Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-21 screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.

Regulating Phase Transition in Neurodegenerative Diseases by Nuclear Import Receptors.

RNA-binding proteins (RBPs) with a low-complexity prion-like domain (PLD) can undergo aberrant phase transitions and have been implicated in neurodegenerative diseases such as ALS and FTD. Several nuclear RBPs mislocalize to cytoplasmic inclusions in disease conditions. Impairment in nucleocytoplasmic transport is another major event observed in ageing and in neurodegenerative disorders. Nuclear import receptors (NIRs) regulate the nucleocytoplasmic transport of different RBPs bearing a nuclear localization signal by restoring their nuclear localization. NIRs can also specifically dissolve or prevent the aggregation and liquid-liquid phase separation of wild-type or disease-linked mutant RBPs, due to their chaperoning activity. This review focuses on the LLPS of intrinsically disordered proteins and the role of NIRs in regulating LLPS in neurodegeneration. This review also discusses the implication of NIRs as therapeutic agents in neurogenerative diseases.

Poly(ADP-ribose) promotes toxicity of C9ORF72 arginine-rich dipeptide repeat proteins.

Arginine-rich dipeptide repeat proteins (R-DPRs), abnormal translational products of a GGGGCC hexanucleotide repeat expansion in C9ORF72, play a critical role in C9ORF72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the most common genetic form of the disorders (c9ALS/FTD). R-DPRs form liquid condensates in vitro, induce stress granule formation in cultured cells, aggregate, and sometimes coaggregate with TDP-43 in postmortem tissue from patients with c9ALS/FTD. However, how these processes are regulated is unclear. Here, we show that loss of poly(ADP-ribose) (PAR) suppresses neurodegeneration in c9ALS/FTD fly models and neurons differentiated from patient-derived induced pluripotent stem cells. Mechanistically, PAR induces R-DPR condensation and promotes R-DPR-induced stress granule formation and TDP-43 aggregation. Moreover, PAR associates with insoluble R-DPR and TDP-43 in postmortem tissue from patients. These findings identified PAR as a promoter of R-DPR toxicity and thus a potential target for treating c9ALS/FTD.

Role of CNC1 gene in TDP-43 aggregation-induced oxidative stress-mediated cell death in S. cerevisiae model of ALS.

TDP-43 protein is found deposited as inclusions in the amyotrophic lateral sclerosis (ALS) patient's brain. The mechanism of neuron death in ALS is not fully deciphered but several TDP-43 toxicity mechanisms such as mis-regulation of autophagy, mitochondrial impairment and generation of oxidative stress etc., have been implicated. A predominantly nuclear protein, Cyclin C, can regulate the oxidative stress response via transcription of stress response genes and also by translocation to the cytoplasm for the activation of mitochondrial fragmentation-dependent cell death pathway. Using the well-established yeast TDP-43 proteinopathy model, we examined here whether upon TDP-43 aggregation, cell survival depends on the CNC1 gene that encodes the Cyclin C protein or other genes which encode proteins that function in conjunction with Cyclin C, such as DNM1, FIS1 and MED13. We show that the TDP-43's toxicity is significantly reduced in yeast deleted for CNC1 or DNM1 genes and remains unaltered by deletions of genes, FIS1 and MED13. Importantly, this rescue is observed only in presence of functional mitochondria. Also, deletion of the YBH3 gene involved in the mitochondria-dependent apoptosis pathway reduced the TDP-43 toxicity. Deletion of the VPS1 gene involved in the peroxisomal fission pathway did not mitigate the TDP-43 toxicity. Strikingly, Cyclin C-YFP was observed to relocate to the cytoplasm in response to TDP-43's co-expression which was prevented by addition of an anti-oxidant molecule, N-acetyl cysteine. Overall, the Cyclin C, Dnm1 and Ybh3 proteins are found to be important players in the TDP-43-induced oxidative stress-mediated cell death in the S. cerevisiae model.

Computational insights into mechanism of AIM4-mediated inhibition of aggregation of TDP-43 protein implicated in ALS and evidence for in vitro inhibition of liquid-liquid phase separation (LLPS) of TDP-432C-A315T by AIM4.

