High throughput screening of a new fluorescent G-quadruplex ligand having telomerase inhibitory activity in human A549 cells.

Identification of a new G-quadruplex ligand having anti-telomerase activity would be a promising strategy for cancer therapy. The screened compound from ZINC database using docking studies was experimentally verified for its binding with three different telomeric G-quadruplex DNA sequences and anti-telomerase activity in A549 cells. Identified compound is an intrinsic fluorescent molecule, permeable to live cells and has a higher affinity to 22AG out of three different telomeric G-quadruplex DNA. It showed cytotoxicity and a significant reduction of telomerase activity in human A549 cells at a very low dose. So, this compound has a good anti-cancer effect.

Investigation of the effect of UV-B light on Arabidopsis MYB4 (AtMYB4) transcription factor stability and detection of a putative MYB4-binding motif in the promoter proximal region of AtMYB4.

Here, we have investigated the possible effect of UV-B light on the folding/unfolding properties and stability of Arabidopsis thaliana MYB4 (AtMYB4) transcription factor in vitro by using biophysical approaches. Urea-induced equilibrium unfolding analyses have shown relatively higher stability of the wild-type recombinant AtMYB4 protein than the N-terminal deletion forms after UV-B exposure. However, as compared to wild-type form, AtMYB4Δ2 protein, lacking both the two N-terminal MYB domains, showed appreciable alteration in the secondary structure following UV-B exposure. UV-B irradiated AtMYB4Δ2 also displayed higher propensity of aggregation in light scattering experiments, indicating importance of the N-terminal modules in regulating the stability of AtMYB4 under UV-B stress. DNA binding assays have indicated specific binding activity of AtMYB4 to a putative MYB4 binding motif located about 212 bp upstream relative to transcription start site of AtMYB4 gene promoter, while relatively weak DNA binding activity was detected for another putative MYB4 motif located at -908 bp in AtMYB4 promoter. Gel shift and fluorescence anisotropy studies have shown increased binding affinity of UV-B exposed AtMYB4 to the promoter proximal MYB4 motif. ChIP assay has revealed binding of AtMYB4 to the promoter proximal (-212 position) MYB4 motif (ACCAAAC) in vivo. Docking experiments further revealed mechanistic detail of AtMYB4 interaction with the putative binding motifs. Overall, our results have indicated that the N-terminal 62-116 amino acid residues constituting the second MYB domain plays an important role in maintaining the stability of the C-terminal region and the overall stability of the protein, while a promoter proximal MYB-motif in AtMYB4 promoter may involve in the regulation of its own expression under UV-B light.

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

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

An Engineered Variant of the B1 Domain of Protein G Suppresses the Aggregation and Toxicity of Intra- and Extracellular Aß42.

Intra- and extraneuronal deposition of amyloid ß (Aß) peptides have been linked to Alzheimer's disease (AD). While both intra- and extraneuronal Aß deposits affect neuronal cell viability, the molecular mechanism by which these Aß structures, especially when intraneuronal, do so is still not entirely understood. This makes the development of inhibitors challenging. To prevent the formation of toxic Aß structural assemblies so as to prevent neuronal cell death associated with AD, we used a combination of computational and combinatorial-directed evolution approaches to develop a variant of the HTB1 protein (HTB1M2). HTB1M2 inhibits in vitro self-assembly of Aß42 peptide and shifts the Aß42 aggregation pathway to the formation of oligomers that are nontoxic to neuroblastoma SH-SY5Y cells overexpressing or treated with Aß42 peptide. This makes HTB1M2 a potential therapeutic lead in the development of AD-targeted drugs and a tool for elucidating conformational changes in the Aß42 peptide.

An Aß42 variant that inhibits intra- and extracellular amyloid aggregation and enhances cell viability.

