Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species

Cu,Zn superoxide dismutase (SOD1) is a dimeric metal-binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to properties of the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are > 95% folded at neutral pH and 37 °C, A4V, L38V, G93A, and L106V ranged from 50% to ∼ 90% unfolded. The reduction of the disulfide bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range, where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the native dimeric state.     

Structural instability and Cu-dependent pro-oxidant activity acquired by the apo form of mutant SOD1 associated with amyotrophic lateral sclerosis

Cu,Zn-superoxide dismutase (SOD1) is a cytosolic antioxidant enzyme, and its mutation has been implicated in amyotrophic lateral sclerosis (ALS), a disease causing a progressive loss of motor neurons. Although the pathogenic mechanism of ALS remains unclear, it is hypothesized that some toxic properties acquired by mutant SOD1 play a role in the development of ALS. We have examined the structural and catalytic properties of an ALS-linked mutant of human SOD1, His43Arg (H43R), which is characterized by rapid disease progression. As revealed by circular dichroism spectroscopy, H43R assumes a stable β-barrel structure in the Cu(2+),Zn(2+)-bound holo form, but its metal-depleted apo form is highly unstable and readily unfolds or misfolds into an irregular structure at physiological temperature. The conformational change occurs as a two-state transition from a nativelike apo form to a denatured apo form with a half-life of ～0.5 h. At the same time as the denaturation, the apo form of H43R acquires pro-oxidant potential, which is fully expressed in the presence of Cu(2+) and H(2)O(2), as monitored with a fluorogenic probe for detecting pro-oxidant activity. Comparison of d-d absorption bands suggests that the Cu(2+) binding mode of the denatured apo form is different from that of the native holo form. The denatured apo form of H43R is likely to provide non-native Cu(2+) binding sites where the Cu(2+) ion is activated to catalyze harmful oxidation reactions. This study raises the possibility that the structural instability and the resultant Cu-dependent pro-oxidant activity of the apo form of mutant SOD1 may be one of the pathogenic mechanisms of ALS.     

Dimerization,Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.     

Molecular Chaperone Mediated Late-Stage Neuroprotection in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1) are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2), mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a) in vivo in motor neurons of SOD1^G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1^G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1^G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM) dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.     

Solvent sensitivity of protein aggregation in Cu,Zn superoxide dismutase: a molecular dynamics simulation study

Misfolding and aggregation of Cu, Zn Superoxide Dismutase (SOD1) is often found in amyotrophic lateral sclerosis (ALS) patients. The central apo SOD1 barrel was involved in protein maturation and pathological aggregation in ALS. In this work, we employed atomistic molecular dynamics (MD) simulations to study the conformational dynamics of SOD1^barrel monomer in different concentrations of trifluoroethanol (TFE). We find concentration dependence unusual structural and dynamical features, characterized by the local unfolding of SOD1^barrel. This partially unfolded structure is characterized by the exposure of hydrophobic core, is highly dynamic in nature, and is the precursor of aggregation seen in SOD1^barrel. Our computational studies supports the hypothesis of the formation of aggregation ‘building blocks’ by means of local unfolding of apo monomer as the mechanism of SOD1 fibrillar aggregation. The non-monotonic TFE concentration dependence of protein conformational changes was explored through simulation studies. Our results suggest that altered protein conformation and dynamics within its structure may underlie the aggregation of SOD1 in ALS.     

