Phosphorylated TAR DNA-binding protein-43: Aggregation and antibody-based inhibition.

TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of ß-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 µg/mL: 45 µg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition.

Superoxide dismutase 1 (SOD1) and cadmium: A three models approach to the comprehension of its neurotoxic effects.

Cadmium (Cd) is a widespread toxic environmental contaminant, released by anthropogenic activities. It interferes with essential metal ions homeostasis and affects protein structures and functions by substituting zinc, copper and iron. In this study, the effect of cadmium on SOD1, a CuZn metalloenzyme catalyzing superoxide conversion into hydrogen peroxide, has been investigated in three different biological models. We first evaluated the effects of cadmium combined with copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21. The enzyme activity and expression were investigated in the presence of fixed copper and/or zinc doses with different cadmium concentrations, in the cellular medium. Cadmium caused a dose-dependent reduction in SOD1 activity, while the expression remains constant. Similar results were obtained in the cellular model represented by the human SH-SY5Y neuronal cell line. After cadmium treatment for 24 and 48 h, SOD1 enzymatic activity decreased in a dose- and time-dependent way, while the protein expression remained constant. Finally, a 16 h cadmium treatment caused a 25 % reduction of CuZn-SOD activity without affecting the protein expression in the Caenorhabditis elegans model. Taken together our results show an inhibitory effect of cadmium on SOD1 enzymatic activity, without affecting the protein expression, in all the biological models used, suggesting that cadmium can displace zinc from the enzyme catalytic site.

The Dual Effects of Reactive Oxygen Species on the Mandibular Alveolar Bone Formation in SOD1 Knockout Mice: Promotion or Inhibition.

The status of reactive oxygen species (ROS) correlates closely with the normal development of the oral and maxillofacial tissues. Oxidative stress caused by ROS accumulation not only affects the development of enamel and dentin but also causes pathological changes in periodontal tissues (periodontal ligament and alveolar bone) that surround the root of the tooth. Although previous studies have shown that ROS accumulation plays a pathologic role in some oral and maxillofacial tissues, the effects of ROS on alveolar bone development remain unclear. In this study, we focused on mandibular alveolar bone development of mice deficient in superoxide dismutase1 (SOD1). Analyses were performed using microcomputerized tomography (micro-CT), TRAP staining, immunohistochemical (IHC) staining, and enzyme-linked immunosorbent assay (ELISA). We found for the first time that slightly higher ROS in mandibular alveolar bone of SOD1(-/-) mice at early ages (2-4 months) caused a distinct enlargement in bone size and increased bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2), and osteopontin (OPN). With ROS accumulation to oxidative stress level, increased trabecular bone separation (Tb.Sp) and decreased expression of ALP, Runx2, and OPN were found in SOD1(-/-) mice at 6 months. Additionally, dosing with N-acetylcysteine (NAC) effectively mitigated bone loss and normalized expression of ALP, Runx2, and OPN. These results indicate that redox imbalance caused by SOD1 deficiency has dual effects (promotion or inhibition) on mandibular alveolar bone development, which is closely related to the concentration of ROS and the stage of growth. We present a valuable model here for investigating the effects of ROS on mandibular alveolar bone formation and highlight important roles of ROS in regulating tissue development and pathological states, illustrating the complexity of the redox signal.

Human superoxide dismutase 1 attenuates quinoneimine metabolite formation from mefenamic acid.