TDP-43 is an RNA/DNA-binding protein which is also implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) disease. TDP-43's cytoplasmic mis-localization, liquid-liquid phase separation (LLPS) due to RNA depletion and aggregation, are proposedly important TDP-43-toxicity causing mechanisms. So far, therapeutic options for ALS are extremely ineffective hence, multi-faceted approaches such as targeting the oxidative stress and inhibiting the TDP-43's aggregation, are being actively pursued. Recently, we have identified an acridine derivative, AIM4, as an anti-TDP-43 aggregation molecule however, its mechanism is not deciphered. Here, we have utilized computational tools to examine binding site(s) of AIM4 in the TDP-43 structure and compared with other relevant compounds. We find that AIM4 has a binding site in the C-terminal amyloidogenic region (aa: 288-319), with Gly-288 & Phe-289 residues which are also important for TDP-43's LLPS. Importantly, alike to previously reported effects of RNA, AIM4 could also inhibit the in vitro LLPS of a C-terminal fragment TDP-432C bearing an A315T familial mutation. Furthermore, isothermal titration calorimetry (ITC) data also support the binding of AIM4 to TDP-432C-A315T. This antagonism of AIM4 towards TDP-43's LLPS and presence of binding site of AIM4 on TDP-43 support AIM4's potential to be an important molecule towards ALS therapeutic research.

Molecular Mechanisms of TDP-43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis.

TAR DNA binding protein 43 (TDP-43) is a versatile RNA/DNA binding protein involved in RNA-related metabolism. Hyper-phosphorylated and ubiquitinated TDP-43 deposits act as inclusion bodies in the brain and spinal cord of patients with the motor neuron diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While the majority of ALS cases (90-95%) are sporadic (sALS), among familial ALS cases 5-10% involve the inheritance of mutations in the TARDBP gene and the remaining (90-95%) are due to mutations in other genes such as: C9ORF72, SOD1, FUS, and NEK1 etc. Strikingly however, the majority of sporadic ALS patients (up to 97%) also contain the TDP-43 protein deposited in the neuronal inclusions, which suggests of its pivotal role in the ALS pathology. Thus, unraveling the molecular mechanisms of the TDP-43 pathology seems central to the ALS therapeutics, hence, we comprehensively review the current understanding of the TDP-43's pathology in ALS. We discuss the roles of TDP-43's mutations, its cytoplasmic mis-localization and aberrant post-translational modifications in ALS. Also, we evaluate TDP-43's amyloid-like in vitro aggregation, its physiological vs. pathological oligomerization in vivo, liquid-liquid phase separation (LLPS), and potential prion-like propagation propensity of the TDP-43 inclusions. Finally, we describe the various evolving TDP-43-induced toxicity mechanisms, such as the impairment of endocytosis and mitotoxicity etc. and also discuss the emerging strategies toward TDP-43 disaggregation and ALS therapeutics.

The amyloidogenicity of a C-terminal region of TDP-43 implicated in Amyotrophic Lateral Sclerosis can be affected by anions, acetylation and homodimerization.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease associated with accumulation of hyper-phosphorylated, and ubiquitinated TAR DNA-binding protein-43 (TDP-43) as inclusion deposits in neuronal cells. Recently, amyloid-like fibrillar aggregates of TDP-43 have been reported from several ALS patients. The C-terminal region of TDP-43 is central to TDP-43's pathological aggregation and most of the familial ALS mutations in the encoding TARDBP gene are located in this domain. Also, aberrant proteolytic cleavages of TDP-43 produce cytotoxic C-terminal fragments of ∼15-35 kDa. The C-terminal end harbours a glycine-rich region and a Q/N rich prion-like aggregation-prone domain which has been shown to form amyloid-like fibrillar aggregates in vitro. Previously, TDP-43 protein has also been shown to undergo several other post-translational modifications such as acetylation and dimerization, however, their effects on TDP-43's amyloid-like in vitro aggregation have not been examined. Towards this, we have here examined effects of anions, acetylation and homodimerization on the in vitro aggregation of a C-terminal fragment (amino acid: 193-414) of TDP-43 termed TDP-432C. We find that kosmotropic anions greatly accelerate whereas chaotropic anions impede its aggregation. Also, we show that acetylation of certain lysines in C-terminal fragments significantly reduces the TDP-432C's amyloid-like aggregation. Furthermore, we separated spontaneously formed cysteine-linked homodimers of the recombinantly purified TDP-432C using size-exclusion chromatography and found that these dimers retain amyloidogenicity. These findings would be of significance to the TDP-43 aggregation-induced pathology in ALS.

Q-Rich Yeast Prion [PSI+] Accelerates Aggregation of Transthyretin, a Non-Q-Rich Human Protein.

