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• W2230622832 abstract "Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids. Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids. The extracellular matrix (ECM) 5The abbreviations used are: ECM, extracellular matrix; TEM, transmission electron microscopy; CR, Congo Red; ThT, thioflavin T; FAK, focal adhesion kinase; Sub P, substance P; α-Syn, α-synuclein; PAA, polyamino acid; PLL, poly-l-lysine; PE, polyglutamic acid; PLA, poly-l-alanine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; RBC, red blood cell; ROCK, Rho-associated kinase; MLCK, myosin light chain kinase; VIP, vasoactive intestinal peptide; GRP, gastrin releasing peptide; GRF, growth hormone releasing factor; GIP, glucose-dependent insulinotropic polypeptide; oCRF, ovine corticotropin-releasing factor. composed of proteins, minerals, and carbohydrates (1Lord E.M. Sanders L.C. Roles for the extracellular matrix in plant development and pollination: a special case of cell movement in plants.Dev. Biol. 1992; 153: 16-28Crossref PubMed Scopus (0) Google Scholar) provides physical support to individual cells and facilitates interactions between cells and links them into functional tissue (2Daley W.P. Yamada K.M. ECM-modulated cellular dynamics as a driving force for tissue morphogenesis.Curr. Opin. Genet. Dev. 2013; 23: 408-414Crossref PubMed Scopus (118) Google Scholar). Depending on the source of the tissue, the difference in the composition and organization of the proteins in the ECM dictate the physical properties of that particular tissue. For example, bones consist of highly mineralized ECM that supports and resist compression (3Sapir-Koren R. Livshits G. Bone mineralization and regulation of phosphate homeostasis.IBMS BoneKEy. 2011; 8: 286-300Crossref Google Scholar), whereas skin has dense interpenetrating matrix, which is elastic, strong, and regenerative (4Watt F.M. Fujiwara H. Cell-extracellular matrix interactions in normal and diseased skin.Cold Spring Harb. Perspect. Biol. 2011; (10.1101/cshperspect.a005124)Crossref PubMed Scopus (152) Google Scholar). Cells within the tissue sense the physicochemical properties of the ECM and bind to each other through a gamut of different cell adhesion molecules, including integrins, cadherins, and trans-membrane proteoglycans, and regulate their own functions (5Choi Y. Chung H. Jung H. Couchman J.R. Oh E.S. Syndecans as cell surface receptors: unique structure equates with functional diversity.Matrix Biol. 2011; 30: 93-99Crossref PubMed Scopus (109) Google Scholar). These interactions between the cell and its surrounding ECM through integrins and other receptors directly control cell adhesion, cell proliferation, and tissue organization (6Geiger B. Bershadsky A. Pankov R. Yamada K.M. Transmembrane crosstalk between the extracellular matrix-cytoskeleton crosstalk.Nat. Rev. Mol. Cell Biol. 2001; 2: 793-805Crossref PubMed Scopus (1709) Google Scholar). Amyloids are unique protein folds responsible for both disease and function in host organisms (7Chiti F. Dobson C.M. Protein misfolding, functional amyloid, and human disease.Annu. Rev. Biochem. 2006; 75: 333-366Crossref PubMed Scopus (4634) Google Scholar). Although the initial identification of amyloids emerged from their association with several human diseases, including Alzheimer disease and Parkinson disease, many amyloids with native functions in the host were also discovered from unicellular to multicellular organisms, including mammals (7Chiti F. Dobson C.M. Protein misfolding, functional amyloid, and human disease.Annu. Rev. Biochem. 2006; 75: 333-366Crossref PubMed Scopus (4634) Google Scholar, 8Fowler D.M. Koulov A.V. Balch W.E. Kelly J.W. Functional amyloid: from bacteria to humans.Trends Biochem. Sci. 2007; 32: 217-224Abstract Full Text Full Text PDF PubMed Scopus (751) Google Scholar). For instance, in many organisms like Escherichia coli, yeast, and barnacle, amyloids help the host organism with their adhesion to surfaces (9Chapman M.R. Robinson L.S. Pinkner J.S. Roth R. Heuser J. Hammar M. Normark S. Hultgren S.J. Role of Escherichia coli curli operons in directing amyloid fiber formation.Science. 