Mefenamic acid (MFA), one of the nonsteroidal anti-inflammatory drugs (NSAIDs), sometimes causes liver injury. Quinoneimines formed by cytochrome P450 (CYP)-mediated oxidation of MFA are considered to be causal metabolites of the toxicity and are detoxified by glutathione conjugation. A previous study reported that NAD(P)H:quinone oxidoreductase 1 (NQO1) can reduce the quinoneimines, but NQO1 is scarcely expressed in the human liver. The purpose is to identify enzyme(s) responsible for the decrease in MFA-quinoneimine formation in the human liver. The formation of MFA-quinoneimine by recombinant CYP1A2 and CYP2C9 was significantly decreased by the addition of human liver cytosol, and the extent of the decrease in the metabolite formed by CYP1A2 was larger than that by CYP2C9. By column chromatography, superoxide dismutase 1 (SOD1) was identified from the human liver cytosol as an enzyme decreasing MFA-quinoneimine formation. Addition of recombinant SOD1 into the reaction mixture decreased the formation of MFA-quinoneimine from MFA by recombinant CYP1A2. By a structure-activity relationship study, we found that SOD1 decreased the formation of quinoneimines from flufenamic acid and tolfenamic acid, but did not affect those produced from acetaminophen, amodiaquine, diclofenac, and lapatinib. Thus, SOD1 may selectively decrease the quinoneimine formation from fenamate-class NSAIDs. To examine whether SOD1 can attenuate cytotoxicity caused by MFA, siRNA for SOD1 was transfected into CYP1A2-overexpressed HepG2 cells. The leakage of lactate dehydrogenase caused by MFA treatment was significantly increased by knockdown of SOD1. In conclusion, we found that SOD1 can serve as a detoxification enzyme for quinoneimines to protect from drug-induced toxicity.

A Cerium Vanadate Nanozyme with Specific Superoxide Dismutase Activity Regulates Mitochondrial Function and ATP Synthesis in Neuronal Cells.

Nanoparticles that functionally mimic the activity of metal-containing enzymes (metallo-nanozymes) are of therapeutic importance for treating various diseases. However, it is still not clear whether such nanozymes can completely substitute the function of natural enzymes in living cells. In this work, we show for the first time that a cerium vanadate (CeVO4 ) nanozyme can substitute the function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the neuronal cells even when the natural enzyme is down-regulated by specific gene silencing. The nanozyme prevents the mitochondrial damage in SOD1- and SOD2-depleted cells by regulating the superoxide levels and restores the physiological levels of the anti-apoptotic Bcl-2 family proteins. Furthermore, the nanozyme effectively prevents the mitochondrial depolarization, leading to a significant improvement in the cellular levels of ATP under oxidative stress.

Inhibition of TXNRD or SOD1 overcomes NRF2-mediated resistance to ß-lapachone.

Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent ß-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to ß-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of ß-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to ß-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to ß-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to ß-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to ß-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations.

Cytotoxic Activity of LCS-1 is not Only due to Inhibition of SOD1.

BACKGROUND: The cytotoxic activity of the pyridazin-3-one derivative LCS-1 was previously suggested to be due to the inhibition of superoxide dismutase 1 (SOD1). However, no direct evidence was provided that LCS-1 inhibits SOD1 within cells. METHODS: In this study, we investigated the cytotoxic activity of LCS-1 against bloodstream forms of Trypanosoma brucei, a protozoan parasite that does not express copper/zinc-containing SOD1, but an iron-containing superoxide dismutase (FeSOD). RESULTS: At 250 µM, LCS-1 did not inhibit the activity of FeSOD in cell lysates of bloodstream forms of T. brucei, confirming that the compound is a specific inhibitor of SOD1. However, LCS-1 displayed substantial trypanocidal activity with a minimum inhibitory concentration of 10 µM and a half-maximal effective concentration of 1.36 µM, indicating that the cytotoxic action of the compound cannot solely be due to inhibition of SOD1. CONCLUSION: The results of this study is an important finding as it shows that LCS-1 has more than one cytotoxic mode of action.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing Bcl2-like 12 [BCL2L12] and superoxide dismutase 1 [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA. Toward this goal, we identified an aliphatic lipid-grafted low-molecular weight polyethylenimine (PEI) that accommodated both pDNA and siRNA in a single complex. The co-delivery of pTRAIL with BCL2L12- or SOD1-specific siRNAs resulted more significant cell death in different breast cancer cells compared with separate delivery without affecting nonmalignant cells viability. Ternary complexes of lipopolymer with pTRAIL and BCL2L12 siRNA significantly retarded the growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. The lipid-grafted PEIs capable of co-delivering multiple types of nucleic acids can serve as powerful carriers for more effective complementary therapeutics. Graphical Abstract [Figure: see text].

PM2.5 inhibits SOD1 expression by up-regulating microRNA-206 and promotes ROS accumulation and disease progression in asthmatic mice.