Interactions amongst different amyloid proteins have been proposed as a probable mechanism of aggregation and thus an important risk factor for the onset as well as progression of various neurodegenerative disorders including Alzheimer's, Parkinson's, Huntington's, and Amyotrophic Lateral Sclerosis. Evidences suggest that transthyretin (TTR), a plasma protein associated with transthyretin amyloidosis or familial polyneuropathy (FAP) interacts with heterologous amyloid proteins including amyloid beta and islet amyloid polypeptide. In addition, recent clinical studies have revealed the presence of systemic polyneuropathy associated with FAP mutations in patients with spinocerebral ataxia, amyotrophic lateral sclerosis, and new familial systematic prion disease. Hence, it is important to investigate the interactions amongst different amyloid proteins to gain better insight into the pathology of amyloid disorders. Yeast has been an excellent model system to study interaction/ cross-seeding between heterologous amyloid proteins, more because of presence of endogenous yeast prions. Here, we examined interactions of non-glutamine (non-Q)-rich transthyretin, with glutamine (Q)-rich yeast prion protein Sup35. We established aggregation of an engineered double (F87M/L110M) mutant M-TTR-GFP in yeast. This mutant is monomeric and readily formed aggregates compared to WT-TTR-GFP in yeast at acidic pH. Interestingly, aggregation of M-TTR-GFP was significantly enhanced in presence of [PSI+], an endogenous prion form of Sup35. Different variants of [PSI+] seeded M-TTR-GFP with different efficiencies and curing of [PSI+] (losing the prion form) in these strains reduced aggregation. Moreover, overexpression of prion domain of Sup35 fused to RFP (NM-RFP) also increased M-TTR-GFP aggregation. M-TTR-GFP and NM-RFP aggregates co-localized in perivacuolar and juxtranuclear region. Sup35 protein was even immunocaptured in M-TTR-GFP aggregates. However, M-TTR-GFP overexpression did not induce Sup35 aggregation. Thus, it appears to be a unidirectional interaction between these two amyloid proteins. However, no affect on M-TTR-GFP aggregation was observed due to another yeast prion, [PIN+]. Our findings thus show the molecular interaction of transthyretin with yeast prion and support that sequence similarity is not the prime requirement for heterologous amyloid interactions.

Synthesis and implication of novel poly(acrylic acid)/nanosorbent embedded hydrogel composite for lead ion removal.

Lead stands second among the deadly heavy metal pollutants owing to the incompetent mechanism possessed by the human body for its removal. A polymeric hydrogel in the form of composite was prepared using acrylic acid (monomer) and novel nanofiller that possess super adsorbent properties with restricted gel seepage into flowing ionic liquid. The filler used is an adsorbent which is biocompatible, biodegradable, economical, abundant, non-hazardous and easy to synthesize. The invariably porous nanofiller, the Nanobentonite(clay), was synthesized using ion exchange reaction by creating acidic environment for accelerated dispersion with exfoliation by CTAB to enhance cation exchange capacity. NanobentoFnite was capable of removing >97% lead ion in batch adsorption study and followed pseudo-second order kinetic model. Freundlich isotherm suggested a removal capacity of ~20 mg/g. Thus, the successfully experimented adsorbent was implicated as filler to form polyacrylic acid nanoclay hydrogel polymerized in ultrasonic bath. The amount of filler was varied from 0.25 to 2 wt% to get 94% removal, analyzed using ICP-OES. The prepared adsorbents were characterized before and after adsorption using TEM, FESEM, XRD, FTIR and DSC to understand the structural changes and metal-sorbent interaction. Thus, the novel nanosorbent/composite are promiscuous and competent in terms of availability, reusability and longevity to remove heavy metal ions.

A Protocol of Using White/Red Color Assay to Measure Amyloid-induced Oxidative Stress in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae (S. cerevisiae) harboring ade1 or ade2 mutations manifest red colony color phenotype on rich yeast medium YPD. In these mutants, intermediate metabolites of adenine biosynthesis pathway are accumulated. Accumulated intermediates, in the presence of reduced glutathione, are transported to the vacuoles, whereupon the development of the red color phenotype occurs. Here, we describe a method to score for presence of oxidative stress upon expression of amyloid-like proteins that would convert the red phenotype of ade1/ade2 mutant yeast to white. This assay could be a useful tool for screening for drugs with anti-amyloid aggregation or anti-oxidative stress potency.