2002; 295: 851-855Crossref PubMed Scopus (833) Google Scholar10Ramsook C.B. Tan C. Garcia M.C. Fung R. Soybelman G. Henry R. Litewka A. O'Meally S. Otoo H.N. Khalaf R.A. Dranginis A.M. Gaur N.K. Klotz S.A. Rauceo J.M. Jue C.K. Lipke P.N. Yeast cell adhesion molecules have functional amyloid-forming sequences.Eukaryot. Cell. 2010; 9: 393-404Crossref PubMed Scopus (97) Google Scholar, 11Sullan R.M. Gunari N. Tanur A.E. Chan Y. Dickinson G.H. Orihuela B. Rittschof D. Walker G.C. Nanoscale structures and mechanics of barnacle cement.Biofouling. 2009; 25: 263-275Crossref PubMed Scopus (60) Google Scholar12Barlow D.E. Dickinson G.H. Orihuela B. Kulp J.L. Rittschof D. Wahl K.J. Characterization of the adhesive plaque of the barnacle balanus amphitrite: amyloid-like nanofibrils are a major component.Langmuir. 2010; 26: 6549-6556Crossref PubMed Scopus (0) Google Scholar). Recently in mammals, amyloids have been found to have a functional role in melanin synthesis and hormone storage (13Fowler D.M. Koulov A.V. Alory-Jost C. Marks M.S. Balch W.E. Kelly J.W. Functional amyloid formation within mammalian tissue.PLoS Biol. 2006; 4: e6Crossref PubMed Scopus (606) Google Scholar, 14Maji S.K. Perrin M.H. Sawaya M.R. Jessberger S. Vadodaria K. Rissman R.A. Singru P.S. Nilsson K.P. Simon R. Schubert D. Eisenberg D. Rivier J. Sawchenko P. Vale W. Riek R. Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.Science. 2009; 325: 328-332Crossref PubMed Scopus (658) Google Scholar). Due to their self-replication capability (15Cohen F.E. Prusiner S.B. Pathologic conformations of prion proteins.Annu. Rev. Biochem. 1998; 67: 793-819Crossref PubMed Scopus (460) Google Scholar, 16Jarrett J.T. Lansbury Jr., P.T. Seeding “one-dimensional crystallization” of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie?.Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1804) Google Scholar) and their function as biological and chemical catalysts (17Rufo C.M. Moroz Y.S. Moroz O.V. Stöhr J. Smith T.A. Hu X. DeGrado W.F. Korendovych I.V. Short peptides self-assemble to produce catalytic amyloids.Nat. Chem. 2014; 6: 303-309Crossref PubMed Scopus (278) Google Scholar), amyloid folds are now considered as one of the primitive protein folds (18Greenwald J. Riek R. On the possible amyloid origin of protein folds.J. Mol. Biol. 2012; 421: 417-426Crossref PubMed Scopus (71) Google Scholar, 19Carny O. Gazit E. A model for the role of short self-assembled peptides in the very early stages of the origin of life.FASEB J. 2005; 19: 1051-1055Crossref PubMed Scopus (73) Google Scholar). Amyloid fibrils possess a highly repetitive structure along with unique surface properties consisting of a combination of charged and hydrophobic surfaces, depending on the sequence, making them sticky (20Nelson R. Eisenberg D. Recent atomic models of amyloid fibril structure.Curr. Opin. Struct. Biol. 2006; 16: 260-265Crossref PubMed Scopus (295) Google Scholar, 21Wasmer C. Lange A. Van Melckebeke H. Siemer A.B. Riek R. Meier B.H. Amyloid fibrils of the HET-s(218–289) prion form a β solenoid with a triangular hydrophobic core.Science. 2008; 319: 1523-1526Crossref PubMed Scopus (769) Google Scholar22Maji S.K. Wang L. Greenwald J. Riek R. Structure-activity relationship of amyloid fibrils.FEBS Lett. 2009; 583: 2610-2617Crossref PubMed Scopus (86) Google Scholar) and thereby enabling them to bind to both small molecules and large macromolecules/polymers (23Nilsson K.P. Small organic probes as amyloid specific ligands: past and recent molecular scaffolds.FEBS Lett. 2009; 583: 2593-2599Crossref PubMed Scopus (0) Google Scholar24Calamai M. Kumita J.R. Mifsud J. Parrini C. Ramazzotti M. Ramponi G. Taddei N. Chiti F. Dobson C.M. Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes.Biochemistry. 