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Application of Photochemical Cross-linking to the Study of Oligomerization of Amyloidogenic Proteins

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Amyloid Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 849))

Abstract

Assembly of amyloidogenic proteins into toxic oligomers and fibrils is an important pathogenic feature of over 30 amyloid-related diseases. Understanding the structures and mechanisms involved in the assembly process is necessary for rational approaches geared at inhibiting formation of these toxic species. Here, we review the application of photo-induced cross-linking of unmodified proteins (PICUP) to two disease-related amyloidogenic proteins (1) islet amyloid polypeptide (IAPP), whose toxic oligomers are thought to cause the demise of pancreatic β-cells in type-2 diabetes mellitus and (2) α-synuclein, which aggregates into toxic oligomers and precipitates in Lewy bodies in Parkinson’s disease. PICUP is an effective method allowing chemical “freezing” of dynamically changing oligomers and subsequent study of the oligomer size distribution that existed before cross-linking. The method has provided insights into the factors controlling early oligomerization, which could not be obtained by other means. We discuss sample preparation, experimental details, optimization of parameters, and troubleshooting.

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Notes

  1. 1.

    Measuring the concentration immediately after dissolution is essential because as proteins aggregate, the absorbance values vary compared with the freshly dissolved proteins. Hence, for aggregating proteins, it is recommended to measure the concentration immediately after dissolution in appropriate buffer.

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Acknowledgments

This work was supported by grants from American Health Assistance Foundation (A2008-350), the Jim Easton Consortium for Alzheimer’s Drug Discovery and Biomarker Development at UCLA, UCLA Center for Gene-Environment Studies in Parkinson’s Disease, and the Michael J. Fox Foundation.

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Authors and Affiliations

  1. Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Dahabada H. J. Lopes, Sharmistha Sinha, Clark Rosensweig & Gal Bitan

  2. Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, USA

    Sharmistha Sinha

Authors
  1. Dahabada H. J. Lopes
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  2. Sharmistha Sinha
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  3. Clark Rosensweig
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  4. Gal Bitan
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Corresponding author

Correspondence to Gal Bitan .

Editor information

Editors and Affiliations

  1. School of Medicine, Dept. Psychiatry, New York University, First Ave. 550, New York, 10016, New York, USA

    Einar M. Sigurdsson

  2. Centro Nacional de Microbiología, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km 2, Madrid, 28220, Spain

    Miguel Calero

  3. Inst. Química Física, Rocasolano, CSIC Madrid, C/ Serrano 119, Madrid, 28006, Spain

    María Gasset

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Lopes, D.H.J., Sinha, S., Rosensweig, C., Bitan, G. (2012). Application of Photochemical Cross-linking to the Study of Oligomerization of Amyloidogenic Proteins. In: Sigurdsson, E., Calero, M., Gasset, M. (eds) Amyloid Proteins. Methods in Molecular Biology, vol 849. Humana Press. https://doi.org/10.1007/978-1-61779-551-0_2

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  • DOI: https://doi.org/10.1007/978-1-61779-551-0_2

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-550-3

  • Online ISBN: 978-1-61779-551-0

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