Genotype-Property Patient-Phenotype Relations Suggest that Proteome Exhaustion Can Cause Amyotrophic Lateral Sclerosis

Late-onset neurodegenerative diseases remain poorly understood as search continues for the perceived pathogenic protein species. Previously, variants in Superoxide Dismutase 1 (SOD1) causing Amyotrophic Lateral Sclerosis (ALS) were found to destabilize and reduce net charge, suggesting a pathogenic aggregation mechanism. This paper reports analysis of compiled patient data and experimental and computed protein properties for variants of human SOD1, a major risk factor of ALS. Both stability and reduced net charge correlate significantly with disease, with larger significance than previously observed. Using two independent methods and two data sets, a probability < 3% (t-statistical test) is found that ALS-causing mutations share average stability with all possible 2907 SOD1 mutations. Most importantly, un-weighted patient survival times correlate strongly with the misfolded/unfolded protein copy number, expressed as an exponential function of the experimental stabilities (R ² = 0.31, p = 0.002), and this phenotype is further aggravated by charge (R ² = 0.51, p = 1.8 x 10−5). This finding suggests that disease relates to the copy number of misfolded proteins. Exhaustion of motor neurons due to expensive protein turnover of misfolded protein copies is consistent with the data but can further explain e.g. the expression-dependence of SOD1 pathogenicity, the lack of identification of a molecular toxic mode, elevated SOD1 mRNA levels in sporadic ALS, bioenergetic effects and increased resting energy expenditure in ALS patients, genetic risk factors affecting RNA metabolism, and recent findings that a SOD1 mutant becomes toxic when proteasome activity is recovered after washout of a proteasome inhibitor. Proteome exhaustion is also consistent with energy-producing mitochondria accumulating at the neuromuscular junctions where ALS often initiates. If true, this exhaustion mechanism implies a complete change of focus in treatment of ALS towards actively nursing the energy state and protein turnover of the motor neurons.     

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

Using yeast models to probe the molecular basis of amyotrophic lateral sclerosis

ALS (amyotrophic lateral sclerosis) is a fatal neurodegenerative disease attributable to the death of motor neurons. Associated with ALS are mutations in the genes encoding SOD1 (superoxide dismutase 1), FUS (fused in Sarcoma) protein and TDP-43 (TAR DNA-binding protein-43) each of which leads to aggregation of the respective protein. For example, the ALS-associated mutations in the hSOD1 (human SOD1) gene typically destabilize the native SOD homodimer, leading to misfolding, aggregation and degradation of SOD1. The ALS-associated pathology is not a consequence of the functional inactivation of SOD1 itself, but is rather due to a toxic gain-of-function triggered by mutant SOD1. Recently, the molecular basis of a number of human neurodegenerative diseases resulting from protein misfolding and aggregation, including fALS (familial ALS), was probed by using the baker's yeast, Saccharomyces cerevisiae, as a highly tractable model. Such studies have, for example, identified novel mutant SOD1-specific interactions and demonstrated that mutant SOD1 disrupts mitochondrial homoeostasis. Features of ALS associated with TDP-43 aggregation have also been recapitulated in S. cerevisiae including the identification of modulators of the toxicity of TDP-43. In this paper, we review recent studies of ALS pathogenesis using S. cerevisiae as a model organism and summarize the potential mechanisms involved in ALS progression.     

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Background

Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.

Methodology/Principal Findings

As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn₂-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu²⁺ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu²⁺: a moderate-affinity site (10⁶ M^-1) and a high-affinity site (10⁸ M^-1). Furthermore, Cu²⁺ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.

Conclusions/Significance

We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu²⁺ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.     

Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo- and reduced form of SOD1, leading to unfolding and oxidative aggregation

Mutant forms of Cu,Zn-superoxide dismutase (SOD1) that cause familial amyotrophic lateral sclerosis (ALS) exhibit toxicity that promotes the death of motor neurons. Proposals for the toxic properties typically involve aberrant catalytic activities or protein aggregation. The striking thermodynamic stability of mature forms of the ALS mutant SOD1 (Tm>70 degrees C) is not typical of protein aggregation models that involve unfolding. Over 44 states of the polypeptide are possible, depending upon metal occupancy, disulfide status, and oligomeric state; however, it is not clear which forms might be responsible for toxicity. Recently the intramolecular disulfide has been shown to be required for SOD1 activity, leading us to examine these states of several disease-causing SOD1 mutants. We find that ALS mutations have the greatest effect on the most immature form of SOD1, destabilizing the metal-free and disulfide-reduced polypeptide to the point that it is unfolded at physiological temperatures (Tm<37 degrees C). We also find that immature states of ALS mutant (but not wild type) proteins readily form oligomers at physiological concentrations. Furthermore, these oligomers are more susceptible to mild oxidative stress, which promotes incorrect disulfide cross-links between conserved cysteines and drives aggregation. Thus it is the earliest disulfide-reduced polypeptides in the SOD1 assembly pathway that are most destabilized with respect to unfolding and oxidative aggregation by ALS-causing mutations.     