An acridine derivative, [4,5-bis{(N-carboxy methyl imidazolium)methyl}acridine] dibromide, shows anti-TDP-43 aggregation effect in ALS disease models.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with aggregation of TAR DNA-binding protein-43 (TDP-43) in neuronal cells and manifests as motor neuron dysfunction &muscle atrophy. The carboxyl-terminal prion-like domain of TDP-43 can aggregate in vitro into toxic ß-sheet rich amyloid-like structures. So far, treatment options for ALS are very limited and Riluzole, which targets glutamate receptors, is the only but highly ineffective drug. Therefore, great interest exists in developing molecules for ALS treatment. Here, we have examined certain derivatives of acridine containing same side chains at position 4 &5, for inhibitory potential against TDP-43 aggregation. Among several acridine derivatives examined, AIM4, which contains polar carboxyl groups in the side arms, significantly reduces TDP-43-YFP aggregation in the powerful yeast model cell and also abolishes in vitro amyloid-like aggregation of carboxyl terminal domain of TDP-43, as observed by AFM imaging. Thus, AIM4 can be a lead molecule potentiating further therapeutic research for ALS.

Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid-induced oxidative stress in saccharomyces cerevisiae.

Mutations in adenine biosynthesis pathway genes ADE1 and ADE2 have been conventionally used to score for prion [PSI+ ] in yeast. If ade1-14 mutant allele is present, which contains a premature stop codon, [psi- ] yeast appear red on YPD medium owing to accumulation of a red intermediate compound in vacuoles. In [PSI+ ] yeast, partial inactivation of the translation termination factor, Sup35 protein, owing to its amyloid aggregation allows for read-through of the ade1-14 stop codon and the yeast appears white as the red intermediate pigment is not accumulated. The red colour development in ade1 and ade2 mutant yeast requires reduced-glutathione, which helps in transport of the intermediate metabolite P-ribosylaminoimidazole carboxylate into vacuoles, which develops the red colour. Here, we hypothesize that amyloid-induced oxidative stress would deplete reduced-glutathione levels and thus thwart the development of red colour in ade1 or ade2 yeast. Indeed, when we overexpressed amyloid-forming human proteins TDP-43, Aß-42 and Poly-Gln-103 and the yeast prion protein Rnq1, the otherwise red ade1 yeast yielded some white colonies. Further, the white colour eventually reverted back to red upon turning off the amyloid protein's expression. Also, the aggregate-bearing yeast have increased oxidative stress and white phenotype yeast revert to red when grown on media with reducing agent. Furthermore, the red/white assay could also be emulated in ade2-1, ade2Δ, and ade1Δ mutant yeast and also in an ade1-14 mutant with erg6 gene deletion that increases cell-wall permeability. This model would be useful tool for drug-screening against general amyloid-induced oxidative stress and toxicity. Copyright © 2016 John Wiley & Sons, Ltd.

Virtual Screening Approaches in Identification of Bioactive Compounds Akin to Delphinidin as Potential HER2 Inhibitors for the Treatment of Breast Cancer.

Small molecule tyrosine kinase inhibitors targeting HER 2 receptors have emerged as an important therapeutic approach in inhibition of downstream proliferation and survival signals for the treatment of breast cancers. Recent drug discovery efforts have demonstrated that naturally occurring polyphenolic compounds like delphinidin have potential to inhibit proliferation and promote apoptosis of breast cancer cells by targeting HER2 receptors. While delphinidin may thus reduce tumour size, it is associated with serious side effects like dysphonia. Owing to the narrow therapeutic window of delphinidin, the present study aimed to identify high affinity compounds targeting HER2 with safer pharmacological profiles than delphinidin through virtual screening approaches. Delphinidin served as the query parent for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. The compounds retrieved were further subjected to Lipinski and Verber's filters to obtain drug like agents, then further filtered by diversity based screens with a cut off of 0.6. The compound with Pubchem ID: 91596862 was identified to have higher affinity than its parent. In addition it also proved to be non-toxic with a better ADMET profile and higher kinase activity. The compound identified in the study can be put to further in vitro drug testing to complement the present study.

High Affinity Pharmacological Profiling of Dual Inhibitors Targeting RET and VEGFR2 in Inhibition of Kinase and Angiogeneis Events in Medullary Thyroid Carcinoma.

