Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors
The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders.
Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest.
Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides.
- Chiti F, Dobson CM: Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 2006, 75:333–366. CrossRef
- Fernandez-Busquets X, de Groot NS, Fernandez D, Ventura S: Recent structural and computational insights into conformational diseases. Curr Med Chem 2008,15(13):1336–1349. CrossRef
- Friedman R: Aggregation of amyloids in a cellular context: modelling and experiment. Biochem J 2011,438(3):415–426.
- Ross CA, Poirier MA: Protein aggregation and neurodegenerative disease. Nat Med 2004,10(Suppl):S10-S17. CrossRef
- Lee LL, Ha H, Chang YT, DeLisa MP: Discovery of amyloid-beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility. Protein Sci 2009,18(2):277–286. CrossRef
- Morell M, de Groot NS, Vendrell J, Aviles FX, Ventura S: Linking amyloid protein aggregation and yeast survival. Mol Biosyst 2011,7(4):1121–1128. CrossRef
- Amijee H, Madine J, Middleton DA, Doig AJ: Inhibitors of protein aggregation and toxicity. Biochem Soc Trans 2009,37(Pt 4):692–696. CrossRef
- Hardy J, Selkoe DJ: The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 2002,297(5580):353–356. CrossRef
- Kuperstein I, Broersen K, Benilova I, Rozenski J, Jonckheere W, Debulpaep M, Vandersteen A, Segers-Nolten I, Van Der Werf K, Subramaniam V, et al.: Neurotoxicity of Alzheimer's disease Abeta peptides is induced by small changes in the Abeta42 to Abeta40 ratio. EMBO J 2010,29(19):3408–3420. CrossRef
- Karran E, Mercken M, De Strooper B: The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics. Nat Rev Drug Discov 2011,10(9):698–712. CrossRef
- Bonda DJ, Lee HG, Blair JA, Zhu X, Perry G, Smith MA: Role of metal dyshomeostasis in Alzheimer's disease. Metallomics 2011,3(3):267–270. CrossRef
- Jomova K, Vondrakova D, Lawson M, Valko M: Metals, oxidative stress and neurodegenerative disorders. Mol Cell Biochem 2010,345(1–2):91–104. CrossRef
- de Groot NS, Espargaro A, Morell M, Ventura S: Studies on bacterial inclusion bodies. Future Microbiol 2008, 3:423–435. CrossRef
- Ventura S, Villaverde A: Protein quality in bacterial inclusion bodies. Trends Biotechnol 2006,24(4):179–185. CrossRef
- Carrio M, Gonzalez-Montalban N, Vera A, Villaverde A, Ventura S: Amyloid-like properties of bacterial inclusion bodies. J Mol Biol 2005,347(5):1025–1037. CrossRef
- Morell M, Bravo R, Espargaro A, Sisquella X, Aviles FX, Fernandez-Busquets X, Ventura S: Inclusion bodies: specificity in their aggregation process and amyloid-like structure. Biochim Biophys Acta 2008,1783(10):1815–1825. CrossRef
- de Groot NS, Sabate R, Ventura S: Amyloids in bacterial inclusion bodies. Trends Biochem Sci 2009,34(8):408–416. CrossRef
- Wang L, Maji SK, Sawaya MR, Eisenberg D, Riek R: Bacterial inclusion bodies contain amyloid-like structure. PLoS Biol 2008,6(8):e195. CrossRef
- Sabate R, de Groot NS, Ventura S: Protein folding and aggregation in bacteria. Cell Mol Life Sci 2010,67(16):2695–2715. CrossRef
- Garcia-Fruitos E, Sabate R, de Groot NS, Villaverde A, Ventura S: Biological role of bacterial inclusion bodies: a model for amyloid aggregation. FEBS J 2011,278(14):2419–2427. CrossRef
- Lotti M: Bacterial inclusion bodies as active and dynamic protein ensembles. FEBS J 2011,278(14):2407. CrossRef
- Villar-Pique A, de Groot NS, Sabate R, Acebron SP, Celaya G, Fernandez-Busquets X, Muga A, Ventura S: The Effect of Amyloidogenic Peptides on Bacterial Aging Correlates with Their Intrinsic Aggregation Propensity. J Mol Biol 2011.
- Dasari M, Espargaro A, Sabate R, Lopez Del Amo JM, Fink U, Grelle G, Bieschke J, Ventura S, Reif B: Bacterial Inclusion Bodies of Alzheimer's Disease beta-Amyloid Peptides Can Be Employed To Study Native-Like Aggregation Intermediate States. Chem Bio Chem 2011,12(3):407–423.