Rational discovery of a SOD1 tryptophan oxidation inhibitor with therapeutic potential for amyotrophic lateral sclerosis

Abstract

Formation of Cu, Zn superoxide dismutase 1 (SOD1) protein inclusions within motor neurons is one of the principal characteristics of SOD1-related amyotrophic lateral sclerosis (ALS). A hypothesis as to the nature of SOD1 aggregation implicates oxidative damage to a solvent-exposed tryptophan as causative. Here, we chart the discovery of a phenanthridinone based compound (Lig9) from the NCI Diversity Set III by rational methods by in silico screening and crystallographic validation. The crystal structure of the complex with SOD1, refined to 2.5 Å, revealed that Lig9 binds the SOD1 β-barrel in the β-strand 2 and 3 region which is known to scaffold SOD1 fibrillation. The phenanthridinone moiety makes a substantial π–π interaction with Trp32 of SOD1. The compound possesses a significant binding affinity for SOD1 and inhibits oxidation of Trp32; a critical residue for SOD1 aggregation. Thus, Lig9 is a good candidate from which to develop a new library of SOD1 aggregation inhibitors through protection of Trp32 oxidation.

Communicated by Ramaswamy H. Sarma     

SOD1 gene mutations in patients with amyotrophic lateral sclerosis: Potential of method of molecular modeling

Molecular modeling is a promising method for assessing protein structures that is capable of presenting an energetically beneficial protein conformation with atomic precision. This method is of great importance for studying molecular interactions and confirming the pathogenic significance of the changes in protein structures caused by particular mutations. In this study, we used molecular modeling to assess mutations in the SOD1 gene in patients with amyotrophic lateral sclerosis (ALS), a severe neurodegenerative disorder characterized by the loss of spinal and cerebral motor neurons. The product of SOD1 is a cytosolic dimeric enzyme Cu/Zn superoxide dismutase (SOD1) responsible for the detoxification of cellular superoxide radicals. We showed that all eight revealed coding-point mutations of the gene led to moderate or significant changes in SOD1 protein energy. The mutation His49Arg increased protein energy, and the reconstruction of the respective model indicated the spatial destabilization of the molecule and abnormal interactions with the metal ion inside the active center. Conversely, the other seven mutations (Gly17Ala, Leu85Val, Asn87Ser, Asp91Ala, Ser106Leu, Glu134Gly, and Leu145Phe) led to a decrease in protein energy and an increase in the spatial stability of SOD 1, which is usually accompanied by an increased tendency for the inert mutant molecule to misfold and demonstrate cellular aggregation. Therefore, the results of the in silico analysis of the SOD1 gene mutations confirms that ALS belongs to the class of the so-called conformational diseases of the central nervous system, a characteristic feature of which is the formation of cytotoxic, insoluble protein inclusions in neurons.     