2006; 45: 12806-12815Crossref PubMed Scopus (105) Google Scholar, 25Ghosh D. Dutta P. Chakraborty C. Singh P.K. Anoop A. Jha N.N. Jacob R.S. Mondal M. Mankar S. Das S. Malik S. Maji S.K. Complexation of amyloid fibrils with charged conjugated polymers.Langmuir. 2014; 30: 3775-3786Crossref PubMed Scopus (23) Google Scholar26Solomon J.P. Bourgault S. Powers E.T. Kelly J.W. Heparin binds 8 kDa gelsolin cross-beta-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension.Biochemistry. 2011; 50: 2486-2498Crossref PubMed Scopus (0) Google Scholar). Various microorganisms, for their surface attachment and colonization, exploit this sticky property of amyloids. Amyloid fibrils have been reported to be a part of the biofilms in numerous microorganisms, including harpins of Xanthomonas campestris and Pseudomonas syringae (27Oh J. Kim J.G. Jeon E. Yoo C.H. Moon J.S. Rhee S. Hwang I. Amyloidogenesis of type III-dependent harpins from plant pathogenic bacteria.J. Biol. Chem. 2007; 282: 13601-13609Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), pili from Mycobacterium tuberculosis (28Alteri C.J. Xicohténcatl-Cortes J. Hess S. Caballero-Olín G. Girón J.A. Friedman R.L. Mycobacterium tuberculosis produces pili during human infection.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5145-5150Crossref PubMed Scopus (134) Google Scholar), chaplins from Streptomyces coelicolor (29Claessen D. Rink R. de Jong W. Siebring J. de Vreugd P. Boersma F.G. Dijkhuizen L. Wosten H.A. A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils.Genes Dev. 2003; 17: 1714-1726Crossref PubMed Scopus (259) Google Scholar), and hydrophobins from fungi (30Wösten H.A. Hydrophobins: multipurpose proteins.Annu. Rev. Microbiol. 2001; 55: 625-646Crossref PubMed Scopus (462) Google Scholar). These amyloidogenic ECM components help these organisms to adhere onto the surface and form colonies. The above studies suggest that amyloids possess many ECM-like features and may be capable of supporting cell adhesion. In this context, amyloid fibrils functionalized with cell-adhesive RGD motifs were shown to support cell adhesion (31Gras S.L. Surface- and solution-based assembly of amyloid fibrils for biomedical and nanotechnology applications.Adv. Chem. Engineer. 2009; 35: 161-209Crossref Scopus (28) Google Scholar, 32Gras S.L. Tickler A.K. Squires A.M. Devlin G.L. Horton M.A. Dobson C.M. MacPhee C.E. Functionalised amyloid fibrils for roles in cell adhesion.Biomaterials. 2008; 29: 1553-1562Crossref PubMed Scopus (137) Google Scholar). Recent studies also suggest that amyloid fibrils alone (without any functionalization) are also capable of supporting cell adhesion due to their unique nanotopographic features (33Reynolds N.P. Charnley M. Mezzenga R. Hartley P.G. Engineered lysozyme amyloid fibril networks support cellular growth and spreading.Biomacromolecules. 2014; 15: 599-608Crossref PubMed Scopus (49) Google Scholar34Reynolds N.P. Styan K.E. Easton C.D. Li Y. Waddington L. Lara C. Forsythe J.S. Mezzenga R. Hartley P.G. Muir B.W. Nanotopographic surfaces with defined surface chemistries from amyloid fibril networks can control cell attachment.Biomacromolecules. 2013; 14: 2305-2316Crossref PubMed Scopus (40) Google Scholar, 35Li C. Born A.K. Schweizer T. Zenobi-Wong M. Cerruti M. Mezzenga R. Amyloid-hydroxyapatite bone biomimetic composites.Adv. Mater. 2014; 26: 3207-3212Crossref PubMed Scopus (107) Google Scholar, 36Yan H. Nykanen A. Ruokolainen J. Farrar D. Gough J.E. Saiani A. Miller A.F. Thermo-reversible protein fibrillar hydrogels as cell scaffolds.Faraday Discuss. 2008; 139 (discussion 105–128, 419–120): 71-84Crossref PubMed Scopus (34) Google Scholar37Jacob R.S. Ghosh D. Singh P.K. Basu S.K. Jha N.N. Das S. Sukul P.K. Patil S. Sathaye S. Kumar A. Chowdhury A. Malik S. Sen S. Maji S.K. Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation.Biomaterials. 