermediates in the presence and absence of 5-CN-Trp. Our findings indicate a significantly increased probability of protein aggregate formation in 5CN-Trp-modified ensembles compared to wildtype. Dimeric ß-sheets of different natures were observed exclusively in the 5CN-Trp-modified peptides, contrasting with wildtype simulations. Free-energy calculations and detailed analyses of the dimer structure revealed augmented interstrand interactions attributed to 5-CN-Trp, which contributed more to peptide affinity than any other residues. These results explored the key events critical for the early nucleation of amyloid-prone proteins and also shed light on the practice of using noncanonical derivatives to study the aggregation mechanism.

Versatile Fluorescence Lifetime-Based Copper Probe to Quantify Mitochondrial Membrane Potential and Reveal Its Interaction with Protein Aggregation.

Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.

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.

Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ µM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

Implications of ALS-Associated Mutations on Biochemical and Biophysical Features of hSOD1 and Aggregation Formation.

One of the recognized motor neuron degenerative disorders is amyotrophic lateral sclerosis (ALS). By now, several mutations have been reported and linked to ALS patients, some of which are induced by mutations in the human superoxide dismutase (hSOD1) gene. The ALS-provoking mutations are located throughout the structure of hSOD1 and promote the propensity to aggregate. Despite numerous investigations, the underlying mechanism related to the toxicity of mutant hSOD1 through the gain of a toxic function is still vague. We surveyed two mutant forms of hSOD1 by removing and adding cysteine at positions 146 and 72, respectively, to investigate the biochemical characterization and amyloid formation. Our findings predicted the harmful and destabilizing impact of two SOD1 mutants using multiple programs. The specific activity of the wild-type form was about 1.42- and 1.92-fold higher than that of C146R and G72C mutants, respectively. Comparative structural studies using CD spectropolarimetry, and intrinsic and ANS fluorescence showed alterations in secondary structure content, exposure of hydrophobic patches, and structural compactness of WT-hSOD1 vs. mutants. We demonstrated that two mutants were able to promote amyloid-like aggregates under amyloid induction circumstances (50-mM Tris-HCl pH 7.4, 0.2-M KSCN, 50-mM DTT, 37 °C, 190 rpm). Monitoring aggregates were done using an enhancement in thioflavin T fluorescence and alterations in Congo red absorption. The mutants accelerated fibrillation with subsequently greater fluorescence amplitude and a shorter lag time compared to WT-SOD1. These findings support the aggregation of ALS-associated SOD1 mutants as an integral part of ALS pathology.

In silico analysis of SOD1 aggregation inhibition modes of tertiary amine pyrazolone and pyrano coumarin ferulate as ALS drug candidates.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, the familial form (fALS) of which is often cognate to mutations in the antioxidant enzyme Cu/Zn superoxide dismutase 1 (SOD1) leading to misfolding and aggregation. Two small molecules, a tertiary amine pyrazolone (TAP) and a pyrano coumarin ferulate (PCF) were suggested to be ALS drug candidates following experimental observation of their ability to inhibit SOD1 protein misfolding and aggregation. The present work aims at computational investigation of these experimentally proposed drug candidates to gain insight into their mechanism of SOD1 misfolding and aggregation inhibition. On the basis of molecular docking, molecular dynamics simulation, MM-PBSA and per-residue energy decomposition analysis, we examined the specific interactions of TAP and PCF with three probable binding sites of SOD1, namely, dimeric interface cavity, W32 and, UMP binding sites. Results suggest that the binding of TAP at W32 and at UMP sites are least probable due to absence of any favorable interaction. The binding of TAP to dimeric cavity is also unstable due to strong unfavorable interactions. In case of PCF, binding at the UMP site is least probable while binding at dimeric cavity is accompanied by unfavorable interactions. PCF, however, exhibits stable binding with the W32 binding site of SOD1 by stabilizing the solvent accessible hydrophobic residues, which otherwise would have acted as contact points for aggregation. Thus the results imply that compound PCF functions as an inhibitior of SOD1 misfolding/aggregation through direct interaction with the protein SOD1 at the W32 binding site. However, TAP is likely to act as an inhibitor through a different mechanism rather than direct interaction with the protein SOD1. These results apart from reinforcing previous experimental findings, shed light on the probable mechanism of action of the proposed drug candidates.

Polyphenolic flavonoid compounds act as the inhibitory potential of aggregation process: Implications for the prevention and therapeutics against FALS-associated D101G SOD1 mutant.

Aggregation of proteins is a biological phenomenon caused by misfolded proteins. Human superoxide dismutase (hSOD1) misfolding and aggregation underlie the neurological illness amyotrophic lateral sclerosis (ALS). The most significant contributing factor to ALS is genetic point mutations in SOD1. particularly, D101G mutant is the most harmful because it significantly reduces the life expectancy of patients. Subsequently, the use of natural polyphenolic flavonoids is strongly recommended to reduce the amyloidogenic behavior of protopathic proteins. In this study, using computational parameters such as protein-ligand interaction and molecular dynamics (MD) simulation analyses, we are trying to identify a pharmacodynamically promising flavonoid compound that can effectively inhibit the pathogenic behavior of the D101G mutant. Epigallocatechin-gallate (EGCG), Hesperidin, Isorhamnetin, and Diosmetin were identified as potential leads in a preliminary screening of flavonoids to anti-amyloid action. The results of MD showed that the binding of flavonoids to D101G mutant caused changes in stability, hydrophobicity of protein, and flexibility, as well as significantly led to the restoration of lost hydrogen bonds. Secondary structure analysis showed that protein destabilization and the increased propensity of ß-sheet caused by the mutation were restored to the wild-type state upon binding of flavonoids. Besides, to differentiate aggregation, we elucidated alterations in the free energy landscape (FEL) and dynamic cross-correlation matrix (DCCM) of WT-SOD1 and mutant (unbound /bound) states. Among flavonoids, Epigallocatechin-gallate and Hesperidin had the most therapeutic efficacy against the D101G mutant. Therefore, Epigallocatechin-gallate and Hesperidin promise considerable therapeutic potential to develop highly effective inhibitors in reducing fatal and irreversible ALS.