Aggregation and accumulation of the 42-residue amyloid ß peptide (Aß42) in the extracellular matrix and within neuronal cells is considered a major cause of neuronal cell cytotoxicity and death in Alzheimer's disease (AD) patients. Therefore, molecules that bind to Aß42 and prevent its aggregation are therapeutically promising as AD treatment. Here, we show that a non-self-aggregating Aß42 variant carrying two surface mutations, F19S and L34P (Aß42DM), inhibits wild-type Aß42 aggregation and significantly reduces Aß42-mediated cell cytotoxicity. In addition, Aß42DM inhibits the uptake and internalization of extracellularly added pre-formed Aß42 aggregates into cells. This was the case in both neuronal and non-neuronal cells co-expressing Aß42 and Aß42DM or following pre-treatment of cells with extracellular soluble forms of the two peptides, even at high Aß42 to Aß42DM molar ratios. In cells, Aß42DM associates with Aß42, while in vitro, the two soluble recombinant peptides exhibit nano-molar binding affinity. Importantly, Aß42DM potently suppresses Aß42 amyloid aggregation in vitro, as demonstrated by thioflavin T fluorescence and transmission electron microscopy for detecting amyloid fibrils. Overall, we present a new approach for inhibiting Aß42 fibril formation both within and outside cells. Accordingly, Aß42DM should be evaluated in vivo for potential use as a therapeutic lead for treating AD.

MIF inhibits the formation and toxicity of misfolded SOD1 amyloid aggregates: implications for familial ALS.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)-a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties-inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.

p-Benzoquinone-induced aggregation and perturbation of structure and chaperone function of α-crystallin is a causative factor of cigarette smoke-related cataractogenesis.

Cigarette smoking is a significant risk factor for cataract. However, the mechanism by which cigarette smoke (CS) causes cataract remains poorly understood. We had earlier shown that in CS-exposed guinea pig, p-benzoquinone (p-BQ) derived from CS in the lungs is carried by the circulatory system to distant organs and induces various smoke-related pathogeneses. Here, we observed that CS exposure caused accumulation of the p-BQ-protein adduct in the eye lens of guinea pigs. We also observed accumulation of the p-BQ-protein adduct in resected lens from human smokers with cataract. No such accumulation was observed in the lens of never smokers. p-BQ is a strong arylating agent that forms Michael adducts with serum albumin and haemoglobin resulting in alterations of structure and function. A major protein in the mammalian eye lens is αA-crystallin, which is a potent molecular chaperone. αA-crystallin plays a key role in maintaining the integrity and transparency of the lens. SDS-PAGE indicated that p-BQ induced aggregation of αA-crystallin. Various biophysical techniques including UV-vis spectroscopy, fluorescence spectroscopy, FT-IR, bis-ANS titration suggested a perturbation of structure and chaperone function of αA-crystallin upon p-BQ modification. Our results indicate that p-BQ is a causative agent involved in the modification of αA-crystallin and pathogenesis of CS-induced cataract. Our findings would educate public about the impacts of smoking on eye health and help to discourage them from smoking. The study might also help scientists to develop new drugs for the intervention of CS-induced cataract at an early stage.

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.

Superoxide Dismutase 1 (SOD1)-Derived Peptide Inhibits Amyloid Aggregation of Familial Amyotrophic Lateral Sclerosis SOD1 Mutants.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the death of the upper and lower motor neurons. Superoxide dismutase 1 (SOD1) is an ALS pathogenic protein, whose misfolding results in the formation of amyloid aggregates. The mechanism underlying SOD1 pathogenesis in ALS remains obscure, but one possible mechanism involves gain-of-interaction, in which the misfolded soluble SOD1 forms abnormal protein-protein interactions (PPIs) with various cellular proteins, including with other SOD1 molecules, thereby interfering with their function. The structural basis of this gain-of-interaction mechanism is unknown. Here, we characterized the backbone dynamics landscape of misfolded SOD1 to pinpoint surface areas predisposed to aberrant PPIs. This analysis enabled us to formulate a working hypothesis for the mechanism of the gain-of-function of misfolded SOD1, according to which an abnormal PPI potential results from the increased mobility of the SOD1 surface backbone. Guided by the backbone dynamics landscape, we have identified a SOD1-derived peptide that can bind SOD1 proteins and divert the typical amyloid aggregation of ALS-related SOD1 mutants toward a potentially less toxic amorphous aggregation pathway.