Bronchial asthma is the most common chronic respiratory disease. Chronic airway inflammation, airflow restriction and airway hyper-responsiveness are its main manifestations. In recent decades, the prevalence and mortality of asthma have been increasing all over the world, which seriously threatens public health. Research suggests that air pollution is associated with the increased incidence of asthma. PM2.5 is one of the most complex pollutants in the atmospheric environment and harmful to human health. It is related to the incidence of asthma. However, the molecular mechanism of PM2.5 in the development of asthma is still unclear. In this study, we established a mouse model of asthma using CRE to observe the effect of PM2.5 on the symptoms of asthmatic mice and its possible molecular mechanism. The results showed that PM2.5 could significantly increase airway resistance and pulmonary inflammation, increase the number of inflammatory cells, eosinophils, macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid in asthmatic mice. Moreover, PM2.5 could reduce the contents of antioxidant enzymes such as CAT, GSH, GSH-Px and T-SOD in lung tissue of mice, and increase the ROS level. PM2.5 can promote the expression of microRNA-206 in lung tissue of mice. miR-206 can target the 3'-UTR of SOD1 to inhibit SOD1 expression, which leads to the increase of ROS level and aggravates pulmonary inflammatory response and asthma symptoms in asthmatic mice. This study found the possible molecular mechanism of PM2.5 aggravating asthma, and miR-206 may be a potential target for asthma treatment.

p-Coumaric Acid Has Protective Effects against Mutant Copper-Zinc Superoxide Dismutase 1 via the Activation of Autophagy in N2a Cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons. In previous our study, an ethanol extract of Brazilian green propolis (EBGP) prevented mutant copper-zinc superoxide dismutase 1 (SOD1mut)-induced neurotoxicity. This paper aims to reveal the effects of p-coumaric acid (p-CA), an active ingredient contained in EBGP, against SOD1mut-induced neurotoxicity. We found that p-CA reduced the accumulation of SOD1mut subcellular aggregation and prevented SOD1mut-associated neurotoxicity. Moreover, p-CA attenuated SOD1mut-induced oxidative stress and endoplasmic reticulum stress, which are significant features in ALS pathology. To examine the mechanism of neuroprotective effects, we focused on autophagy, and we found that p-CA induced autophagy. Additionally, the neuroprotective effects of p-CA were inhibited by chloroquine, an autophagy inhibiter. Therefore, these results obtained in this paper suggest that p-CA prevents SOD1mut-induced neurotoxicity through the activation of autophagy and provides a potential therapeutic approach for ALS.

Virtual screening identification of novel chemical inhibitors for aberrant interactions between pathogenic mutant SOD1 and tubulin.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease caused by selective motor neuron death. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) belong to one of the four major mutation clusters responsible for pathogenesis of ALS. Toxic gain-of-function (not loss-of-function) of SOD1 mutants causes motor neuron degeneration. Aberrant protein-protein interactions (PPI) between mutant SOD1 and other proteins are involved in this toxic gain-of-function. Therefore, PPI inhibitors of mutant SOD1 not only increase understanding of ALS pathogenesis, but can also be used as novel therapeutics for ALS. Although it is challenging to identify PPI inhibitors, prior knowledge of the binding site can increase success probability. We have previously reported that tubulin interacts with N-terminal residues 1-23 of mutant SOD1. In the present study, we performed virtual screening by docking simulation of 32,791 compounds using this N-terminal binding site as prior knowledge. An established assay system for interaction inhibition between mutant SOD1-tubulin was used as an in-house model system to identify mutant SOD1 PPI inhibitors, with the goal of developing novel therapeutics for ALS. Consequently, five of six assay-executable compounds among top-ranked compounds during docking simulation inhibited the mutant SOD1-tubulin interaction in vitro. Binding mode analysis predicted that some inhibitors might bind the tubulin binding site of G85R SOD1 by pi electron interaction with the aromatic ring of the Trp32 residue of G85R SOD1. Our screening methods may contribute to the identification of lead compounds for treating ALS.

The Specific Inhibition of SOD1 Selectively Promotes Apoptosis of Cancer Cells via Regulation of the ROS Signaling Network.

