Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.
References
[1]
Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75: 333–366.
[2]
Herczenik E, Gebbink MF (2008) Molecular and cellular aspects of protein misfolding and disease. FASEB J 22: 2115–2133.
[3]
Chiti F, Webster P, Taddei N, Clark A, Stefani M, et al. (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc Natl Acad Sci U S A 96: 3590–3594.
[4]
Dobson CM (1999) Protein misfolding, evolution and disease. Trends Biochem Sci 24: 329–332.
[5]
Dobson CM (2001) The structural basis of protein folding and its links with human disease. Philos Trans R Soc Lond B Biol Sci 356: 133–145.
[6]
Fandrich M, Fletcher MA, Dobson CM (2001) Amyloid fibrils from muscle myoglobin. Nature 410: 165–166.
[7]
Dobson CM (2003) Protein folding and misfolding. Nature 426: 884–890.
[8]
Lee CC, Nayak A, Sethuraman A, Belfort G, McRae GJ (2007) A three-stage kinetic model of amyloid fibrillation. Biophys J 92: 3448–3458.
[9]
Bouchard M, Zurdo J, Nettleton EJ, Dobson CM, Robinson CV (2000) Formation of insulin amyloid fibrils followed by FTIR simultaneously with CD and electron microscopy. Protein Sci 9: 1960–1967.
[10]
Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB, et al. (2003) Amyloid beta -protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways. Proc Natl Acad Sci U S A 100: 330–335.
[11]
Jimenez JL, Nettleton EJ, Bouchard M, Robinson CV, Dobson CM, et al. (2002) The protofilament structure of insulin amyloid fibrils. Proc Natl Acad Sci U S A 99: 9196–9201.
[12]
Smith MI, Sharp JS, Roberts CJ (2008) Insulin fibril nucleation: the role of prefibrillar aggregates. Biophys J 95: 3400–3406.
[13]
Smith MI, Sharp JS, Roberts CJ (2007) Nucleation and growth of insulin fibrils in bulk solution and at hydrophobic polystyrene surfaces. Biophys J 93: 2143–2151.
[14]
Kourie JI, Henry CL (2002) Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: the role of dangerous unchaperoned molecules. Clin Exp Pharmacol Physiol 29: 741–753.
[15]
Rao RV, Bredesen DE (2004) Misfolded proteins, endoplasmic reticulum stress and neurodegeneration. Curr Opin Cell Biol 16: 653–662.
[16]
Lashuel HA (2005) Membrane permeabilization: a common mechanism in protein-misfolding diseases. Sci Aging Knowledge Environ 2005: pe28.
[17]
Zhu X, Su B, Wang X, Smith MA, Perry G (2007) Causes of oxidative stress in Alzheimer disease. Cell Mol Life Sci 64: 2202–2210.
[18]
Campioni S, Mannini B, Zampagni M, Pensalfini A, Parrini C, et al. (2010) A causative link between the structure of aberrant protein oligomers and their toxicity. Nat Chem Biol 6: 140–147.
[19]
Morishima Y, Gotoh Y, Zieg J, Barrett T, Takano H, et al. (2001) Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of Fas ligand. J Neurosci 21: 7551–7560.
[20]
Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med (Berl) 81: 678–699.
[21]
Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, et al. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416: 507–511.
[22]
Meratan AA, Ghasemi A, Nemat-Gorgani M (2011) Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products. J Mol Biol 409: 826–838.
[23]
Austen BM, Paleologou KE, Ali SA, Qureshi MM, Allsop D, et al. (2008) Designing peptide inhibitors for oligomerization and toxicity of Alzheimer’s beta-amyloid peptide. Biochemistry 47: 1984–1992.
[24]
Karp G (2005) Cell and molecular biology: concepts and experiments. Hoboken, NJ: John Wiley and Sons. 864 p.
Dische FE, Wernstedt C, Westermark GT, Westermark P, Pepys MB, et al. (1988) Insulin as an amyloid-fibril protein at sites of repeated insulin injections in a diabetic patient. Diabetologia 31: 158–161.
[27]
Gibson TJ, Murphy RM (2006) Inhibition of insulin fibrillogenesis with targeted peptides. Protein Sci 15: 1133–1141.
[28]
Wang SS, Liu KN, Han TC (2010) Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants. Biochim Biophys Acta 1802: 519–530.
[29]
Arora A, Ha C, Park CB (2004) Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures. Protein Sci 13: 2429–2436.
[30]
Nielsen L, Frokjaer S, Carpenter JF, Brange J (2001) Studies of the structure of insulin fibrils by Fourier transform infrared (FTIR) spectroscopy and electron microscopy. J Pharm Sci 90: 29–37.
[31]
Ahmad A, Millett IS, Doniach S, Uversky VN, Fink AL (2004) Stimulation of insulin fibrillation by urea-induced intermediates. J Biol Chem 279: 14999–15013.