Exposure of Hydrophobic Surfaces Initiates Aggregation of Diverse ALS-Causing Superoxide Dismutase-1 Mutants

The copper-zinc superoxide dismutase-1 (SOD1) is a highly structured protein and, a priori, one of the least likely proteins to be involved in a misfolding disease. However, more than 140, mostly missense, mutations in the SOD1 gene cause aggregation of the affected protein in familial forms of amyotrophic lateral sclerosis (ALS). The remarkable diversity of the effects of these mutations on SOD1 properties has suggested that they promote aggregation by a variety of mechanisms. Experimental assessment of surface hydrophobicity using a sensitive fluorescent-based assay, revealed that diverse ALS-causing mutations provoke SOD1 aggregation by increasing their propensity to expose hydrophobic surfaces. These findings could not be anticipated from analysis of the amino acid sequence. Our results uncover the biochemical nature of the misfolded aggregation-prone intermediate and reconcile the seemingly diverse effects of ALS-causing mutations into a unifying mechanism. Furthermore, the method we describe here will be useful for investigating and interfering with aggregation of various proteins and thereby provide insight into the molecular mechanisms underlying many neurodegenerative diseases.     

Destabilizing Protein Polymorphisms in the Genetic Background Direct Phenotypic Expression of Mutant SOD1 Toxicity

Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS) cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles.     

Prion-like activity of Cu/Zn superoxide dismutase

Neurodegenerative diseases belong to a larger group of protein misfolding disorders, known as proteinopathies. There is increasing experimental evidence implicating prion-like mechanisms in many common neurodegenerative disorders, including Alzheimer disease, Parkinson disease, the tauopathies, and amyotrophic lateral sclerosis (ALS), all of which feature the aberrant misfolding and aggregation of specific proteins. The prion paradigm provides a mechanism by which a mutant or wild-type protein can dominate pathogenesis through the initiation of self-propagating protein misfolding. ALS, a lethal disease characterized by progressive degeneration of motor neurons is understood as a classical proteinopathy; the disease is typified by the formation of inclusions consisting of aggregated protein within and around motor neurons that can contribute to neurotoxicity. It is well established that misfolded/oxidized SOD1 protein is highly toxic to motor neurons and plays a prominent role in the pathology of ALS. Recent work has identified propagated protein misfolding properties in both mutant and wild-type SOD1, which may provide the molecular basis for the clinically observed contiguous spread of the disease through the neuroaxis. In this review we examine the current state of knowledge regarding the prion-like properties of SOD1 and comment on its proposed mechanisms of intercellular transmission.     

Bicarbonate-dependent peroxidase activity of human Cu,Zn-superoxide dismutase induces covalent aggregation of protein: intermediacy of tryptophan-derived oxidation products

This study addresses the mechanism of covalent aggregation of human Cu,Zn-superoxide dismutase (hSOD1WT) induced by bicarbonate (HCO3-)-mediated peroxidase activity. Higher molecular weight species (apparent dimers and trimers) of hSOD1WT were formed from incubation mixtures containing hSOD1WT, H2O2, and HCO3-. HCO3--dependent peroxidase activity and covalent aggregation of hSOD1WT were mimicked by UV photolysis of hSOD1-WT in the presence of a [Co(NH3)5CO3]+ complex that generates the carbonate radical anion (CO3.). Human SOD1WT has but one aromatic residue, a tryptophan residue (Trp-32) on the surface of the protein. Substitution of Trp-32 with phenylalanine produced a mutant (hSOD1W32F) that exhibits HCO3--dependent peroxidase activity similar to wild-type enzyme. However, unlike hSOD1WT, incubations containing hSOD1W32F,H2O2, and HCO3-did not result in covalent aggregation of SOD1. These findings indicate that Trp-32 is crucial for CO3.-induced covalent aggregation of hSOD1WT. Spin-trapping results revealed the formation of the Trp-32 radical from hSOD1WT, but not from hSOD1W32F. Spin traps also inhibited the covalent aggregation of hSOD1WT. Fluorescence experiments revealed that Trp-32 was further oxidized by CO3., forming kynurenine-type products in the presence of oxygen. Molecular oxygen was needed for HCO3-/H2O2-dependent aggregation of hSOD1WT, implicating a role for a Trp-32-dependent peroxidative reaction in the covalent aggregation of hSOD1WT. Taken together, these results indicate that Trp-32 oxidation is crucial for covalent aggregation of hSOD1. Implications of HCO3--dependent SOD1 peroxidase activity in amyotrophic lateral sclerosis disease are discussed.     