Multiple signaling pathways including ERK, PI3K-Akt, and NF-κB, which are essential for onset and development of cancer, can be activated by intracellularly sustained high levels of H2O2 provided by elevated activity and expression of copper/zinc superoxide dismutase (SOD1) that catalyzes the dismutation of O2 •- into H2O2. Here, tests performed by the utilization of our designed specific SOD1 inhibitor LD100 on cancer and normal cells reveal that the signaling pathways and their crosstalk to support cancer cell growth are repressed, but the signaling pathways to promote cancer cell cycle arrest and apoptosis are stimulated by specific SOD1 inhibition-mediated ROS changes. These regulated pathways constitute an ROS signaling network that determines the fate of cancer cells. This ROS signaling network is also regulated in SOD1 knockdown cells. These findings might facilitate disclosure of action mechanisms by copper-chelating anticancer agents and design of SOD1-targeting and ROS signaling pathway-interfering anticancer small molecules.

Cadmium effects on superoxide dismutase 1 in human cells revealed by NMR.

Cadmium is a toxic pollutant that in recent decades has become more widespread in the environment due to anthropogenic activities, significantly increasing the risk of exposure. Concurrently, a continually growing body of research has begun to enumerate the harmful effects that this heavy metal has on human health. Consequently, additional research is required to better understand the mechanism and effects of cadmium at the molecular level. The main mechanism of cadmium toxicity is based on the indirect induction of severe oxidative stress, through several processes that unbalance the anti-oxidant cellular defence system, including the displacement of metals such as zinc from its native binding sites. Such mechanism was thought to alter the in vivo enzymatic activity of SOD1, one of the main antioxidant proteins of many tissues, including the central nervous system. SOD1 misfolding and aggregation is correlated with cytotoxicity in neurodegenerative diseases such as amyotrophic lateral sclerosis. We assessed the effect of cadmium on SOD1 folding and maturation pathway directly in human cells through in-cell NMR. Cadmium does not directly bind intracellular SOD1, instead causes the formation of its intramolecular disulfide bond in the zinc-bound form. Metallothionein overexpression is strongly induced by cadmium, reaching NMR-detectable levels. The intracellular availability of zinc modulates both SOD1 oxidation and metallothionein overexpression, strengthening the notion that zinc-loaded metallothioneins help maintaining the redox balance under cadmium-induced acute stress.

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

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 ß-barrel in the ß-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.

Shenxian-Shengmai Oral Liquid Reduces Myocardial Oxidative Stress and Protects Myocardium from Ischemia-Reperfusion Injury.

BACKGROUND/AIMS: Shenxian-shengmai (SXSM) oral liquid, a Chinese patent compound medicine, has been used to treat sinus bradyarrhythmias induced by mild sick sinus syndrome in clinical practice. Myocardial ischemia, in particular in serious or right coronary-related heart diseases, can cause bradyarrhythmias and cardiac dysfunction. Moreover, reperfusion of ischemic myocardium is associated with additional myocardial damage known as myocardial ischemia-reperfusion (I/R) injury. This study was designed to evaluate the effects of SXSM on bradyarrhythmias and cardiac dysfunction induced by myocardial I/R injury, and to explore the underlying mechanisms. METHODS: Administration of SXSM to adult male Sprague Dawley (SD) rats was achieved orally by gavage and control rats were given equivalent deionized water every day for 14 days. After the last administration, the heart was connected with the Langendorff perfusion apparatus and both groups were subjected to ischemia for 20 min followed by reperfusion for 40 min to induce myocardial I/R injury. Heart rate (HR), left ventricular developed pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax) were recorded by a physiological signal acquisition system. The heart treated with ischemic preconditioning (IPC) for 3 times at a range of 5 min/time before ischemia served as a positive control group. The hearts without I/R injury served as control group. After reperfusion, superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in the myocardium were determined by appropriate assay kits. Myocardial SOD1 and glutamate cysteine ligase catalytic subunit (GCLC) expression were assessed by western blot analysis. For the in vitro study, SXSM serum was prepared according to the serum pharmacological method and neonatal rat cardiomyocytes were isolated from the heart of new born SD rats. Neonatal rat cardiomyocytes were pretreated with SXSM serum and subjected to H2O2 or anoxia/ reoxygenation (A/R) treatment to induce oxidative damage. Cell viability was evaluated using a Cell Counting Kit-8 (CCK8) assay. Levels of reactive oxygen species (ROS), SOD, GSH and GSH-Px in cardiomyocytes were determined by appropriate assay kits. SOD1 and GCLC expression were assessed by western blot analysis. Buthionine-[S, R]-sulfoximine (BSO), a GCLC inhibitor, and SOD1 siRNA were also used for identifying the cardiac protective targets of SXSM. RESULTS: SXSM and ischemic preconditioning (IPC) significantly increased heart rate during myocardial reperfusion and protected cardiac function against myocardial I/R injury, including an increase in left ventricular diastolic pressure (LVDP), the maximal increase rate of left ventricular pressure (+dp/dtmax) and the maximal decrease rate of left ventricular pressure (-dp/dtmax). We also found that SXSM and IPC improved the expansion of myocardial interstitium, the structural abnormality and morphological changes of cardiomyocytes induced by I/R injury. Meanwhile, SXSM protected cardiomyocytes against the oxidative damage induced by H2O2 and A/R injury through reducing intracellular ROS levels. Moreover, SXSM increased SOD activity through enhancing SOD1 expression and increased GSH content through promoting GCLC expression as well as GSH-Px activity. BSO and SOD1 siRNA counteracted anti-arrhythmic and cardiac protective effect of SXSM, suggesting that the therapeutic targets of SXSM might be SOD1 and GCLC. CONCLUSION: SXSM is effective in protecting the myocardium from I/R injury, with myocardial SOD1 and GCLC being the potential therapeutic targets.