Understanding the Physical and Molecular Basis of Stability of Arabidopsis DNA Pol λ under UV-B and High NaCl Stress.

Here, we have investigated the physical and molecular basis of stability of Arabidopsis DNA Pol λ, the sole X family DNA polymerase member in plant genome, under UV-B and salinity stress in connection with the function of the N-terminal BRCT (breast cancer-associated C terminus) domain and Ser-Pro rich region in the regulation of the overall structure of this protein. Tryptophan fluorescence studies, fluorescence quenching and Bis-ANS binding experiments using purified recombinant full length Pol λ and its N-terminal deletion forms have revealed UV-B induced conformational change in BRCT domain deficient Pol λ. On the other hand, the highly conserved C-terminal catalytic core PolX domain maintained its tertiary folds under similar condition. Circular dichroism (CD) and fourier transform infrared (FT-IR) spectral studies have indicated appreciable change in the secondary structural elements in UV-B exposed BRCT domain deficient Pol λ. Increased thermodynamic stability of the C-terminal catalytic core domain suggested destabilizing effect of the N-terminal Ser-Pro rich region on the protein structure. Urea-induced equilibrium unfolding studies have revealed increased stability of Pol λ and its N-terminal deletion mutants at high NaCl concentration. In vivo aggregation studies using transient expression systems in Arabidopsis and tobacco indicated possible aggregation of Pol λ lacking the BRCT domain. Immunoprecipitation assays revealed interaction of Pol λ with the eukaryotic molecular chaperone HSP90, suggesting the possibility of regulation of Pol λ stability by HSP90 in plant cell. Overall, our results have provided one of the first comprehensive information on the biophysical characteristics of Pol λ and indicated the importance of both BRCT and Ser-Pro rich modules in regulating the stability of this protein under genotoxic stress in plants.

Structure and functional properties of a multimeric protein αA-Crystallin adsorbed on silver nanoparticle surface.

Proteins adsorb onto a nanoparticle surface to form a protein-nanoparticle corona which becomes the identity of the nanoparticle in the cellular environment. Conformation of the protein at the interface influences the cellular uptake of the nanoparticle. Hence, interaction of proteins with nanomaterials is of special significance in the field of biotechnology. Adsorption of protein on the nanoparticle surface is a complex process that depends on the dielectric properties and pH of the medium, surface morphology and surface heterogeneity of the nanoparticle, and the quaternary structure of the protein. Thus, interaction of a large multimeric protein with a nanoparticle will be different from that of small oligomeric proteins. In this article we report the conformational and functional properties of a large oligomeric protein αA-Crystallin, a major constituent of the mammalian eye lens, adsorbed onto silver nanoparticle surface. Selective alkylation of the two cysteine residues at the α-Crystallin domain, followed by ITC study showed that these residues play crucial roles in the interaction process. The chaperone function and the refolding capacity of the protein, which is primarily governed by the α-Crystallin domain, are lost to a significant extent when adsorbed onto AgNP surface. The protein in the interface also shows loss of oligomerization that is linked to the biological activity of the protein. Nonetheless, the protein at bio-nano interface shows resistance to urea unfolding process as compared to protein in the solution phase. This might be due to the coordination of AgNP with two cysteine residues of ß8 and ß9 region of the α-Crystallin domain that imparts extra stability. The compactness in the structure of the adsorbed protein reduces the dynamics of the subunit exchange, which was confirmed by the FRET study. The secondary structure of αA-Crystallin bound to AgNP at substoichiometric ratio remained native-like.

Critical role of zinc ion on E. coli glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) structure and function.

Glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) and glutamyl-tRNA synthetase (GluRS), differ widely by their function although they share close structural resemblance within their catalytic core of GluRS. In particular both Escherichia coli GluRS and Glu-Q-RS contain a single zinc-binding site in their putative tRNA acceptor stem-binding domain. It has been shown that the zinc is crucial for correct positioning of the tRNA(Glu) acceptor-end in the active site of E. coli GluRS. To address the role of zinc ion in Glu-Q-RS, the C101S/C103S Glu-Q-RS variant is constructed. Energy dispersive X-ray fluorescence show that the zinc ion still remained coordinated but the variant became structurally labile and acquired aggregation capacity. The extent of aggregation of the protein is significantly decreased in presence of the small substrates and more particularly by adenosine triphosphate. Addition of zinc increased significantly the solubility of the variant. The aminoacylation assay reveals a decrease in activity of the variant even after addition of zinc as compared to the wild-type, although the secondary structure of the protein is not altered as shown by the Fourier transform infrared spectroscopy study.

Spectroscopic studies of the unfolding of a multimeric protein α-crystallin.

α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol(-1) on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol(-1) on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.

Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

Interaction of silver nanoparticles with proteins: a characteristic protein concentration dependent profile of SPR signal.

Silver nanoparticles are finding increasing applications in biological systems, for example as antimicrobial agents and potential candidates for control drug release systems. In all such applications, silver nanoparticles interact with proteins and other biomolecules. Hence, the study of such interactions is of considerable importance. While BSA has been extensively used as a model protein for the study of interaction with the silver nanoparticles, studies using other proteins are rather limited. The interaction of silver nanoparticles with light leads to collective oscillation of the conducting electrons giving rise to surface plasmon resonance (SPR). Here, we have studied the protein concentration dependence of the SPR band profiles for a number of proteins. We found that for all the proteins, with increase in concentration, the SPR band intensity initially decreased, reaching minima and then increased again leading to a characteristic "dip and rise" pattern. Minimum point of the pattern appeared to be related to the isoelectric point of the proteins. Detailed dynamic light scattering and transmission electron microscopy studies revealed that the consistency of SPR profile was dependent on the average particle size and state of association of the silver nanoparticles with the change in the protein concentration. Fluorescence spectroscopic studies showed the binding constants of the proteins with the silver nanoparticles were in the nano molar range with more than one nanoparticle binding to protein molecule. Structural studies demonstrate that protein retains its native-like structure on the nanoparticle surface unless the molar ratio of silver nanoparticles to protein exceeds 10. Our study reveals that nature of the protein concentration dependent profile of SPR signal is a general phenomena and mostly independent of the size and structure of the proteins.

Modulation of pathway of insulin fibrillation by a small molecule helix inducer 2,2,2-trifluoroethanol.

Many proteins form ordered irreversible structural aggregates called amyloid fibrils, which are associated with numerous neurodegenerative diseases. Insulin, a largely α-helical protein associated with type II diabetes, self-assembles to form amyloid fibrils in vitro. Insulin fibrillation goes through a number of intermediate phases that includes a soluble oligomeric phase believed to be the most toxic phase. Small molecules may play a very important role in modulating the fibrillation pathways. It is possible to induce and stabilize helix structures in proteins by a fluorinated alcohol 2,2,2-trifluoro ethanol (TFE). Since fibrillation process of many proteins is associated with conversion of α-helical structures into ß-sheet configuration, we thought it would be interesting to study the effect of TFE on the fibrillation of insulin. In absence of TFE, soluble protofibrillar oligomeric intermediates formed directly from the insulin trimer. The protofibrillar aggregates transformed into mature fibrils over time. We demonstrated that although TFE did not prevent the appearance of matured amyloid fibrils, it prevented the appearance of soluble aggregates of insulin. TFE converted the insulin trimer into monomers and fibril formation proceeded from the monomeric state in a cooperative way avoiding the soluble oligomeric phase. At 25% TFE, distinct morphological changes resulting in more discrete fibrils were visible. The effect of the small molecule TFE on the avoidance of the formation soluble oligomeric state during fibrillation may have considerable implications in reducing cellular toxicity.