- Garcia-Fruitos E, Gonzalez-Montalban N, Morell M, Vera A, Ferraz RM, Aris A, Ventura S, Villaverde A: Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins. Microb Cell Fact 2005, 4:27. CrossRef
- de Groot NS, Aviles FX, Vendrell J, Ventura S: Mutagenesis of the central hydrophobic cluster in Abeta42 Alzheimer's peptide, Side-chain properties correlate with aggregation propensities. FEBS J 2006,273(3):658–668. CrossRef
- de Groot NS, Ventura S: Protein activity in bacterial inclusion bodies correlates with predicted aggregation rates. J Biotechnol 2006,125(1):110–113. CrossRef
- de Groot NS, Ventura S: Effect of temperature on protein quality in bacterial inclusion bodies. FEBS Lett 2006, 580:6471–6476. CrossRef
- Kiefhaber T, Rudolph R, Kohler HH, Buchner J: Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation. Bio/Technology 1991,9(9):825–829. CrossRef
- Jin L, Wu WH, Li QY, Zhao YF, Li YM: Copper inducing Abeta42 rather than Abeta40 nanoscale oligomer formation is the key process for Abeta neurotoxicity. Nanoscale 2011,3(11):4746–4751. CrossRef
- Zhang X, Smith DL, Meriin AB, Engemann S, Russel DE, Roark M, Washington SL, Maxwell MM, Marsh JL, Thompson LM, et al.: A potent small molecule inhibits polyglutamine aggregation in Huntington's disease neurons and suppresses neurodegeneration in vivo. Proc Natl Acad Sci U S A 2005,102(3):892–897. CrossRef
- Chen J, Armstrong AH, Koehler AN, Hecht MH: Small molecule microarrays enable the discovery of compounds that bind the Alzheimer's Abeta peptide and reduce its cytotoxicity. J Am Chem Soc 2010,132(47):17015–17022. CrossRef
- Hegde ML, Bharathi P, Suram A, Venugopal C, Jagannathan R, Poddar P, Srinivas P, Sambamurti K, Rao KJ, Scancar J, et al.: Challenges associated with metal chelation therapy in Alzheimer's disease. J Alzheimer's Dis 2009,17(3):457–468.
- Necula M, Kayed R, Milton S, Glabe CG: Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem 2007,282(14):10311–10324. CrossRef
- Necula M, Breydo L, Milton S, Kayed R, van der Veer WE, Tone P, Glabe CG: Methylene blue inhibits amyloid Abeta oligomerization by promoting fibrillization. Biochemistry 2007,46(30):8850–8860. CrossRef
- Kim H, Park BS, Lee KG, Choi CY, Jang SS, Kim YH, Lee SE: Effects of naturally occurring compounds on fibril formation and oxidative stress of beta-amyloid. J Agric Food Chem 2005,53(22):8537–8541. CrossRef
- Jagota S, Rajadas J: Effect of Phenolic Compounds Against Abeta Aggregation and Abeta-Induced Toxicity in Transgenic C. elegans. Neurochem Res 2012,37((1):40–48. CrossRef
- Matsuzaki K, Noguch T, Wakabayashi M, Ikeda K, Okada T, Ohashi Y, Hoshino M, Naiki H: Inhibitors of amyloid beta-protein aggregation mediated by GM1-containing raft-like membranes. Biochim Biophys Acta 2007,1768(1):122–130. CrossRef
- Chen WT, Liao YH, Yu HM, Cheng IH, Chen YR: Distinct effects of Zn2+, Cu2+, Fe3+, and Al3+ on amyloid-beta stability, oligomerization, and aggregation: amyloid-beta destabilization promotes annular protofibril formation. J Biol Chem 2011,286(11):9646–9656. CrossRef
- De Felice FG, Vieira MN, Saraiva LM, Figueroa-Villar JD, Garcia-Abreu J, Liu R, Chang L, Klein WL, Ferreira ST: Targeting the neurotoxic species in Alzheimer's disease: inhibitors of Abeta oligomerization. FASEB J 2004,18(12):1366–1372. CrossRef
- Rodriguez-Rodriguez C, Sanchez De Groot N, Rimola A, Alvarez-Larena A, Lloveras V, Vidal-Gancedo J, Ventura S, Vendrell J, Sodupe M, Gonzalez-Duarte P: Design, selection, and characterization of thioflavin-based intercalation compounds with metal chelating properties for application in Alzheimer's disease. J Am Chem Soc 2009,131(4):1436–1451. CrossRef
- Hilario E, Romero I, Celis H: Determination of the physicochemical constants and spectrophotometric characteristics of the metallochromic Zincon and its potential use in biological systems. J Biochem Biophys Methods 1990,21(3):197–207. CrossRef
- Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
Microbial Cell Factories
- Online Date
- May 2012
- Online ISSN
- BioMed Central
- Additional Links
- Inclusion bodies
- Protein folding
- Protein aggregation
- Author Affiliations
- 1. Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, E-08193, Bellaterra, Spain
- 2. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, E-08193, Bellaterra, Spain
- 3. Present address: Departament de Fisicoquímica, Facultat de Farmàcia, Universitat de Barcelona, Avda. Joan XXIII, 08028, Barcelona, Spain
- 4. Present address: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, United Kingdom