2015; 54: 97-105Crossref PubMed Scopus (81) Google Scholar). However, it remains unclear whether this cell-adhesive property is dependent on the sequence composition or is a consequence of the amyloid nature. Here we demonstrate that irrespective of the sequence, amyloid fibrils are capable of supporting cell adhesion. Unless specified, all chemicals and reagents were purchased from Sigma. Water was double-distilled and deionized using a Milli-Q system (Millipore Corp., Bedford, MA). All of the peptide hormones except human galanin and somatostatin were a kind gift from Prof. Roland Riek (ETH Zurich). Somatostatin was purchased from BACHEM, and human galanin peptides were custom synthesized by USV Ltd. (Mumbai, India) with >95% purity. Purity of all of these peptides was further confirmed by MALDI-TOF mass spectrometry. To test the adhesion of cells on amyloid fibrils, the amyloid fibrils were prepared by dissolving the peptides of kassinin (2 mg/ml), GLP 1 (0.25 mg/ml), rat UCN (2 mg/ml), oCRF (2 mg/ml), glucagon (2 mg/ml), GIP (2 mg/ml), mouse UCN III (2 mg/ml), and Aβ(25–35) (1 mg/ml) in 5% d-mannitol with 0.01% sodium azide and incubated at 37 °C with slight rotation. The peptides of somatostatin (2 mg/ml), human GRF (2 mg/ml), bombesin (2 mg/ml), VIP (2 mg/ml), helodermin (2 mg/ml), GRP (2 mg/ml), galanin (1 mg/ml), β-endorphin (2 mg/ml), and Sub P (1 mg/ml) were also similarly dissolved in 5% d-mannitol with 0.01% sodium azide and incubated in the presence of 400 μm low molecular weight heparin at 37 °C in 1.5-ml Eppendorf tubes. α-Synuclein (α-Syn) protein was expressed and purified according to the protocol described by Volles and Lansbury (38Volles M.J. Lansbury Jr., P.T. Relationships between the sequence of α-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity.J. Mol. Biol. 2007; 366: 1510-1522Crossref PubMed Scopus (146) Google Scholar) in E. coli BL21 (DE3) strain. For α-Syn, 30 mg/ml lyophilized protein was dissolved in 20 mm MES buffer, pH 6.0, and low molecular weight α-Syn was prepared by passing the dissolved protein through 100 kDa cut-off membrane as described before (39Singh P.K. Kotia V. Ghosh D. Mohite G.M. Kumar A. Maji S.K. Curcumin modulates α-synuclein aggregation and toxicity.ACS Chem. Neurosci. 2013; 4: 393-407Crossref PubMed Scopus (158) Google Scholar). The Eppendorf tubes containing peptide/protein solutions were placed into an EchoTherm model RT11 rotating mixture (Torrey Pines Scientific) at 50 rpm inside a 37 °C incubator. At suitable intervals, thioflavin T (ThT), circular dichroism (CD), and transmission electron microscopy (TEM) were performed to analyze the aggregation. CD spectroscopy is a commonly used technique to monitor the secondary structural transitions during protein/peptide aggregation studies (40Whitmore L. Wallace B.A. Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases.Biopolymers. 2008; 89: 392-400Crossref PubMed Scopus (1571) Google Scholar). To study the conformational changes during the aggregation of proteins/peptides, 15 μl of peptide solutions was diluted in 5% d-mannitol to 200 μl such that the final peptide concentration was of 20 μm. For α-Syn, 10 μl of protein solution was diluted in 20 mm MES buffer, pH 6.0, to 200 μl such that the final concentration was 15 μm. The protein/peptide solution was placed into a 0.1-cm path length quartz cell (Hellma, Forest Hills, NY), and the spectra were acquired using a JASCO 810 instrument. All measurements were done at 25 °C. Spectra were recorded over the wavelength range of 198–260 nm. Three independent experiments were performed with each sample. Raw data were processed by smoothing and subtraction of buffer spectra as per the manufacturer's instructions. ThT is an amyloid detection dye widely used to probe amyloid formation during protein aggregation (41LeVine 3rd, H. Thioflavine T interaction with synthetic Alzheimer's disease β-amyloid peptides: detection of amyloid aggregation in solution.Protein Sci. 1993; 2: 404-410Crossref PubMed Google Scholar). In order to