Targeting of Disordered Proteins by Small Molecules in Neurodegenerative Diseases.

The formation of protein aggregates and inclusions in the brain and spinal cord is a common neuropathological feature of a number of neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and many others. These are commonly referred as neurodegenerative proteinopathies or protein-misfolding diseases. The main characteristic of protein aggregates in these disorders is the fact that they are enriched in amyloid fibrils. Since protein aggregation is considered to play a central role for the onset of neurodegenerative proteinopathies, research is ongoing to develop strategies aimed at preventing or removing protein aggregation in the brain of affected patients. Numerous studies have shown that small molecule-based approaches may be potentially the most promising for halting protein aggregation in neurodegenerative diseases. Indeed, several of these compounds have been found to interact with intrinsically disordered proteins and promote their clearing in experimental models. This notwithstanding, at present small molecule inhibitors still awaits achievements for clinical translation. Hopefully, if we determine whether the formation of insoluble inclusions is effectively neurotoxic and find a valid biomarker to assess their protein aggregation-inhibitory activity in the human central nervous system, the use of small molecule inhibitors will be considered as a cure for neurodegenerative protein-misfolding diseases.

Regression model for predicting pathogenic properties of SOD1 mutants based on the analysis of conformational stability and conservation of hydrogen bonds.

Intracellular aggregation of proteins is thought to be involved in the aetiology of various neurodegenerative diseases. In particular, mutations in the SOD1 gene are linked to the familial form of amyotrophic lateral sclerosis (ALS). Recently, we developed a regression model for estimating the survival time of ALS patients carrying mutations in SOD1. This model was built based on an analysis of the stability of hydrogen bonds formed in SOD1 mutant proteins during a molecular dynamics (MD) simulation. In the present paper, the regression model was improved by taking into account a new hydrogen-bond property that reflects the conservation measure of a hydrogen bond in the space of protein conformational states. Conformational conservation of hydrogen bonds, being obtained with elastic network (EN) models, allowed us to find eight hydrogen bonds that might affect the pathogenic SOD1 mutants' properties in addition to the bonds that were found via MD in our previous work. The correlation coefficient between survival time of patients with ALS-linked mutations in SOD1 predicted within the improved model and that observed in the literature was 0.91. SOD1 amino acid residues forming these pathogenic hydrogen bonds are found in zinc-binding and electrostatic loops as well as at zinc-binding sites and are in contact with SOD1 aggregates, which implies that these regions are sensitive to perturbations from pathogenic mutations.

A computational combinatorial approach identifies a protein inhibitor of superoxide dismutase 1 misfolding, aggregation, and cytotoxicity.