[32]
Nielsen L, Khurana R, Coats A, Frokjaer S, Brange J, et al. (2001) Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry 40: 6036–6046.
[33]
Zako T, Sakono M, Hashimoto N, Ihara M, Maeda M (2009) Bovine insulin filaments induced by reducing disulfide bonds show a different morphology, secondary structure, and cell toxicity from intact insulin amyloid fibrils. Biophys J 96: 3331–3340.
[34]
Grudzielanek S, Velkova A, Shukla A, Smirnovas V, Tatarek-Nossol M, et al. (2007) Cytotoxicity of insulin within its self-assembly and amyloidogenic pathways. J Mol Biol 370: 372–384.
[35]
Nieto-Suárez M, Vila-Romeu N, Prieto I (2008) Behaviour of insulin Langmuir monolayers at the air-water interface under various conditions. Thin Solid Films 516: 8873–8879.
[36]
Lin F, Kwok D, Policova Z, Zingg W, Neumann A (1995) The effect of ph and concentration on the surface tension of adsorbed layers of various insulin preparations. Colloids and Surfaces B: Biointerfaces 3: 281–286.
[37]
Kim JR, Gibson TJ, Murphy RM (2003) Targeted control of kinetics of beta-amyloid self-association by surface tension-modifying peptides. J Biol Chem 278: 40730–40735.
[38]
Arora A, Ha C, Park CB (2004) Inhibition of insulin amyloid formation by small stress molecules. FEBS letters 564: 121–125.
[39]
Laughlin SB, Sejnowski TJ (2003) Communication in neuronal networks. Science 301: 1870–1874.
[40]
Sporns O, Tononi G, Edelman GM (2000) Connectivity and complexity: the relationship between neuroanatomy and brain dynamics. Neural Netw 13: 909–922.
[41]
Eisert R, Felau L, Brown LR (2006) Methods for enhancing the accuracy and reproducibility of Congo red and thioflavin T assays. Anal Biochem 353: 144–146.
[42]
Messina P, Prieto G, Dodero V, Cabrerizo Vílchez M, Maldonado Valderrama J, et al. (2006) Surface characterization of human serum albumin and sodium perfluorooctanoate mixed solutions by pendant drop tensiometry and circular dichroism. Biopolymers 82: 261–271.
[43]
Narsimhan G, Uraizee F (1992) Kinetics of Adsorption of Globular Proteins at an Air Water Interface. Biotechnology prog 8: 187–196.
[44]
Lin TY, Timasheff SN (1996) On the role of surface tension in the stabilization of globular proteins. Protein Sci 5: 372–381.
[45]
Treuheit MJ, Kosky AA, Brems DN (2002) Inverse relationship of protein concentration and aggregation. Pharm res 19: 511–516.
[46]
Oliveberg M, Tan YJ, Fersht AR (1995) Negative activation enthalpies in the kinetics of protein folding. Proc Natl Acad Sci U S A 92: 8926–8929.
[47]
Makievski A, Fainerman V, Bree M, Wüstneck R, Kr？gel J, et al. (1998) Adsorption of proteins at the liquid/air interface. J Phys Chem B 102: 417–425.
[48]
Kaufmann WE, Moser HW (2000) Dendritic anomalies in disorders associated with mental retardation. Cereb Cortex 10: 981–991.
[49]
Knobloch M, Mansuy IM (2008) Dendritic spine loss and synaptic alterations in Alzheimer’s disease. Mol Neurobiol 37: 73–82.
[50]
Marwarha G, Dasari B, Prasanthi JR, Schommer J, Ghribi O (2010) Leptin reduces the accumulation of Abeta and phosphorylated tau induced by 27-hydroxycholesterol in rabbit organotypic slices. J Alzheimers Dis 19: 1007–1019.
[51]
Liu X, Xu K, Yan M, Wang Y, Zheng X (2010) Protective effects of galantamine against Abeta-induced PC12 cell apoptosis by preventing mitochondrial dysfunction and endoplasmic reticulum stress. Neurochem Int 57: 588–599.
[52]
Tusi SK, Khalaj L, Ashabi G, Kiaei M, Khodagholi F (2011) Alginate oligosaccharide protects against endoplasmic reticulum-and mitochondrial-mediated apoptotic cell death and oxidative stress. Biomaterials 32: 5438–5458.
[53]
Dobson CM (2004) Principles of protein folding, misfolding and aggregation. Semin Cell Dev Biol 15: 3–16.
[54]
Stefani M (2010) Biochemical and biophysical features of both oligomer/fibril and cell membrane in amyloid cytotoxicity. FEBS J 277: 4602–4613.
[55]
Caughey B, Lansbury PT (2003) Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu Rev Neurosci 26: 267–298.