track amyloid formation in the aggregating mixtures of proteins/peptides, a 10-μl aliquot of peptide/protein samples was diluted to 500 μl in 5% d-mannitol containing 0.01% (w/v) sodium azide such that the final concentration of the peptide/protein was of 8 μm. For α-Syn, 10 μl of protein solution was diluted to 500 μl such that the final concentration was of 6 μm. These solutions were then mixed with 2 μl of 1 mm ThT prepared in 10 mm Tris-HCl, pH 8.0. Fluorescence was measured immediately after the addition of ThT. The fluorescence experiment was carried out using a Fluoromax 4 spectrofluorometer (Horiba Jobin Yvon Inc.), with excitation at 450 nm and emission from 460 to 500 nm. The intensity values at 480 nm were plotted. Three independent experiments were performed for each sample. To examine the morphology of the protein/peptide amyloid fibrils under EM, aliquots of peptide/protein samples were diluted in distilled water to obtain a final concentration of ∼50 μm. The diluted solutions were spotted on a glow-discharged, carbon-coated Formvar grid (Electron Microscopy Sciences, Fort Washington, PA) and incubated for 5 min. The grids were then washed with distilled water and stained with 1% (w/v) aqueous uranyl formate solution. Uranyl formate solution was freshly prepared and filtered through 0.22-μm sterile syringe filters (Millipore). EM analysis was performed using an FEI Tecnai G2 12-electron microscope at 120 kV with nominal magnifications in the range of 26,000–60,000. Images were recorded digitally by using the SIS Megaview III imaging system. At least two independent experiments were carried out for each sample. The images obtained were analyzed in ImageJ (National Institutes of Health) to calculate fibril diameter. It was suggested previously that BSA could form amyloid fibrils at low pH and in the presence of high salt conditions (42Bhattacharya M. Jain N. Mukhopadhyay S. Insights into the mechanism of aggregation and fibril formation from bovine serum albumin.J. Phys. Chem. B. 2011; 115: 4195-4205Crossref PubMed Scopus (110) Google Scholar, 43Usov I. Adamcik J. Mezzenga R. Polymorphism complexity and handedness inversion in serum albumin amyloid fibrils.ACS Nano. 2013; 7: 10465-10474Crossref PubMed Scopus (76) Google Scholar). To further examine the conditions required for BSA amyloid formation, lyophilized BSA (Sigma) was dissolved in PBS, pH 7.4, with a concentration of 21 mg/ml, and the concentration was determined by UV absorption measurement at 280 nm, considering the molar absorptivity of BSA as 43,824 m−1 cm−1. The 300 μm stock solution of BSA was used for the further study. The 300 μm BSA and 5 m NaCl stock solutions were used to search the optimal conditions for BSA amyloid formation. The concentration of BSA in the reaction was varied from 5 to 100 μm using 20 mm glycine-NaOH buffer, pH 3.0, and the NaCl concentration was varied from 0 to 300 μm. All solutions were incubated at 65 °C, and the aggregation and amyloid formation were monitored using ThT binding studies (data not shown). The condition that showed the highest ThT binding was considered as the amyloid-forming condition and was used for further studies. The secondary structural changes were determined by CD spectroscopy, and morphological analysis was done using an electron microscope. Further amyloid formation was confirmed using Congo Red (CR) binding by UV spectral assay and birefringence study. CR is a dye routinely used for detecting amyloid formation in protein/peptide aggregation studies (44Klunk W.E. Jacob R.F. Mason R.P. Quantifying amyloid by Congo red spectral shift assay.Methods Enzymol. 