Clinical evidence shows that dual inhibition of kinases as well angiogenesis provides ideal therapeutic option in the treatment of medullary thyroid carcinoma (MTC) than inhibiting either of these with the events separately. Although treatment with dual inhibitors has shown good clinical responses in patients with MTC, it has been associated with serious side effects. Some inhibitors are active agents for both angiogenesis or kinase activity. Owing to narrow therapeutic window of established inhibitors, the present study aims to identify high affinity dual inhibitors targeting RET and VEGFR2 respectively for kinase and angiogenesis activity. Established inhibitors like Vandetanib, Cabozantinib, Motesanib, PP121, RAF265 and Sunitinib served as query parent compounds for identification of structurally similar compounds by Tanimoto-based similarity searching with a threshold of 95% against the PubChem database. All the parent inhibitors and respective similar compounds were docked against RET and VEGFR2 in order to retrieve high affinity compounds with these two proteins. AGN-PC-0CUK9P PubCID: 59320403 a compound related to PPI21 showed almost equal affinity for RET and VEGFR2 and unlike other screened compounds with no apparent bias for either of the receptors. Further, AGN- PC-0CUK9P demonstrated appreciable interaction with both RET and VEGFR2 and superior kinase activity in addition to showed optimal ADMET properties and pharmacophore features. From our in silico investigation we suggest AGN-PC-0CUK9P as a superior dual inhibitor targeting RET and VEGFR2 with high efficacy which should be proposed for pharmacodynamic and pharmacokinetic studies for improved treatment of MTC.

Computer aided identification of sodium channel blockers in the clinical treatment of epilepsy using molecular docking tools.

UNLABELLED: Phenytoin (PHT) and Carbamazepine (CBZ) are excellent sodium channel blockers administered in clinical treatment of epileptic seizures. However, the narrow therapeutic range and limited pharmacokinetics of these drugs have raised serious concerns in the proper management of epilepsy. To overcome this, the present study attempts to identify a candidate molecule with superior pharmacological profile than PHT and CBZ through In silico approaches. PHT and CBZ served as query small molecules for Tanimoto based similarity search with a threshold of 95% against PubChem database. Aided by MolDock algorithm, high affinity similar compound against each query was retrieved. PHT and CBZ and their respective similar were further tested for toxicity profiles, LC 50 values and biological activity. Compounds, NSC403438 and AGN-PC-0BPCBP respectively similar to PHT and CBZ demonstrated higher affinity to sodium channel protein than their respective leads. Of particular relevance, NSC403438 demonstrated highest binding affinity bestowed with least toxicity, better LC 50 values and optimal bioactivity. NSC403438 was further mapped for its structure based pharmacophoric features. In the study, we report NSC403438 as potential sodium channel blocker as a better candidate than PHT and CBZ which can be put forth for pharmacodynamic and pharmacokinetic studies. ABBREVIATIONS: AEDs - Antiepileptic drugs, BLAST - Basic Local Alignment Search Tool, CBZ - Carbamazepine, GEFS+ - Generalized Epilepsy with Febrile Seizures Plus, GPCR - G Protein Coupled Receptor, Nav - Sodium channel with specific voltage conduction, PDB - Protein Data Bank, PHT - Phenytoin, PIR - Protein Information resources, SAVES - Structural Analysis and Verification Server, VGSC - Voltage-gated Sodium channels.

Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist.

Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2 agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085 (Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN 9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation.

Implications of a novel Pseudomonas species on low density polyethylene biodegradation: an in vitro to in silico approach.

Degradation of Petroleum-plastics like Low Density Polyethylene (LDPE) is a budding challenge due to increasing white pollution. The present investigation has focused the aspect through microbial assisted biodegradation. Various indigenous microorganisms were isolated from collected municipal landfill soil. Growth medium enriched with 0.2 g of LDPE powder was used to screen the soil bacteria with biodegradation potential. The screened bacteria were subjected to biodegradation assay in presence of LDPE sheets in growth medium. Four strains gave 5%, 17.8%, 0.9% and 0.6% degradation rate based on weight loss in the conducted in vitro assay for four days. The maximum degraded sheet was analyzed through Scanning Electron Microscopy, Fourier transform infrared spectroscopy and Thermogravimetry, taking undegraded LDPE sheet as control. Results illustrated one-step weight loss with control and three-step weight loss with test. Thus, it proved the efficacy of isolated strain. The strain identification was carried out by genomic DNA isolation followed by PCR and 16S rRNA sequencing. Genotypic identification revealed the bacterium as Pseudomonas citronellolis. BLAST gave a similarity with the database of 96%, thus phylogenetic assessment clarified the bacterium as a novel strain. The isolate was named as Pseudomonas citronellolis EMBS027 and sequence was deposited as LDPE degrading species, in GenBank with accession number KF361478.