Molecular agents that specifically bind and neutralize misfolded and toxic superoxide dismutase 1 (SOD1) mutant proteins may find application in attenuating the disease progression of familial amyotrophic lateral sclerosis. However, high structural similarities between the wild-type and mutant SOD1 proteins limit the utility of this approach. Here we addressed this challenge by converting a promiscuous natural human IgG-binding domain, the hyperthermophilic variant of protein G (HTB1), into a highly specific aggregation inhibitor (designated HTB1M) of two familial amyotrophic lateral sclerosis-linked SOD1 mutants, SOD1G93A and SOD1G85R We utilized a computational algorithm for mapping protein surfaces predisposed to HTB1 intermolecular interactions to construct a focused HTB1 library, complemented with an experimental platform based on yeast surface display for affinity and specificity screening. HTB1M displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation in vitro, prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1G93A expressed in motor neuron-like cells. Competition assays and molecular docking simulations suggested that HTB1M binds to SOD1 via both its α-helical and ß-sheet domains at the native dimer interface that becomes exposed upon mutated SOD1 misfolding and monomerization. Our results demonstrate the utility of computational mapping of the protein-protein interaction potential for designing focused protein libraries to be used in directed evolution. They also provide new insight into the mechanism of conversion of broad-spectrum immunoglobulin-binding proteins, such as HTB1, into target-specific proteins, thereby paving the way for the development of new selective drugs targeting the amyloidogenic proteins implicated in a variety of human diseases.

Probing the inhibitory activity of epigallocatechin-gallate on toxic aggregates of mutant (L84F) SOD1 protein through geometry based sampling and steered molecular dynamics.

Amyloid formation and protein aggregation are considered to be at the core of the disease pathology for the various neurodegenerative disorders such as Amyotrophic lateral sclerosis (ALS). Considerable experimental reports have suggested that epigallocatechin-gallate (EGCG), a natural polyphenol from the green tea inhibits the amyloid formation in multiple neurodegenerative disease. Mutations in SOD1 protein are considered to a key factor that contributes towards the rapid disease progression and the pathogenesis in both, the sporadic and familial form. In our study, we computationally examined the inhibitory action of EGCG against the native and the mutant SOD1 through molecular docking, steered molecular dynamics and conformational sampling methods From the outcome, we could conjecture that the protein destabilization and increased ß-sheet propensity that occurred due to mutation were regained upon the binding of EGCG. Moreover, the concepts of the free energy landscape analysis are introduced to establish the visual appearance of protein aggregation upon mutation. Altogether, we come to know that the binding of EGCG on mutant SOD1 has reduced the formation of the toxic aggregates upon mutation. Hence, our study could be an initiative in deciphering the inhibitory action of EGCG against the aggregated mutant SOD1, which could be a therapeutic potential against the treatment for the incurable neurodegenerative disorder (ALS) affecting the mankind.

The synthetic lethal killing of RAD54B-deficient colorectal cancer cells by PARP1 inhibition is enhanced with SOD1 inhibition.

Colorectal cancer (CRC) is a leading cause of cancer-related death throughout the world. Despite improved screening efforts, most CRCs are diagnosed at late stages when surgery alone is not curative. Moreover, the low 5-year survival rate (~8-13%) for those living with stage IV CRC highlights the need for better treatment options. Many current chemotherapeutic approaches are non-specific and associated with side effects due to their tendency to target both normal and cancer cells. To address this issue, synthetic lethal (SL) approaches are now being explored in cancer and are defined as the lethal combination of two independently viable mutations/deletions. From a therapeutic perspective, SL interactors of genes mutated in cancer serve as candidate drug targets. The present study focuses on RAD54B, a gene that is aberrantly expressed in many cancer types, including CRC. We show that PARP1 silencing or inhibition (BMN673 or Olaparib) leads to selective killing within RAD54B-deficient cells relative to controls, and is accompanied by increases in γ-H2AX (a surrogate marker of DNA double strand breaks) and cleaved Caspase-3 (an apoptotic indicator). We further show that BMN673 synergizes with LCS-1 (an inhibitor of an established RAD54B SL interactor) to induce enhanced killing in RAD54B-deficient cells. Collectively, these data identify RAD54B and PARP1 as SL interactors, and thus reveal PARP1 as a novel candidate drug target in RAD54B-deficient CRCs. These findings further show that combinatorial chemotherapies involving multiple SL targets may promote synergistic killing within cancer cells, a strategy that may hold potential in many cancer contexts.