1999; 309: 285-305Crossref PubMed Scopus (286) Google Scholar). In order to confirm the amyloid nature of BSA aggregates, CR binding studies were performed. For CR binding, a 10-μl aliquot of aggregated BSA (of 100 μm concentration) was mixed with 160 μl of PBS buffer containing 10% ethanol. Then 30 μl of 100 μm CR solution in PBS (containing 10% ethanol) was added. After a 5-min incubation in the dark at room temperature, CR absorbance was measured in the range of 300–700 nm using a JASCO V-650 spectrophotometer. Similarly, the CR alone spectrum was also recorded as a control. Three independent experiments were performed for each sample. To further confirm the binding of CR to amyloid fibrils, CR birefringence was performed. 50 μl of incubated solutions of protein/peptide aggregates was subjected to ultracentrifugation (Optima Max-XP, Beckman Coulter) at 90,000 rpm for 1 h and was washed once by distilled water. The pellets obtained were then used for performing CR birefringence. To do that, pellets were stained with CR solution for 20 min during continuous vortexing. The mixtures were again centrifuged at 90,000 rpm for 1 h, and pellets were washed twice with 500 μl of 20% ethanol. The pellets were resuspended in Milli-Q water and then spread evenly onto glass slides and air-dried at room temperature. The slides were analyzed using a microscope (Olympus SZ61 stereo zoom) equipped with two polarizer equipped with a CCD camera. The preparation of polyamino acid (PAA) amyloids was reported previously (45Fändrich M. Dobson C.M. The behaviour of polyamino acids reveals an inverse side chain effect in amyloid structure formation.EMBO J. 2002; 21: 5682-5690Crossref PubMed Scopus (428) Google Scholar, 46Arnott S. Dover S.D. Elliott A. Structure of β-poly-l-alanine: refined atomic co-ordinates for an anti-parallel β-pleated sheet.J. Mol. Biol. 1967; 30: 201-208Crossref PubMed Google Scholar47Fulara A. Lakhani A. Wójcik S. Nieznańska H. Keiderling T.A. Dzwolak W. Spiral superstructures of amyloid-like fibrils of polyglutamic acid: an infrared absorption and vibrational circular dichroism study.J. Phys. Chem. B. 2011; 115: 11010-11016Crossref PubMed Scopus (46) Google Scholar). Briefly, the 1 mg of lyophilized powder of positively charged poly-l-lysine (PLL) (Mr 70,000–150,000; Sigma) and negatively charged polyglutamic acid (PE) (Mr 2000–15,000; Sigma) were separately dissolved in 1 ml of sterile Milli-Q water. The pH of PLL solution was adjusted to 11.1 by adding a few drops of 10 mm NaOH, and the pH of PE was adjusted to pH 4.1 with dilute HCl. These freshly prepared PAAs were then incubated at 65 °C for 48 h. For preparing poly-l-alanine (PLA) (Mr 1000–5000; MP Biomedicals) amyloid, 2 mg of PLA was dissolved in 100 μl of trifluoroacetic acid (TFA) because it does not dissolve in most of the other solvents studied due to the highly hydrophobic nature of PLA. 10 μl of PLA solution was then gradually added into 190 μl of sterile Milli-Q water casted on a clean coverslip, and the entire setup was dried in steam. The resultant dried aggregates were collected and were used for the study. The secondary structure of PAA aggregates was analyzed using FTIR, and morphological analysis was done using an electron microscope. Further amyloid formation was confirmed using a CR binding by birefringence study and x-ray diffraction studies. For secondary structural analysis of the aggregated PAAs, FTIR spectroscopy was performed. FTIR spectra of the PAAs were recorded using a Vertex-80 FTIR instrument (Bruker, Germany) equipped with a deuterated triglycine sulfate detector. 10 μl of each aggregated PAA was spotted and dried on translucent KBr pellet that was made prior to the experiment by compressing ground KBr powder. 10 μl of sterile Milli-Q water was spotted on another KBr pellet and used for the background spectrum. FTIR spectra (1500–1800 cm−1) were recorded as an average of 32 scans, and raw data corresponding to the amide-I region (1600–1700 cm−1) was deconvoluted by the Fourier self-deconvolution method. The deconvoluted spectra were then subjected to a Lorenzian curve-fitting procedure using Opus-65 software. Two independent experiments were performed for each sample. For x-ray diffraction studies, sample solutions were dried either in glass capillary tubes or at the center of two glass rods arranged end to end. The resulting film was mounted parallel to the x-ray beam. The images were obtained using a Rigaku R Axis IV++ detector (Rigaku, Japan) mounted on a rotating anode. The sample to detector distance was 300 mm, and the image files were analyzed using Adxv software. Lyophilized powder of Aβ42 was a kind gift from Prof. Sudipta Maiti, Tata Institute of Fundamental Research (Navy Nagar, India). 