Summary
The ultimate goal of proteomics is to characterize the function of all proteins in parallel and in the most physiologically relevant settings possible. A step toward this goal has been the introduction of activity-based proteomics. The simultaneous detection of individual protein activities can be facilitated directly in the proteome using specific activity-based probes consisting of a recognition site targeting a certain enzyme species, a properly positioned reactive site which forms a covalent bond with the target and a reporter tag for visualization and/or purification of the covalently bound target. As properties like polarity, size, charge, structure, and chemical reactivity of the reporter tag have a large impact on the reactivity of the probes toward the target enzymes probes suitable for reporter tagging after the enzyme-activity probe-binding event were designed. These probes resemble the natural substrates more closely and are small and hydrophobic enough to cross the membrane of living cells. Here the methodology for detection of lipolytic activities in intact living cells, including synthesis of probe and reporter, labeling procedure, and detection of target enzymes is described.
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References
Birner-Gruenberger, R, and Hermetter, A. (2007) Activity-based proteomics of lipolytic enzymes. Curr Drug Discov Technol 4(1):1–11.
Schmidinger, H., Birner-Gruenberger, R., Riesenhuber, G., Saf, R., Susani-Etzerodt, H., Hermetter, A. (2005) Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases. Chembiochem 6(10):1776–1781.
Susani-Etzerodt, H., Schmidinger, H., Riesenhuber, G, Birner-Gruenberger, R., Hermetter, A. (2006) A versatile library of activity-based probes for fluorescence detection and/or affinity isolation of lipolytic enzymes. Chem Phys Lipids 144(1):60–68.
Speers, A.E., Adam, G.C. and Cravatt, B.F. (2003) Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition. J Am Chem Soc 125(16):4686–4687.
Speers, A.E., and Cravatt, B.F. (2004) Profiling enzyme activities in vivo using click chemistry methods. Chem Biol 11(4):535–546.
Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V.V., Sharpless, K.B. and Finn, M.G. (2003) Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J Am Chem Soc 125(11):3192–3193.
Zimmermann, R., Strauss, J.G., Haemmerle, G., et al. (2004) Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306(5700):1383–1386.
Birner-Gruenberger, R., Susani-Etzerodt, H., Waldhuber, M., et al. (2005) The lipolytic proteome of mouse adipose tissue. Mol Cell Proteom 4(11):1710–1717.
Student, A.K., Hsu, R.Y. and Lane, M.D. (1980) Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. J Biol Chem 255(10):4745–4750.
Bradford, M.M. (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72(1–2):248–254.
Lim, K., and Chae, C.B. (1989) A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase. Biotechniques 7(6):576–579.
Greenspan, P., Mayer, E.P. and Fowler, S.D. (1985) Nile red: A selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100(3):965–973.
Holm, C., Olivecrona, G. and Ottosson, M. (2001) Assays of lipolytic enzymes. Methods in Molecular Biology (Totowa, NJ, United States) 155(Adipose Tissue Protocols): 97–119.
Acknowledgment
This work was supported by the “Click it: In vivo functional proteomic screening and cell imaging of lipases” project in the framework of GEN-AU (GENome Research in AUstria; http://www.gen-au.at/), funded by the Austrian Federal Ministry of Science and Research.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Schicher, M., Jesse, I., Birner-Gruenberger, R. (2009). Activity-Based Profiling of Lipases in Living Cells. In: Armstrong, D. (eds) Lipidomics. Methods in Molecular Biology™, vol 580. Humana Press. https://doi.org/10.1007/978-1-60761-325-1_14
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DOI: https://doi.org/10.1007/978-1-60761-325-1_14
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