Conformational transitions induced by in vitro macromolecular crowding lead to the amyloidogenesis of buffalo heart cystatin

The biological cells and extracellular matrix exhibit a highly crowded environment, called as macromolecular crowding. Crowding significantly influences protein structure and may lead to its aggregation. In the present study, buffalo heart cystatin (BHC), after purification from buffalo heart tissue, has been used as a model protein for studying effect of macromolecular crowding in the presence of high concentrations of bovine serum albumin (BSA), poly‐ethylene glycol‐1000 (PEG‐1000), and poly‐ethylene glycol‐4000 (PEG‐4000). Cystatins are thiol protease inhibitors and found to be involved in various important physiological processes. Functional inactivation of BHC was observed upon crowding, which varied as a function of concentration and molecular weight of crowding agents as well as incubation time. Structural changes of BHC at tertiary and secondary level were detected with the help of fluorescence and CD spectroscopy. CD analysis showed changes of α‐helix to β‐sheet, which could be due to aggregation. The ANS‐fluorescence study suggested the unfolding and presence of some partially folded intermediates. Increase in ThT‐fluorescence and absorption of Congo red spectra with red shift, confirmed the amyloid type aggregation of BHC in the presence of various crowding agents. Finally, electron microscopy provided the physical evidence about the formation of amyloid fibrils. Results suggested that among the various crowding agents used, amyloidogenesis of BHC was maximal in case of BSA followed by PEG‐4000 and least for PEG‐1000. The present work makes an important contribution in crowding mediated protein aggregation, which can have implications of potential interest. Copyright © 2015 John Wiley & Sons, Ltd.     

Cysteine to Serine Conversion at 111th Position Renders the Disaggregation and Retains the Stabilization of Detrimental SOD1 A4V Mutant Against Amyotrophic Lateral Sclerosis in Human—A Discrete Molecular Dynamics Study

Protein aggregation is a hallmark of various neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS) in humans. Mutations in Cu/Zn superoxide dismutase (SOD1) protein were found to be a prominent cause behind the majority of the familial ALS cases with abnormal protein aggregates. Herein, we report the biophysical characterization of the beneficial mutation C111S that stabilizes the SOD1 harboring A4V mutation, one of the most lethal diseases causing mutant that leads to protein destabilization and aggregation. In this study, we utilized discrete molecular dynamics (DMD) simulations, which stipulated an outlook over the systematic action of C111S mutation in the A4V mutant that stabilizes the protein and impedes the formation of protein aggregation. Herewith, the findings from our study manifested that the mutation of C111S in SOD1 could aid in regaining the protein structural conformations that protect against the formation of toxic aggregates, thereby hindering the disease pathogenicity subtly. Hence, our study provides a feasible pharmaceutical strategy in developing the treatment for incurable ALS affecting the mankind.     

Conformational change of a G-quadruplex under molecular crowding conditions

Abstract

This study examined the influence of the molecular crowding condition induced by polyethylene glycol (PEG) on the G-quadruplex structure of the thrombin-binding aptamer sequence, 5′-GGGTTGGGTGTGGGTTGGG (G3), in a solution containing a sufficient concentration of mono cations (K⁺ and Na⁺). Although the G3 sequence preferably formed the antiparallel type G-quadruplex structure in a Na⁺ solution, conversion to the parallel type occurred when PEG was added. The antiparallel type was maintained at low PEG concentrations. When the PEG concentration reached 30%, the antiparallel type and parallel type coexist. At PEG concentrations above 40%, the G-quadruplex structure adopted the parallel type completely. In the presence of K⁺ ions, G3 showed a parallel conformation and remained as a parallel conformation with increasing PEG concentration. The dissociation temperature increased with increasing PEG concentration in all cases, suggesting that the G-quadruplex conformation is more stable under molecular crowding conditions.

Communicated by Ramaswamy H. Sarma