1 mg of Aβ42 fibril was suspended in 50 mm sodium phosphate buffer, pH 7.4, and the peptide was dissolved by adding a few drops of 10 mm NaOH into it, after which the pH of the solution was brought back to pH 7.4. The final concentration of this solution was 300 μm. Aβ(25–35) was purchased from BACHEM. For amyloid fibril preparation, 0.5 mg of this peptide was dissolved in 500 μl of sterile Milli-Q water to obtain 1 mm solution. Both of the Aβ solutions were incubated at 37 °C, and the aggregation and amyloid formation was monitored using ThT binding studies (data not shown). The secondary structural changes were determined by CD spectroscopy (data not shown), and morphological analysis was done using an electron microscope. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was carried out to evaluate the toxicity of all amyloid fibrils formed in presence and absence of heparin using neuronal cell line SH-SY5Y. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (HiMedia, Mumbai, India) medium supplemented with 10% FBS (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 humidified environment at 37 °C. Cells were seeded at a density of 1 × 104 cells/well on a 96-well plate. After 24 h of incubation, the old culture medium was replaced with fresh medium along with the addition of various amyloid fibrils at 10 μm final concentration, and cells were further incubated for 24 h at 37 °C. After incubation, 10 μl of a 5 mg/ml MTT stock in PBS was added to each well, and the incubation was continued for 4 h. Finally, 100 μl of a solution containing 50% dimethylformamide and 20% SDS (pH 4.7) was added to each well and incubated. After overnight incubation in a 5% CO2 humidified environment at 37 °C, absorption values at 560 nm were determined with an automatic microplate reader (Spectramax M2, Molecular Devices). For evaluation of cell adhesion on amyloid fibrils, the fibrils were coated on glass coverslips. To do so, round glass coverslips of 12-mm diameter were first washed with 0.1 n NaOH for 30 min and then rinsed with PBS and subsequently sterilized. All coverslips were kept in 24-well plates and coated subsequently with different substrates. Glass coverslips were coated with 10 μg/cm2 of collagen and each amyloid fibril. For coating, each fibril solution was diluted in PBS, pH 7.4, and applied to the surface of the coverslip and kept at 4 °C overnight. The next day, excess solution was removed, and the surfaces were washed with PBS and blocked with 2% F127 Pluronic (Sigma) for 15 min at room temperature before plating the cells. Mouse fibroblasts (L929 and NIH 3T3), neuroblastoma cell SH-SY5Y, and PC12 cells were obtained from the National Centre For Cell Science (Pune, India) and maintained as per established protocols. All cells were maintained at 37 °C with 5% CO2 and plated at a density of 4 × 104 cells/well. For density-dependent experiments, glass coverslips were coated with kassinin at concentrations ranging from 0.5 to 7.5 μm at 37 °C for 2 h, blocked with 2% F127 Pluronic (Sigma) for 15 min at room temperature to prevent nonspecific cell attachment, and UV-sterilized. NIH 3T3 fibroblasts were cultured for 24 h and plated at a seeding density of 4500 cells/cm2 on the coated substrates. For drug studies, cells were cultured for 24 h and then exposed to 10 μm (for NIH" @default.
• W2230622832 created "2016-06-24" @default.
• W2230622832 creator A5013425190 @default.
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• W2230622832 date "2016-03-01" @default.
• W2230622832 modified "2023-10-16" @default.
• W2230622832 title "Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif" @default.
• W2230622832 cites W1523474069 @default.
• W2230622832 cites W1564193520 @default.
• W2230622832 cites W1590619267 @default.
• W2230622832 cites W1691536622 @default.
• W2230622832 cites W1845277443 @default.
• W2230622832 cites W1960543132 @default.
• W2230622832 cites W1966964152 @default.
• W2230622832 cites W1967589959 @default.
• W2230622832 cites W1968470624 @default.
• W2230622832 cites W1968942251 @default.
• W2230622832 cites W1969430572 @default.
• W2230622832 cites W1971060969 @default.
• W2230622832 cites W1971948710 @default.
• W2230622832 cites W1972429626 @default.
• W2230622832 cites W1976061072 @default.
• W2230622832 cites W1976201852 @default.
• W2230622832 cites W1978718923 @default.
• W2230622832 cites W1981545549 @default.
• W2230622832 cites W1984569087 @default.
• W2230622832 cites W1986594503 @default.
• W2230622832 cites W1987568997 @default.
• W2230622832 cites W1988437687 @default.
• W2230622832 cites W1988590842 @default.
• W2230622832 cites W1990542024 @default.
• W2230622832 cites W1991622848 @default.
• W2230622832 cites W1992045220 @default.
• W2230622832 cites W1992430893 @default.
• W2230622832 cites W1993006648 @default.
• W2230622832 cites W1997218140 @default.
• W2230622832 cites W1997937079 @default.
• W2230622832 cites W2000934926 @default.
• W2230622832 cites W2001339111 @default.
• W2230622832 cites W2009827441 @default.
• W2230622832 cites W2010320204 @default.
• W2230622832 cites W2011146030 @default.
• W2230622832 cites W2014290130 @default.
• W2230622832 cites W2020462791 @default.
• W2230622832 cites W2021785060 @default.
• W2230622832 cites W2023113549 @default.
• W2230622832 cites W2025595434 @default.
• W2230622832 cites W2026727321 @default.
• W2230622832 cites W2030749300 @default.
• W2230622832 cites W2030998305 @default.
• W2230622832 cites W2032487213 @default.
• W2230622832 cites W2038559728 @default.
• W2230622832 cites W2038673804 @default.
• W2230622832 cites W2041037209 @default.
• W2230622832 cites W2042036635 @default.
• W2230622832 cites W2043794106 @default.
• W2230622832 cites W2044895919 @default.
• W2230622832 cites W2046093999 @default.
• W2230622832 cites W2050896806 @default.
• W2230622832 cites W2051007768 @default.
• W2230622832 cites W2051908377 @default.
• W2230622832 cites W2052619557 @default.
• W2230622832 cites W2056563854 @default.
• W2230622832 cites W2057223961 @default.
• W2230622832 cites W2059699393 @default.
• W2230622832 cites W2059804089 @default.
• W2230622832 cites W2060393626 @default.
• W2230622832 cites W2063825596 @default.
• W2230622832 cites W2064297038 @default.
• W2230622832 cites W2064367582 @default.
• W2230622832 cites W2064773836 @default.
• W2230622832 cites W2065491098 @default.
• W2230622832 cites W2068157315 @default.
• W2230622832 cites W2072613948 @default.
• W2230622832 cites W2074252927 @default.
• W2230622832 cites W2075417889 @default.
• W2230622832 cites W2075482445 @default.
• W2230622832 cites W2081265873 @default.
• W2230622832 cites W2086052553 @default.
• W2230622832 cites W2092550529 @default.
• W2230622832 cites W2094199966 @default.
• W2230622832 cites W2095782512 @default.
• W2230622832 cites W2096410114 @default.
• W2230622832 cites W2105488386 @default.
• W2230622832 cites W2106638887 @default.
• W2230622832 cites W2107642756 @default.
• W2230622832 cites W2110227826 @default.
• W2230622832 cites W2111471786 @default.
• W2230622832 cites W2113827848 @default.
• W2230622832 cites W2114270638 @default.
• W2230622832 cites W2114286636 @default.
• W2230622832 cites W2119297140 @default.

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